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Chemical Transmitters and Modulation of Sleep vs. Wake Promoting Neurons Mandana Modirrousta M.D. Department ofNeurology and Neurosurgery Neuroscience Program McGiIl University, Montreal, Quebec, Canada December 2005 Supervisor Barbara E. Jones Ph.D. A thesis submitted to the Faculty of Graduate Studies and Research In partial fulfillment of the requirements for the degree Doctor ofPhilosophy © Mandana Modirrousta, 2005 1+1 Library and Archives Canada Bibliothèque et Archives Canada Published Heritage Branch Direction du Patrimoine de l'édition 395 Wellington Street Ottawa ON K1A ON4 Canada 395, rue Wellington Ottawa ON K1A ON4 Canada Your file Votre référence ISBN: 978-0-494-25212-3 Our file Notre référence ISBN: 978-0-494-25212-3 NOTICE: The author has granted a nonexclusive license allowing Library and Archives Canada to reproduce, publish, archive, preserve, conserve, communicate to the public by telecommunication or on the Internet, loan, distribute and sell th es es worldwide, for commercial or noncommercial purposes, in microform, paper, electronic and/or any other formats. AVIS: L'auteur a accordé une licence non exclusive permettant à la Bibliothèque et Archives Canada de reproduire, publier, archiver, sauvegarder, conserver, transmettre au public par télécommunication ou par l'Internet, prêter, distribuer et vendre des thèses partout dans le monde, à des fins commerciales ou autres, sur support microforme, papier, électronique et/ou autres formats. The author retains copyright ownership and moral rights in this thesis. Neither the thesis nor substantial extracts from it may be printed or otherwise reproduced without the author's permission. L'auteur conserve la propriété du droit d'auteur et des droits moraux qui protège cette thèse. Ni la thèse ni des extraits substantiels de celle-ci ne doivent être imprimés ou autrement reproduits sans son autorisation. ln compliance with the Canadian Privacy Act some supporting forms may have been removed from this thesis. Conformément à la loi canadienne sur la protection de la vie privée, quelques formulaires secondaires ont été enlevés de cette thèse. While these forms may be included in the document page count, their removal does not represent any loss of content from the thesis. Bien que ces formulaires aient inclus dans la pagination, il n'y aura aucun contenu manquant. ••• Canada q'o my lius6ana, Œelizaâ ana my ûttfe son, (JJarsa wlio inspirea me witli tlie passion of l'ove ana Eife ana to my supervisor wlio tauglit me tlie meanino ofpatience ana e~ce{fent researcli. II Abstract Aithough evidence has suggested a dual role of the basal forebrain (BF) in arousai and sleep generation, the neurotransmitter identity and activity ofBF neurons serving these different functions have remained uncertain. Furthermore, few studies have been done to clarify how the wake vs. sleep promoting neurons may be modulated to generate their differential activity and sleep-wake cycle. Using a paradigm of sleep deprivation and sleep recovery and examining c-Fos expression as an indicator of cell activity, we found that across the BF and the adjacent preoptic area, more cells including cholinergie neurons were active during waking than during sleep and thus contribute to generating a waking state. On the other hand, the proportion of c-Fos expressing neurons that were GABAergic was higher during sleep recovery than sleep deprivation, indicating that particular GABAergic cells are involved in generating sleep. Aithough the posterior hypothalamus has long been known to play a critical role in the maintenance ofwaking, the neurotransmitter identity ofneurons fulfilling this role was not known. Here using c-Fos, we show that Orexin (Orx) neurons are active during waking. On the other hand, we show that co-distributed cells containing melanin concentrating hormone (MCH) are more active during sleep recovery than sleep deprivation, suggesting an opposite role ofMCH to that of Orx neurons in sleep vs. waking regulation. Sleep vs. wake promoting cells could be differently modulated by noradrenaline (NA) and accordingly would bear different adrenergic receptors. We found that the majority ofGABAergic BF cells expressing c-Fos during sleep bear alpha2 adrenergic III receptors (U2AAR). They would accordingly be inhibited during waking through these receptors. We also found that many Orx cells in the hypothalamus bear UIAAR and thus would be excited by NA during waking. Like the BF GABAergic cells, many MeR neurons were endowed with U2AAR and thus would be inhibited during waking. Activation vs. inhibition of sleep or wake-active cells could also be modulated by changes in the availability of cell surface receptors across behavioral states. We show that following sleep deprivation, the presence and the intensity of GABAARs on the BF cholinergie cell membrane were increased. Such activity dependent changes of GABAARs could underlie homeostatic regulation ofwake promoting cells across the sleep-waking cycle. These studies identify clearly for the first time the specific neurotransmittercontaining neurons that are active during and thus generative for waking vs. sleep through the BF, the preoptic area and the posterior hypothalamus. They moreover illuminate how by bearing different receptors or changing membrane receptor availability, different cells can be modulated to generate the sleep-waking cycle. IV Résumé Bien qu'il ait été suggéré que le télencéphale basal joue un double rôle dans la génération de l'éveil et du sommeil, l'identité des neurotransmetteurs ainsi que l'activité des neurones de cette structure permettant l'établissement de ces différentes fonctions restent à éclaircir. En outre, peu d'études ont été conduites pour clarifier comment les divers neurones impliqués peuvent être modulés pour permettre leurs activités différentielles pour générer le cycle veille-sommeil. Utilisant un modèle de privation et de rétablissement de sommeil et en examinant l'expression de c-Fos comme un indicateur d'activité cellulaire, nous avons trouvé que dans l'ensemble du télencéphale basal et l'aire préoptique adjacente plus de cellules, incluant les neurones cholinergiques, étaient actives durant l'éveil que pendant le sommeil et ainsi contribuent à la génération de l'état d'éveil. D'autre part, parmi les neurones exprimant c-Fos, la proportion des cellules GABAergiques était plus grande pendant le rétablissement que durant la privation de sommeil, indiquant que les cellules GABAergiques sont particulièrement impliquées dans la génération du sommeil. Bien que l'hypothalamus postérieur ait longtemps été reconnu pour jouer un rôle critique dans le maintien de l'éveil, l'identité du neurotransmetteur des neurones remplissant ce rôle n'était pas connue. En utilisant c-Fos, nous avons montré que les neurones exprimant les peptides Orexine (Orx) sont actifs durant l'éveil. Par ailleurs, nous avons montré que les neurones co-distribués contenant 1'hormone de mélanoconcentration (MeR) sont plus actifs durant le rétablissement du sommeil que pendant la v privation de sommeil, suggérant un rôle opposé des neurones à MCH à celui des neurones à Orx dans la régulation du sommeil et de l'éveil. Les cellules à l'origine du sommeil et celles promouvant l'éveil pourraient être différemment modulées par la noradrénaline et porteraient par conséquent des récepteurs adrénergiques différents. Nous avons trouvé que la majorité des neurones GABAergiques du télencéphale basal exprimant c-Fos durant le sommeil porte des récepteurs adrénergiques alpha2 (Œ2AAR). Ces cellules seraient donc inhibées durant l'éveil à travers ces récepteurs. Nous avons aussi trouvé que beaucoup de neurones à Orx dans l'hypothalamus portent ŒIAAR et ainsi seraient excités par noradrénaline pendant l'éveil. Comme les cellules GABAergiques du télencéphale basal, beaucoup de neurones à MCH sont dotés de Œ2AAR et seraient donc inhibés durant l'éveil. L'activation ou l'inhibition des cellules pendant le sommeil et l'éveil pourraient aussi être modulées par des changements de disponibilité des récepteurs membranaires lors des différents états. Nous avons montré que la présence ou le nombre des GABAARs sur la membrane des cellules cholinergiques du télencéphale basal sont augmentés après une période de privation de sommeil. De tels changements de GABAARs qui dépendent de l'activité pourraient être à la base de la régulation des cellules générant l'éveil pendant le cycle veille-sommeil. Ces études identifient clairement pour la première fois, dans le télencéphale basal, l'aire préoptique et l'hypothalamus postérieur, des neurones contenant un neurotransmetteur spécifique et qui favorisent l'éveil ou le sommeil. De plus, elles révèlent comment, en portant des récepteurs différents ou en changeant la disponibilité VI des récepteurs membranaires, des cellules différentes peuvent être modulées pour générer le cycle veille-sommeil. VII Note to examiners This thesis consists of 4 manuscripts, 3 published, 1 in preparation for submission. The author ofthis thesis is the main researcher and first author in 3, and coauthor in 1 manuscript. The original publisher and co-authors have given their written consents that these manuscripts be included in this thesis. The manuscripts citations are: 1. Manns ID, Lee MG, Modirrousta M, Hou YP, Jones BE Alpha 2 adrenergic receptors on GABAergic, putative sleep-promoting basal forebrain neurons. Eur J Neurosci. 2003 Aug;18(3):723-7. 2. Modirrousta, M, Mainville L, Jones BE Gabaergic neurons with alpha2-adrenergic receptors in basal forebrain and preoptic area express c-Fos during sleep. Neuroscience.2004;129(3):803-10. 3. Modirrousta, M, Mainville L, Jones BE Orexin and MeH neurons express c-Fos differently after sleep deprivation vs. recovery and bear different adrenergic receptors. Eur J Neurosci. 2005 May;21(10):2807-16. 4. Modirrousta, M, Mainville L, Jones BE GABAA receptor modifications on basal forebrain cholinergie cells across the sleepwaking cycle (In preparation). VIII Contribution of authors My contribution to the first manuscript was mapping and estimating proportions of GABAergic cens labeled for the U2AAR in the BF and making comments on the text of the manuscript. 1 was the principal investigator of the next three manuscripts. My contributions were designing and performing the experimental paradigm of sleep deprivation and sleep recovery in rats, executing the experiments inc1uding experimental manipulation and observation, immunohistochemical processing ofbrains, image analysis and data analysis using statistics. 1 was also the main author of the three manuscripts. The results in an of the four manuscripts were original and novel and consisted of new findings that have illuminated several important areas in the sleep field. Lynda Mainville, the coauthor in the three manuscripts and the laboratory technician, performed sorne of immunohistochemical staining. IX Acknowledgements Throughout my graduate studies, 1 have had the privilege of the company, support and friendship of a number of individuals. 1 first would like to express my sincerest gratitude to my exceptional supervisor, Dr. Barbara Jones, who with her vast knowledge, experience, kindness and patience guided me through a fruitful and successful graduate pro gram. 1 am especially grateful to her as she taught me that acquiring broad knowledge from different aspects around our theories is the pivotai principles of qualified research. Dr. Jones was always very encouraging whenever 1 asked a question. She not only answered each of my simplest questions with her endless patience, but she also opened new windows of other fascinating and interesting ideas and concepts. Thank you Dr. Jones, my appreciation is boundless. 1 must also thank Lynda Mainville, who, with her excellent skills and knowledge in immunohistochemistry, trained me in a way where 1 finally felt confident in the accuracy ofwhat 1 was doing. 1 thank my colleagues in the lab, the soon to be Dr. Pablo Henny, and Drs Maan Gee Lee, Oum Hassani and Frederic Brischoux, who were always generously available whenever 1 sought help. They also added the flavour of exciting and stimulating scientific discussions in the lab and were always eager to hear about my new results and findings. 1 especially thank Dr. Frederic Brischoux for his great assistance in the French translation of the abstract. 1 extremely thank Naomi Takeda who has been a precious asset in the Complex Neural System Unit. She always found the best possible solutions for my problems and x always kindly provided me with her great help and information. Her presence brings relief to everyone who works with her. 1 thank my advisory committee members, Dr. Edith Hamel, the late Dr. Angel Alonso and my mentor Dr. Andrea Leblanc who provided excellent advice and helped me to obtain a broader view in my field of research. 1 deeply thank my husband, Dr. Behzad Mansouri, who was always there for me, in life and in research as well. 1 thank him for his great help in editing and organizing my thesis. 1 would also like to sincerely thank my parents for their eternal kindness, love and support. XI Abbreviations ACh, acetylcholine AR, adrenergic receptor uIAR, alphal adrenergic receptor u2AR, alpha2 adrenergic receptor BF, basal forebrain ChAT, choline acetyltransferase EEG, e1ectroencephalogram DBB, diagonal band of Broca nuclei DMH, dorsomedial hypothalamic nucleus Dopamine, DA GABA gamma amino butyric acid GAD, glutamic acid decarboxylase GP, globus pallidum Interleukin l, III LC, locus coeruleus LH, lateral hypothalamus LPO, lateral preoptic area MCH, melanin concentrating hormone MCPO, magnocellular preoptic area mIPSCs, miniature Inhibitory Post-Synaptic Currents MnPO, median preoptic nucleus XII MPO, medial preoptic area MS, medial septum NA, noradrenaline Nb, Neurobiotin NDS, nonnal donkey serum Non-REMS, non-rapid eye movement sleep Orx,Orexin PF, perifomical area POA, preoptic area Prostoglandine D2, PGD2 PS, paradoxical sleep REMS, rapid eye movement sleep Rs, receptors SC, sleep control SCN, suprachiasmatic nucleus SD, sleep deprivation SI, substantia innominata SIa, substantia innominata, anterior part SIp, substantia innominata, posterior part SR, sleep recovery SWA, slow wave activity SWS, slow wave sleep TMN, tuberomammillary nucleus VLPO, ventrolateral preoptic area XIII ZI, zona incerta XIV Table of contents Abstract •....••.•.•................................................•........................................................................ III Résumé .............••..............••..•................•.•.•.•.........•.•.••.•...........•........•.•.•.•.•.......•.•...•........•.•....... V Note to examiners ...•.••....•.•.•.•..•.•.•.•.......•.•.•.•.......•.•.•.•........•.•.•.•.•.•....•.•.•..•....•..•.•••.•..•.....•.•.• VIII Contribution of authors •..........••.•.•..............•.•.••.......•.•.•..........•••.•.•........••.•.•.....•.•.•.••.•.•.•.•.•.•. IX A cknowledgements••••••••••••••••••••••.•••••••••••••••••••••••••••.•••••••••••••••••••••••••••••••••••••••••••••••••••••••••• x Abbreviations ..................................•...•...........•...•...•........•.•.•..........••..........................•.•....... XII Table of contents .............................•.•............•.•.•..•.........•.••••...........•............................••....... XV 1. General introduction ...•.••.•.•...•.•..••••••••••.•...•.••••••......••••.••.......••••.•.......•.•••.•...•..•.•.•........ 1 1.1 Sleep vs. wake promoting neurons in the basal forebrain and adjacent preoptic area 5 1.2 VLPO, an area of sleep active cells ........................•...........•..•...........•..••.....•...•••........•.•.•.. 7 1.3 The posterior hypothalamus and sleep vs. wake promoting neurons ........................... 9 1.4 c-Fos expression as an indicator of neuronal activity ................................................... 10 1.5 c-Fos expression in the brain across the sleep-waking cycle ........................................ 12 1.6 Modulation of sleep vs. wake promoting neurons by noradrenaline•.....•.•.•.••.•.....•.•••. 14 1.7 The dynamics of GABAA receptors during sleep vs. waking on cholinergie ceUs of the basal forebrain ............•.•.............................................••.••.........•.•.•••......•...•.•.•.•.•••••••.•..•.•.•.•••.• 15 1.8 The first experiment......................................................................................................... 17 1.9 The second experiment ........•.•.•.......................•.•.•..•.•...•.•.•.•.•••..•..••.•.•.•.•.••••••.••.•..•...•.•.•.• 18 1.10 The third experiment ..................................................................................................... 19 1.11 The fourth experiment .....•.....•.•.•............•••.......................................................•••.......•.• 19 1.12 Preface to chapter 1 •.................•...••......•...•............•.•....................•.................•.•..•......... 21 2. Chapter 1: Alpha 2 Adrenergic Receptors on GABAergic Putative Sleep-Promoting Basal Forebrain Neurons .•......................................................................•.•.......•....•........ 22 2.1 Abbreviations ................................................................................................................... 23 2.2 Abstract •................................•.•...........•...............•.•..........•...•.•.....•....•.......•..•.................... 24 2.3 Introduction ............•..............•...........•............................•.•.•••..........•.•.•.....•.•.•.•••......•••..... 26 2.4 Materials and Methods .........................................•..........•.•........................•...•........•.•..... 28 2.5 Results .....................•.........•....•...............•......................•.•.•..•.....•.•.•.•.•.•.•...•.•......•............. 31 2.6 Discussion...........•.•.•........•.•...•...•..••.•...............•..•........•.•.•................•.........•.........•............ 39 2. 7 Acknowledgements ................................................................•.•....................•.•.•.•......•.•.•.• 42 2.8 Preface to chapter 2 ......................................................................................................... 43 xv 3. Chapter 2: GABAergic Neurons with Alpha 2- Adrenergic Receptors in Basal Forebrain and Preoptic Area Express c-Fos during Sieep •••••••••••••••••••••••••••••••••••••••••••• 45 3.1 Abbreviations ................................................................................................................... 46 3.2 Abstract ............................................................................................................................. 47 3.3 Introduction ...................................................................................................................... 48 3.4 Experimental procedures ................................................................................................ 51 3.5 Results ............................................................................................................................... 55 3.6 Discussion.......................................................................................................................... 68 3.7 Acknowledgments ............................................................................................................ 72 3.8 Preface to chapter 3 ......................................................................................................... 73 4. Chapter 3: Orexin and MCH Neurons Express c-Fos Differently After Sleep Deprivation vs. Recovery and Bear Different Adrenergic Receptors••••••.•.•••••••••••••••••••• 75 4.1 Abbreviations ................................................................................................................... 76 4.2 Abstract ............................................................................................................................. 77 4.3 Introduction ...................................................................................................................... 78 4.4 Material and methods ...................................................................................................... 80 4.5 Results ............................................................................................................................... 84 Sleep deprivation and recovery ........................................................................................................... 84 c-Fos expression in Orx and MCH neurons after sleep deprivation and recovery .............................. 84 Adrenergic receptors on Orx and MCH, including c-Fos expressing, neurons after sleep deprivation and recovery ........................................................................................................................................ 94 4.6 Discussion ........................................................................................................................ 101 c-Fos differentially expressed in Orx and MCH neurons as a function of sleep ............................... 101 Adrenergic receptors differentially distributed on Orx and MCH including c-Fos expressing neurons .......................................................................................................................................................... 104 4. 7 Acknowledgements ......................................................................................................... 106 4.8 Preface to chapter 4 ............................................................................•••.........••.•.•.....•.•• 107 5. Chapter 4: GABAA Receptor Modifications on Basal Forebrain Cholinergie Cells across the Sleep-Waking Cycle ...................................................................................... 109 5.1 Abbreviations ................................................................................................................. 110 5.2 Abstract .......................................•............................•...................................................... 111 5.3 Introduction .................................................................................................................... 112 5.4 Materials and Methods .......................................................•.•.•........•...•........•.•........•.•.•. 114 5.5 Results ............................................................................................................................. 119 Sleep amount in the three experimental groups ................................................................................ 119 Cholinergie cells immunostained with GABAAR ............................................................................. 119 Luminance ofGABAAR membrane staining across conditions ........................................................ 121 5.6 Discussion .................•................•..............•...............................................................•...... 127 5.7 Acknowledgements ........•...............................•.........•.•.•..................•..........•...•................ 131 XVI 6. General Discussion ...............•.............•............•......•.•..••..•.....•.....•....•.•..•....•..........•.. 132 7. Append"ix...................•....................................................•...........•.•.......•..•..•.•............. 152 7.1 Ethics approval •.••.•.•.•.••.•.•.•.••••..•••.•...••.•.•....•.•.•......•.....................••.•..•.•••.••.••••.•••.••.•...... 153 7.2 Consent letters •.•.•..•.•.•.•.•.•••.•••••••••.•••.•.••••.................................•..•••.••••••.•.•.•...•.•............. 154 7. References •.•.•...•.•.•....•.•.•.•.•.....•.....•.•..•.•......•..........•.•.•.•.•...•.•..•••.••••.•..•.•..•............• 159 8. Articles hard prints..............•...•...................•.•...•......•.....•.•.•...•...•.•.•.••••.•........•.•..•.• 190 XVII 1. General introduction Sleep and wakefulness are two different behavioral states that occur in all mammals in a cyc1ical manner. Sleep is characterized by behavioral unresponsiveness together with slow activity in the electroencephalogram (EEG) whereas wakefulness is defined by behavioral responsiveness together with fast activity in the EEG. Since early studies, several regions in the brain have been shown to play roles in the generation of waking. Multiple ascending arousal systems are located in the brainstem. They project forward through a dorsal pathway to the thalamic nuc1ei that form the non-specifie thalamocortical projection system. This system stimulates in tum cortical activation and arousal in a widespread manner (Jones, 2000). In addition, through a second ventral extrathalamic relay pathway, the brainstem arousal systems project through the hypothalamus up to the basal forebrain (BF) from where the basalo-cortical system also projects to the entire cortex and stimulates cortical activation (Starzl et al., 1951, Jones, 2000). In addition to the BF, a critical role of the posterior hypothalamus in maintenance ofwaking has long been known (Jones, 2000). Indeed, the role ofthe posterior hypothalamus in maintenance of waking was first recognized during the large European epidemic of encephalitis lethargica (1916-1927) when von Economo observed that encephalitic lesions in the posterior hypothalamus resulted in hypersomnia, and conversely similar lesions in the anterior hypothalamus were accompanied by insomnia (von Economo, 1931). Since then, it has been suggested that the posterior hypothalamus induces wakefulness whereas the anterior hypothalamus is more important for sleep promotion. Later unit recording revealed that the majority of posterior hypothalamic 1 cells fire during waking (Szymusiak et al., 1989). It appears thus that in addition to the brainstem, the BF and the posterior hypothalamus play important roles in regulation of arousal. With regard to sleep generation, initially it was believed that sleep occurs when the arousal systems are be10w the threshold to become activated. However, studies later suggested that sleep per se is an active process. Early studies showed that lesions in sorne brain regions inc1uding the BF produced insomnia (McGinty and Sterman, 1968). These studies led to the hypothesis that in addition to wake promoting areas, there are specifie nuc1ei in the brain that are involved in sleep generation. Indeed, it was later shown that sorne cells in the BF and the adjacent preoptic areas (POA) are active during sleep and not during waking and therefore could participate in sleep generation and maintenance (Detari et al., 1984, Szymusiak and McGinty, 1986a, Koyama and Hayaishi, 1994). From several pharmacologie al and lesion studies as well as unit recordings, it is now known that different neurotransmitters as well as endogenous factors act on multiple regions to promote and maintain sleep or wakefulness. Accordingly the major ascending arousal systems consist of fibres utilizing different transmitters inc1uding reticular formation fibres that like1y use glutamate (Jones, 1995), locus coeruleus (LC) neurons that contain noreadrenaline (NA), mesencephalic tegmental neurons that synthesize dopamine (DA), acetylcholine (ACh)-containing pontomesencephalic neurons and midline raphe nuc1ei that synthesize and release 5HT. While the re1ease of glutamate is considered to be the major component of cortical activation, it is shown that other neurotransmitters contribute each in a different way to maintain or enhance wakefulness. Pharmacological studies have shown that drugs which increase the NA transmission such 2 as amphetamine either prolong or enhance waking (Hartmann and Cravens, 1976). Consistently, LC noradrenergic neurons discharge maximally during waking and minimally during sleep (Aston-Jones and Bloom, 1981). Similar to LC neurons, 5HT containing cells tire maximally during waking, decrease their discharge during slow wave sleep (SWS) and cease tiring during paradoxical sleep (PS) (Jacobs and Fornal, 1991). However, the role of 5HT in sleep and waking regulation appears to be paradoxical as the raphe neurons have also inhibiting influence on other activating systems including AChcontaining cells (Leonard and Llinas, 1990, Luebke et al., 1992, Cape et al., 1997). Furthermore, tricyclic antidepressant drugs that selective1y inhibit 5HT reuptake have sedative effects (Mayers and Baldwin, 2005). The cholinergie pontomesencephalic neurons of the brainstem can act on different target cell populations to promote cortical activation (Jones, 2005). Finally, DA-containing neurons also participate in the formation of the major ascending arousal projections and their activity is associated with enhanced arousal as well as positive1y rewarding states. In addition to the chemical transmitters of ascending arousal systems, a number of endogenous factors such as adenosine, prostaglandin-D2 (PGD2) and interleukin 1(I11) have been shown to be regulators of the sleep-waking cycle (Lue et al., 1988, Satoh et al., 1996, Hayaishi and Urade, 2002). Both adenosine and PGD2 accumulate in the brain during arousal and sleep deprivation (Ram et al., 1997, Porkka-Heiskanen et al., 2000). The stimulant effect of coffee is large1y due to the antagonistic effect of caffeine on adenosine receptors (Yanik et al., 1987, Virus et al., 1990). Subarachnoid infusion of adenosine A2 receptor agonists promotes sleep (Satoh et al., 1996, Satoh et al., 1998). Similarly, infusion ofPGD2 in the subarachnoid space promotes sleep with a dosedependent increase oflocal adenosine (Matsumura et al., 1994). The involvement of III 3 in the regulation of non-rapid eye movement sleep (non-REMS) has been now suggested by multiple lines of evidence (Obal and Krueger, 2003, Alam et al., 2004). It has been shown that ILl concentrations in the cerebrospinal fluid peak at the onset of sleep (Lue et al., 1988). The sleep modulatory role of III is proposed to be mediated by suppressing the wake-active and activating the sleep-active neurons of the BF and POA (Alam et al., 2004). These factors are likely synthesized and released by glial cells or multiple neurons as a function of activity and diffuse through the cerebrospinal to act upon multiple systems. Despite considerable evidence showing the involvement of particular chemical transmitters or modulators in the generation of sleep-wake states, little has been done to chemically identify neurons of the BF, the POA and the posterior hypothalamus that are specifically involved in sleep or wake generation. Lesion studies in these regions for instance, mostly reveal the significance of the area rather than of a specific cell type within the area in the regulation ofbehavioral states. In most lesion studies, multiple types of cells and fibres are destroyed. Therefore, it would be extremely inappropriate to relate and interpret only a specific neuron or neurotransmitter of that region as due to the consequence oflesions. Unit recording across sleep-wake states bears also sorne limitations; the majority of these studies record the discharge profiles of neurons in a region without chemically identifying them. Furthermore, only a small number of cells are recorded at a time and the activity pattern of all cell types across states can not be easily studied. Understanding the chemical transmitters that regulate sleep vs. waking not only improves our knowledge about the neurophysiology of sleep but it could potentially also illuminate the underlying causes of sleep-wake disorders. 4 1.1 Sleep vs. wake promoting neurons in the basal forebrain and adjacent preoptic area The BF neurons lie in the ventral path of the ascending arousal systems and receive inputs from brainstem noradrenergic and cholinergie cells (Semba et al., 1988, Jones and Cuello, 1989). The BF therefore acts as a re1ay centre for cortical activation. In addition however, electrical stimulations in the BF and adjacent POA also e1icit cortical slow wave activity (SWA) and SWS (Sterman and Clemente, 1962a). Recording studies moreover revealed that in the BF while many cells fire maximally in association with cortical activation and waking, many cells fire maximally during cortical SWA and SWS (Lee et al., 2004). It appears thus that the BF could have a dual role in the regulation of sleep-waking cycle. As for the POA, it has been shown that many neurons in the POA increase their overall rate of discharge during SWS (Findlay and Hayward, 1969, McGinty and Szymusiak, 1988). In paralle1, lesions in the BF and the adjacent POA were found to produce insomnia (Szymusiak and McGinty, 1986b, Sallanon et al., 1989). Lesions in the lateral and the medial preoptic areas (LPO and MPO respective1y) resulted in a long-lasting insomnia, characterized by a significant increase in wakefulness and decrease in SWS (Schmidt et al., 2000, Thomas and Kumar, 2000). The BF consists of different cell types including cholinergie cells that contain ACh, GABAergic cells that contain gamma amino butyric acid (GABA) and glutamatergic cells that contain glutamate (Gritti et al., 1993, Manns et al., 2001). It is thus like1y that the dual role of the BF in sleep vs. waking regulation is performed by neurons containing different neurotransmitters. 5 BF cholinergie cells are distributed through the globus pallidum (GP), magnocellular proptic area (MCPO), substantia innominata (SI), medial septum (MS) and diagonal band of Broca (DBB). Lesions of the BF neurons cause deficits in cortical activation (Stewart et al., 1984). BF cholinergic cells project to the entire neocortex and limbic te1encephalon, exert excitatory effects on target cells and thereby elicit activated EEG patterns characteristic of waking and rapid eye movement sleep REMS (Szymusiak, 1995). Unit recording together with juxtacellular labeling in anesthetized and unanesthetized rats further revealed that all cholinergic cells dis charge in bursts and have their maximal activity during cortical activation characterized by fast waves in the EEG (Lee et al., 2003). Pharmacological and microdialysis studies have moreover e1ucidated the significance of ACh in cortical activation and arousal (Marrosu et al., 1995, Jones, 2004). GABA is the most frequent inhibitory neurotransmitter of the brain. This neurotransmitter is found in the BF and POA neurons projecting to the cortex and the neurons projecting to the posterior hypothalamus (Szymusiak et al., 1989, Gritti et al., 1994, Gritti et al., 1997). In addition, there are sorne other locally projecting presumed GABAergic cells in the BF that may have a local inhibitory role on the BF cholinergic cells (Manns et al., 2000a). In unit recording studies in anesthetized rats the majority of identified or presumably GABAergic cells of the BF and the POA fired maximally in association with cortical SWA (Szymusiak and McGinty, 1986a, Manns et al., 2000a, Lee et al., 2004). Altogether, it is like1y that GABAergic neurons located within the BF and POA mediate sleep promotion. However, no study showed whether the GABAergic cell populations across the BF and POA are active in naturally sleeping animaIs. 6 Moreover, it was not clarified whether the GABAergic cells are the only sleep-active neurons in these regions. Both the BF and POA are comprised of several nuclei including the MCPO, SI, MS, DBB, LPO, ventrolateral preoptic area (VLPO), MPO and median preoptic area (MnPO). The GABAergic cells are intermingled among other cell types across the BF and POA nuclei. Single unit recording also showed that sleep-active neurons are also present and distributed in the nuclei of the BF including the MCPO and SI (Lee et al., 2002) (above). However, sorne studies have shown and argued that sleep-active neurons are mainly confined to specifie nuclei in these areas including most importantly the VLPO and the MnPO rather than being more widely distributed (Scammell et al., 1998, Gong et al., 2000, Gong et al., 2004). 1.2 VLPO, an area of sleep active cells The VLPO was recently found as a small triangular shaped group of cells located lateral to the optic chiasm and rostral to the suprachiasmatic nucleus (SCN) (Sherin et al., 1996). It was first shown that cells in the VLPO cluster expressed c-Fos after sleep and the number of c-Fos expressing cells was positive1y corre1ated with the amount of sleep (Sherin et al., 1996). c-Fos is an immediate early gene whose expression is known to be correlated with neuronal activity. Subsequently by unit recording, it has been observed that the frring rate ofthese neurons increased during both REMS and non-REMS (Szymusiak et al., 1998). Later studies showed that the VLPO lesions caused longlasting and prolonged insomnia whereas the lesions in the area with scattered VLPO 7 neurons medial or dorsal to the VLPO cluster caused smaller changes in non-REMS time and were more close1y associated with loss of REMS (Lu et al., 2000). The majority ofVLPO neurons provide robust innervations to the nuclei ofthe major arousal systems including the hypothalamic tuberomammillary nucleus (TMN), (Sherin et al., 1996, Sherin et al., 1998) and the noradrenergic LC cells (Steininger et al., 2001). The VLPO is dense1y innervated by histaminergic, noradrenergic and serotonergic fibers (Chou et al., 2002). In vitro studies have shown that the majority of VLPO neurons are inhibited by noradrenaline (NA) and ACh, two major transmitters of arousal systems (Gallopin et al., 2000). The VLPO receives moderate or heavy inputs from hypothalamic regions including the MPO, lateral hypothalamic area, and dorsomedial hypothalamic nucleus (DMH), autonomic regions including the infralimbic cortex and parabrachial nucleus, and limbic regions including the lateral septal nucleus and ventral subiculum (Chou et al., 2002). There are light to moderate projections from the posterior hypothalamic orexin (Orx) and me1anin concentrating hormone (MCH) neurons to the VLPO and almost no projection from the BF or the brainstem cholinergic neurons (Chou et al., 2002). In addition, the circadian pacemaker, the SCN, directlyor indirectly, via the DMH projects to the VLPO and might underlie the influence of circadian rhythmicity on sleep. Nonethe1ess, severallines of evidence including lesion and in vivo studies have suggested that the influence of the BF and POA regions on sleep is not exclusively restricted to the VLPO cell cluster (Szymusiak and McGinty, 1986b, Szymusiak and McGinty, 1989, Manns et al., 2000a, Gong et al., 2004). With a precise1y designed paradigm of sleep deprivation (SD) and sleep recovery (SR), we tried to answer whether 8 sleep-active cells are only located in specific nuclei including the VLPO or the MnPO or rather are distributed across the BF and POA. 1.3 The posterior hypothalamus and sleep vs. wake promoting neurons Similar to the BF, the posterior hypothalamus contains different cell types including cells containing the peptides Orx and MCR. These two cell types have been recently found to be exclusively located in the posterior hypothalamus and perifomical areas (Bittencourt et al., 1992, de Lecea et al., 1998). They both have widespread projections to the entire brain including the cortex and the spinal cord (Broberger et al., 1998, Peyron et al., 1998). Since the discovery of Orx neurons, the hypothalamus has been rediscovered as a key regulator of sleep and wakefulness. Many studies have been done to investigate the role of Orx in behavioral states. Quite interestingly, it was found that mice lacking the Orx gene showed syndromes of narcolepsy, a disorder of sleep characterized by an inability to maintain waking and by sudden onset of cataplexy with muscle atonia (Chemelli et al., 1999). They also became obese despite being hypophagic due to a decrease in basal metabolism. Genetic canine narcolepsy was found to be caused by mutations in the Orx2 receptor gene (Lin et al., 1999). Later, it was discovered that human narcolepsy is associated with deficient Orx neurons or peptide (Peyron et al., 2000, Thannickal et al., 2000). Orx neurons have dense projections to all monoaminergic and cholinergic cell groups responsible for arousal (Marcus et al., 2001, Beuckmann and Yanagisawa, 2002, Taheri et al., 2002). These findings brought the intriguing possibility that the critical role of posterior hypothalamus in maintenance of waking is due to the role of Orx neurons. 9 MeR is the other peptide that is found in neurons codistributed in the same region as the Orx cells, yet in distinct cell populations. It has been shown that mice lacking the gene for MeR, in contrast to Orx knockout mice, became lean despite being hyperphagic due to an increase in basal metabolic rate (Shimada et al., 1998). Electrophysiological studies moreover showed that whereas Orx had consistently excitatory effects on postsynaptic targets, MeR had hyperpolarizing effects (Bayer et al., 2001, Eggermann et al., 2001, Gao and van den Pol, 2001, Bayer et al., 2002a, Bayer et al., 2004). Like the opposite roles of Orx vs. MeR on basal metabolism and postsynaptic effects, we thought that Orx and MeR could exert opposite influences in sleep-waking regulation as well. In addition to its pivotaI role in the maintenance of waking, the posterior hypothalamus is involved in the regulation of different neuroendocrine functions as a part ofhypothalamic pituitary axis as well as in the control offeeding and energy expenditure. Thus to find and distinguish the hypothalamic peptide/s or neuronls that might specifically control the sleep-waking cycle would help to functionally differentiate the neurons of the posterior hypothalamus. 1.4 c-Fos expression as an indicator of neuronal activity In order to study the activity of different cell types in the BF, POA and the posterior hypothalamus across the sleep-waking cycle, we used c-Fos expression, together with immunohistochemical techniques for enzymes, neurotransmitters or peptides in a paradigm of sleep deprivation and sleep recovery in rats. c-Fos is expressed in a limited number oftissues, including the central nervous system (Johnson et al., 1992). The expression of c-Fos, an immediate eady gene, induces Fos protein synthesis that goes 10 back to the nucleus after synthesis and acts as a transcription factor. It is known that cFos expression occurs in association with action potentials and Ca++ entry into the cells and can thus reflect cell activation (Morgan and Curran, 1986, Dragunow and Faull, 1989, Fields et al., 1997). c-Fos can be induced by many different stimuli such as classical neurotransmitters, trophic factors, thermal, visual and somatosensory stimuli (Cirelli and Tononi, 2000). cFos expression can be induced within 20 minutes of stimulus onset, whereas the accumulation of Fos protein needs a period of approximately 90 minutes (Sheng and Greenberg, 1990, Morgan and Curran, 1991, Cirelli and Tononi, 2000). High leve1s of Fos protein are generally observed for hours and then decline (Chaudhuri, 1997). Following a light pulse of 5 minutes, Fos protein peaks in the SCN after one to two hours and disappears within six hours (Shiromani and Schwartz, 1995). However, c-Fos data do not always match well with single unit recording data. Many wake-on cells in the BF, for example, did not express c-Fos after sleep deprivation or spontaneous waking (Pompeiano et al., 1992, Pompeiano et al., 1994). During PS rebound after sleep deprivation, only small numbers of cholinergic cells in the dorsal pontine tegmentum expressed c-Fos (Maloney et al., 1999). These observations indicate that increase in firing rate per se may not be enough to induce c-Fos in many neurons. Sorne studies emphasized the role ofCa++ entry as a key factor for c-Fos expression (Ginty, 1997). It has been shown that, glutamate for instance, can induce c-Fos expression in a Ca++ dependent manner (Lerea et al., 1992). Thus, we took into consideration that what we observed as c-Fos expression would like1y reflect only those neuronal discharges that were associated with strong and substantial changes in intracellular Ca++ level. 11 1.5 c-Fos expression in the brain across the sleep-waking cycle The expression of c-Fos together with sorne other genes ofthe immediate early gene family have been shown to be enhanced during spontaneous waking or after sleep deprivation (Pompeiano et al., 1992, Grassi-Zucconi et al., 1993, O'Hara et al., 1993). The pattern of c-Fos expression during total sleep deprivation in the brain is similar to the pattern observed after spontaneous wakefulness (Cirelli et al., 1995). In addition, it has been shown that most genes including c-Fos that are up-regulated by 3-8 hours of sleep deprivation are also up-regulated by spontaneous wakefulness (Cirelli and Tononi, 2000). Moreover, c-Fos expression in the paraventricular hypothalamic nucleus, a key structure known to induce c-Fos in response to stress was minimal after sleep deprivation showing a minimal amount of stress with sleep deprivation (Semba et al., 2001). These findings suggest that the change in gene expression in response to sleep deprivation is probably related to the arousal per se and not to the non-specific effects of stress. Accordingly, to perform our study we applied a paradigm of sleep deprivation to reliably represent a quiet non-stressed waking state in rats. During waking, there is strong c-Fos expression in sorne areas of the forebrain including the MPO, the LPO, the posterior hypothalamus, the DBB, and the intralaminar nuclei of the thalamus (central and lateral) (Cirelli and Tononi, 2000). In most ofthese areas, the highest levels of c-Fos were seen after 3 hours of sleep deprivation showing that the amount of c-Fos expression is not proportional to the amount ofprior wakefulness (Cirelli et al., 1995). With regard to sleep, it was shown that the expression of c-Fos is low throughout the brain during sleep as compared to the waking state (Grassi-Zucconi et al., 1993). 12 There is a negative correlation between the numbers of c-Fos expressing neurons in most brain regions and the amount of total sleep during one to two hours preceding sacrifice (Grassi-Zucconi et al., 1994). As for REMS, it is known that physiological REMS is not associated with significant c-Fos accumulation (Grassi-Zucconi et al., 1994), since, as mentioned before, at least 20 minutes is required for the induction of c-Fos gene and 90 minutes for Fos protein synthesis. Nevertheless, pharrnacological and nonpharrnacological techniques that induced longer REMS like behavior could also result in increased c-Fos in sorne brain regions such as the SeN, dorsal hypothalamus, laterodorsal and pedunculopontine tegrnental nuclei (LDT and PPT respectively) and pontomedullary reticular formation (Merchant-Nancy et al., 1992, Maloney and Jones, 1999, Maloneyet al., 2000). Despite previous studies showing lower amount of c-Fos in almost all brain areas during sleep than during waking, recently it was reported that Fos irnrnunoreactivity in the VLPO was increased during sleep, and the number oflabeled cells was positively correlated with the time spent in sleep (Sherin et al., 1996). Although since the discovery of the VLPO, several other studies have been performed to confirm the original results and the VLPO was later considered by sorne as the sleep nucleus of the brain, we believed that the original and many subsequent studies, using c-Fos expression in the VLPO, were not precisely designed. Particularly in the original experiments, rats were spontaneously asleep for the majority oftime only in the one hour (~>60%) prior to sacrifice (Sherin et al., 1996, Lu et al., 2002) or they were deprived of sleep for 9 or 12 hours and were subsequently allowed to recover from sleep deprivation for 45, 90 and 180 minutes before sacrifice (Sherin et al., 1996). In either case, considering the time needed for Fos protein synthesis, what was observed as an increase in c-Fos in the VLPO 13 could not unequivocally be attributed to prior sleep as opposed to the previous behavioral state ofwaking or deprivation. Moreover, all ofthose studies were based on c-Fos expression alone without chemical identification of the neurotransmitter phenotype of the cells. 1.6 Modulation of sleep vs. wake promoting neurons by noradrenaline Multiple brain regions will probabely utilize different neurotransmitter to regulate sleep wake cycle.Finding the chemical transmitters of individual cells in each region that are responsible for sleep-waking cycle regulation is the first step toward understanding the physiology of sleep. The second step is to know the mechanism of cyclical activation of sleep vs. wake-active cells. It is possible that different cells respond differently to the neurotransmitters of sleep or waking, most importantly to the NA released from the LC brainstem neurons projecting to the BF and POA (Jones and Moore, 1977, Jones and Cuello, 1989, Chou et al., 2002). The release of NA is highest during waking, decreases during non-REMS and complete1y ceases during REMS (Jones, 1989) .. Through alphal adrenergic receptor (aIAR) NA depolarizes, whereas through alpha2 adrenergic receptor (a2AR), NA hyperpolarizes postsynaptic target neurons. Both alAR and a2AR are G protein coupled receptors that act through closing or opening ofK+ channels respectively (McCormick, 1992, Williams and Reiner, 1993, Liu and Alreja, 1998). Indeed, in vitro studies have shown that the BF cholinergie neurons are excited by NA through uIARs (Fort et al., 1995), whereas non-cholinergie cells ofthe BF and the majority of cells in the VLPO are inhibited by NA through u2ARs (Fort et al., 1998, Gallopin et al., 2000). We assumed thus that sleep vs. wake-active cells through bearing different adrenergic 14 receptors become inhibited or disinhibited as the re1ease of NA fluctuates during the sleep-waking cycle. Accordingly, during waking, sleep-active cells would be inhibited whereas wake-active cells would be excited by NA. Reciprocally during sleep, wakeactive cells would no longer be excited whereas sleep-active cells would be disinhibited as the release of NA is lowest. By applying a paradigm of sleep deprivation and recovery in rats we studied the differential adrenergic receptor expression in sleep vs. wake-active neurons across the BF, POA and posterior hypothalamus 1.7 The dynamics ofGABAAreceptors during sleep vs. waking on cholinergie cells of the basal forebrain The concept that sleep vs. wake promoting neurons bear different receptors for the major neurotransmitters of arousal systems could explain how different neurons cyclically become active or inactive during the sleep-waking cycle. Another mechanism however, could potentially be due to differential expression or mobilization of receptors on sleep vs. wake-active neurons depending on the behavioral conditions. In other words, cells might alter the availability of their functional receptors across different conditions in a homeostatic manner and therefore become more or less responsive to a specific neurotransmitter. Indeed, according to the theory of synaptic homeostasis, neurons in order to maintain their functions alter their responses to neurotransmitters partly dependant upon their prior amount of activity (Turrigiano, 1999, Marder and Prinz, 2002). This change occurs in part through changes in receptors associated with depolarizing vs. hyperpolarizing currents. After a period of neuronal activity, cells tend to shift into a more silent state through enhancing the amount of inhibitory synapses. In 15 contrast, after blockade of activity for a short time, cens tend to start up and enhance their dis charge through decreasing inhibitory synapses and/or through increasing excitatory synapses (Turrigiano, 1999, Marder and Prinz, 2002, Mody, 2005). The majority of inhibitory synapses throughout the brain utilize GABA as a neurotransmitter. Through ion-gated channel receptors (Rs), GABAARs and GABAcRs, or G protein coupled receptors, GABABRs, GABA opens cr or K+ channels and consequently hyperpolarizes cens. GABAARs are present and expressed ubiquitously in most neurons of the brain. Severallines of study have found that neuronal susceptibility to the effects of neurotransmitters changes under different circumstances. Activity blockade in rat cultured visual cortex for 2 days, reduced miniature inhibitory post-synaptic currents (mIPSCs), the intensity GABAAR immunostaining and the numbers of synaptic sites that expressed GABAAR (Kilman et al., 2002). Moreover, it has been shown that after 24 hours ofwhisker stimulation in rats, inhibitory synapses in the corresponding barrel field that were detected by e1ectron microscopy increased (Knott et al., 2002). Similarly, pharmacological studies revealed that the application ofbicucunine, a GABAAR antagonist, in hippocampal cultured slices, increased the density of GABAARs (Marty et al., 2004). With regard to sleep and waking, nobody has yet shown whether the phenomenon of synaptic homeostasis happens in a daily and cyc1ical manner. Many cens in order to maintain waking, dis charge in bursts continuously and therefore could be suitable candidates for this study. Accordingly, as it occurs after a period of prolonged neuronal activity, there might be functional changes in GABAARs on activated cells after a period ofwaking. As will be shown in the second chapter, the BF cholinergic cens express cFos during sleep deprivation but not during sleep recovery. Most recently in the current 16 lab, it has been similarly shown that identified BF cholinergic neurons discharge in bursts during waking associated with fast activity in the EEG, and they become silent during sleep associated with slow wave activity (Lee et al., 2003). We thought that numbers, or intensity of GABAARs on the BF cholinergic cells might consequently change during sleep vs. waking. In summary, prior to my research project, few studies had been done to characterize the activity of chemically identified neurons in association with sleep and waking across the BF, POA and the posterior hypothalamus. Moreover, little was known about the mechanism of cyclical regulation of sleep vs. wake promoting neurons. With the goal of understanding chemically identified neurons and their modulation across the sleepwaking cycle we designed several experiments. 1.8 The first experiment In the first chapter, with the aim of identifying putative sleep-active cells, the activity of GABAergic neurons of the BF was studied in urethane-anesthetized rats. By juxtacellular recording, neurobiotin (Nb) labeling and subsequent immunohistochemical identification for glutamic acid decarboxylase (GAD), the dis charge profiles of identified GABAergic neurons in the Mepo and SI was examined in relation to EEG activity. In addition, by using dual-immunostaining for GAD and U2AAR we looked at the distribution of GAD immunoreactive (+) neurons that were also labeled for U2 AAR. Finally in urethane-anesthetized rats, we examined whether Nb-Iabeled, GAD+ cortical activation 'off neurons that discharged maximally in association with cortical SWA, 17 were immunopositive for the a2AAR. This part of study revealed that the majority of GABAergic cells in the MCPO fired maximally in association with SWA and decreased their firing in association with evoked cortical activation. By further labeling for a2AAR, first we found that the majority of GAD+ neurons through the MCPO and SI were labeled for the a2AAR. Second, we found that ail the Nb-Iabe1ed GAD+ cortical activation 'off cells were labe1ed for a2AAR. These results suggested that significant population of GABAergic cells of the BF might be sleep-active cells. It moreover suggested that the common phenotype ofthese sleep-on cells is GABA-containing and a2AAR-bearing. To answer the question ofwhether in naturally sleeping animaIs GABAergic cells of the BF still play roles in sleep promotion, the second experiment was designed. 1.9 The second experiment In the second chapter by using dual-immunostaining for c-Fos and choline acetyl transferase (ChAT) or GAD, we studied the activity of cholinergie and GABAergic cells through the BF and the adjacent POA across conditions of sleep deprivation and sleep recovery. c-Fos study brought the possibility oflooking at different types ofneurons and in different regions at the same time. Moreover, it gave us the chance to study the activity of populations of cells, rather than a few cells, under different conditions. We found that the numbers of cholinergie cells that expressed c-Fos were significantly higher during SD as compared with the SR condition, whereas the proportions of c-Fos expressing cells that were GABAergic were significantly higher after SR as compared 18 with the SD condition. Moreover, consistent with our results in anesthetized rats, we showed that the majority ofGABAergic cens in the BF and POA that expressed c-Fos after SR also bore U2AARs. 1.10 The third experiment In the third chapter, we studied c-Fos expression in Orx and MCR cens of the posterior hypothalamus across conditions of sleep deprivation vs. sleep recovery. We also looked at the incidence of UIAAR vs. U2AAR on Orx and MCR neurons. We found that the numbers of c-Fos expressing Orx cens were significantly higher after SD as compared with SR condition. In contrast, the numbers ofMCR cens that expressed c-Fos were significantly higher after SR as compared with SD condition. In addition, while Orx cens were immunostained with both types of UIAAR and U2AAR, MCR neurons were only immunostained with U2AAR. DifferentiaI adrenergic receptor expression on GABAergic sleep vs. wake-active cens and Orx vs. MCR neurons suggested that bearing different receptors for NA could be one mechanism for cyc1ical activations of sleep vs. wake promoting neurons. 1.11 The fourth experiment In the last chapter, we investigated the possibility of dynamic changes of receptors under conditions of sleep or waking. To test this possibility, by applying immunohistochemistry for ChAT and GABAARs (J32,3) subunits, we quantified and 19 analyzed the intensity of membrane GABAARs on the BF cholinergie cells across different conditions of sleep deprivation and sleep recovery. Since the large majority of GABAARs are localized on somatodendritic membrane and initial segments of axons (Lus cher and Keller, 2004), here we analyzed GABAARs that were located on the soma representing the major GABAARs compartment. We found that both the numbers and the immunofluorescent GABAAR intensity on cholinergie cells were increased after SD as compared with the SR condition. This finding suggested another novel mechanism for modulation of wake promoting cholinergie neurons that in tum could regulate the sleepwaking cycle. 20 1.12 Preface to chapter 1 As reviewed in the Introduction, multiple lines of evidences have suggested that the GABAergic cells might be important for sleep generation andlor maintenance. In the current laboratory, it has been previously shown that the majority of GABAergic neurons in the MCPO nucleus of the BF, fire maximally in association with slow cortical activity whereas their firing is attenuated in association with evoked cortical activation. To test the possibility that these GABAergic putative sleep promoting cells are inhibited by NA during cortical activation and thus perhaps waking, we examined the presence of a2AR on the BF cortical activation-off GABAergic neurons. 21 2. Chapter 1: Alpha 2 Adrenergic Receptors on GABAergic Putative Sleep-Promoting Basal Forebrain Neurons 1 Ian D. Manns, Maan Gee Lee, Mandana Modirrousta, Yiping P. Hou and Barbara E. Jones 1 Eur J Neurosci. 2003 Aug; 18(3 ):723-7 22 2.1 Abbreviations ACh, acetylcholine u2AR, alpha2 adrenergic receptor EEG, electroencephalogram GAD, glutamic acid decarboxylase LC, locus coeruleus MCPO, magnocellular preoptic nucleus NA, noradrenaline Nb, Neurobiotin PS, paradoxical sleep SI, substantia innominata SWS, slow wave sleep VLPO, ventrolateral preoptic region 23 2.2 Abstract The basal forebrain plays an important role in the modulation of cortical activity and sleep-wake states. Yet its role must be multivalent since lesions reportedly diminish cortical fast activity and also cortical slow activity along with slow wave sleep (SWS). Basal forebrain cholinergie versus GABAergic cell groups could differentially influence these processes. By labeling recorded neurons with Neurobiotin (Nb) using the juxtacellular technique and identifying them by immunostaining, we previously found that whereas aIl cholinergic cells increased their firing, the majority of GABAergic neurons decreased their firing in association with evoked cortical activation in urethaneanesthetized rats. Here, we examined the possibility that such GABAergic, cortical activation 'off cells might bear alpha 2 adrenergic receptors (a2AR) through which noradrenaline (NA) could inhibit them during cortical activation. Pirst using simple dual-immunostaining for glutamic acid decarboxylase (GAD) and the a2AAR, we found that the majority (-60%) ofGAD-immunopositive (+) neurons through the magnocellular preoptic nucleus (MCPO) and substantia innominata (SI) were labe1ed for the U2AAR. Second, in urethane-anesthetized rats, we examined whether Nblabeled, GAD+ cortical activation 'off neurons that discharged maximally in association with cortical slow wave activity, were immunopositive for U2AAR. We found that all the Nb+/GAD+ 'off cells were labe1ed for the U2AAR. Such cells could be inhibited in association with cortical activation and waking when noradrenergic locus coeruleus (LC) neurons discharge and be disinhibited with cortical slow waves and SWS when these 24 neurons become inactive. We thus propose that Œ2AR-bearing GABAergic basal forebrain neurons constitute sleep-active and sleep-promoting neurons. 25 2.3 Introduction The basal forebrain plays an important role in modulating cortical activity and sleep-wake states (Jones, 2000). Lesions or inactivation ofthe basal forebrain reportedly diminish cortical fast activity during waking (Stewart et al., 1984, Cape and Jones, 2000) but also diminish cortical slow wave activity and slow wave sleep (SWS) (Szymusiak and McGinty, 1986b). The basal forebrain may thus be comprised of different cell groups that promote different cortical activities and states. In addition to cholinergie neurons that are known to stimulate cortical activation, the basal forebrain contains large numbers ofGABAergic neurons (Gritti et al., 1993). By employingjuxtacellular labeling and immunohistochemical identification of recorded neurons in urethane-anesthetized rats, we recently discovered that GABAergic cells behave very differently as a group than cholinergie cells. Whereas all cholinergie neurons increased their firing with evoked cortical activation, the majority of GABAergic neurons decreased their firing in association with cortical activation and discharged at their highest rate in association with cortical slow wave activity (Manns et al., 2000a). We proposed that such GABAergic cells could be sleep-active neurons. Neurons that discharge at their highest rate during natural SWS have indeed been recorded in the basal forebrain and in the adjacent preoptic area of naturally sleeping-waking rats and cats (Szymusiak and McGinty, 1986a, Koyama and Hayaishi, 1994, Szymusiak et al., 1998). Such SWS-active neurons were inhibited by stimulation of the locus coeruleus (LC) or iontophoretic application of noradrenaline (NA) in vivo (Osaka and Matsumura, 1994, Osaka and Matsumura, 1995). In vitro studies also identified non-cholinergie neurons in the basal forebrain and 26 GABAergic neurons in the ventrolateral preoptic area (VLPO) that are commonly hyperpolarized and inhibited by NA (Fort et al., 1998, Gallopin et al., 2000). Such inhibitory effects of NA are mediated by alpha 2 adrenergic receptors (a2AR) (Osaka and Matsumura, 1995, Bai and Renaud, 1998). The presence of a2AR on basal forebrain neurons might thus identify them as sleep-active cells. The aim of the present study was to determine immunohistochemically whether GABAergic and potentially sleeppromoting, basal forebrain neurons possess a2AR. In a preliminary investigation, we employed dual-immunostaining for glutamic acid decarboxylase (GAD) and the a2AAR to assess if a signiticant proportion of GADimmunopositive (GAD+) neurons in the magnocellular preoptic nucleus (MCPO) or overlying substantia innominata (SI) of the rat were labe1ed for the a 2AAR. We then examined using urethane-anesthetized rats, whether neurons that tire maximally in association with cortical slow wave activity and are GAD+ are labeled for the a2AAR. Recorded within the MCPO or overlying SI, such characterized units were labe1ed with Neurobiotin (Nb) using the juxtacellular technique and subsequently triple-stained for Nb, GAD and the a2AAR. 27 2.4 Materials and Methods For the immunohistochemical study of Œ2AAR on GAD-immunoreactive neurons, 3 male Wistar rats (weighing 200-250 g) were employed for simple perfusion-fixation of the brain under barbiturate anesthesia (Somnotol, 120 mg/kg, i.p.). For the study ofthe receptors on GAD-immunoreactive physiologically characterized celIs, 7 male Long Evans rats (weighing 200-250 g) were employed for acute e1ectrophysiological recording prior to perfusion-fixation ofthe brain. He1d in a stereotaxic frame, they were anesthetized with urethane for the duration of the recording (1.4 g/kg initial dose followed by boosters of .14 g/kg as needed, i.p.) and for the subsequent perfusionfixation (with an additional booster if needed). AlI procedures were approved by the McGill University Animal Care Committee and the Canadian Council on Animal Care. In the electrophysiological experiments, units were recorded in the MCPO or SI using glass micropipettes and characterized in association with electroencephalogram (EEG) or field potentials recorded with surface or deep e1ectrodes from retrosplenial or prefrontal cortex and olfactory bulb, as described in previous publications (Manns et al., 2000a, Manns et al., 2000b). Following isolation of a single unit, its discharge was characterized during baseline cortical irregular slow wave activity and during stimulated cortical activation evoked by tail pinch. Cells that decreased their rate of discharge with cortical activation, previously described as 'off celIs, were se1ected. One such cell per animal was then labe1ed with Neurobiotin (Nb, Vector Laboratories, Burlingame, CA) by applying the 'juxtacellular' technique. As established in our previous publications, 28 modulating the discharge of one recorded unit using 200 ms CUITent pulses over a period of 5 to 30 minutes resulted in labeling of only one neuron. Brains were fixed by intra-aortic perfusion of a paraformaldehyde solution (4% in 0.1 M phosphate buffer, PB, pH 7.4, ~500 ml) preceded by saline (0.9% NaCI, ~50 ml). After removal, aIl brains were placed in a sucrose solution (30% in PB at 4° C) for ~2 days and then frozen. Sections were cut at 25 J.lm thickness in the coronal plane on a freezing microtome. For dual-immunostaining of GAD and Œ2AAR, sections were colIected at 400 J.lm intervals through the basal forebrain. They were incubated for 3 nights at 4° C with an affinity-purified goat polyclonal antibody for the <l2AAR (C-19, sc-1478, Santa Cruz Biotechnology, Santa Cruz, CA) as employed previously (Hou et al., 2002), together with a rabbit polyclonal antibody for GAD67 (1 :3000, Chemicon International, Temecula, CA). The Œ2AAR was revealed using Cy3-conjugated donkey anti-goat antiserum and GAD revealed simultaneously using Cy2-conjugated donkey anti-rabbit antiserum (Jackson Immuno Research, West Grove, PA). GAD-immunoreactive neurons in the MCPO and overlying SI were examined by fluorescence microscopy for the presence of Œ2AAR . For triple staining of recorded celIs for Nb, GAD and Œ2AAR, aIl sections were colIected through the basal forebrain for staining ofthe Nb-Iabe1ed celI using AMCAconjugated streptavidin (Jackson ImmunoResearch Laboratories). After locating the Nblabeled celI, the section containing it was subsequently incubated with the Œ2AAR and GAD antibodies for 3 nights and revealed with Cy2- and Cy3-conjugated secondary antibodies as described above. Nb-Iabe1ed celIs that were also GAD+ were examined by 29 fluorescence microscopy for the presence of a2AAR. To provide images of double labeling, they were superimposed using Adobe Photoshop (v9, Adobe Systems, San Jose, CA). 30 2.5 Results In dual-immunostained series, GAD+ cells within the MCPO and overlying SI were often immunopositive for the a2 AAR (GAD+/a2AAR+, triangles in Fig. 1; filled arrowheads in Fig. 2A'and 2B'). In these cells, the receptor labeling was punctate and distributed over the cell body and proximal dendrites. In many cases, it appeared to be within the cytoplasm, since it surrounded the nucleus of the cell. Punctate or diffuse receptor labeling was also present over blood vessels in the region (bv, Fig. 2A' and B'). In the same area, sorne GAD+ cells were immunonegative for the a2AAR (GAD+/U2AAR-, empty arrowheads, Fig. 2A' and B'). The GAD+/U2AAR+ and GAD+/a2AAR- cells were present in the same vicinity (Fig. 2A and A') and sometimes situated immediately adjacent to one other (Fig. 2B and B'). In a quantitative sampling, a majority of the GAD+ neurons in the MCPO (average of 66%) and a substantial proportion ofthose in the SI (average of38%) or an overall majority in the MCPO-SI (average of 59% in three brains) were judged positively labeled for u2AAR. 31 Figure 1 GAD+/0'2AAR+ .... Nb+/GAD+/0'2AAR+ .. CPu ac 32 Figure 1. GAD+ cells that were labeledfor the a2AAR (gray triangles) are plotted in the BF cholinergic cell area, including the magnocellular preoptic nucleus (MCPO) and substantia innominata (SI) (Gritti et al., 1993). In this same area, units were recorded and selected for juxtacellular labeling and triple-staining for Nb, GAD, and a2AAR. Ali Nb+/GAD+ cortical activation 'off' cells were located in the MCPO and were positively labeledfor a2AAR (black stars). Scale bar = 1 mm. Other abbreviations: ac, anterior commissure; CPu, caudate putamen; GP, globus pallidus; LPO, lateral preoptic area; MPO, medial preoptic area; oc, optic chiasm, OTu, olfactory tubercle. 33 Figure 2 34 Figure 2. Photomicrographs showing GAD immunostaining with Cy2 by green fluorescence emission (A, B, C and D) and Œ2AAR immunostaining with Cy3 by red fluorescence of the same view (A " B', C' and D '). In the left panels, dualimmunostaining reveals that some GAD+ cells (filled arrowhead, A and B) are labeled for the Œ2AAR+ (filled arrowheads, A' and B '), and others are not (open arrowheads, A' and B '). Blood vessels (bv) are also immunostained for the Œ2AAR+. In the right panels, triple-staining reveals that the GAD+ cells (filled arrowheads, C and D) that were physiologically characterized as cortical activation 'off' cells and labeled with Nb in blue fluorescence by AMCA-conjugated streptavidin (C' and D') are also immunopositivefor Œ2AAR (solid arrows, C' and D '). Photomicrographs show a simple image with single green fluorescence emission for GAD immunostaining (left) and superimposed images with blue and red fluorescence for Nb and Œ2AAR immunostaining (right, C' and D '). All Nb+/ GAD+/Œ2AAR+ neurons were located in the MCPO (Fig. 1). Scale bars = 20,Llln. 35 In recording experiments performed under urethane anesthesia, cells were se1ected that decreased their firing with stimulation-evoked cortical activation for labeling with Nb (Manns et al., 2000a). Generally discharging in a tonic irregular manner in association with the baseline irregular cortical slow wave activity, these 'off cells decreased or ceased firing in association with cortical activation (Fig. 3). They subsequently increased their discharge following cessation of the stimulation as cortical slow wave activity retumed to pre-stimulation leve1s (Fig. 3). Across cells, the average discharge rate decreased significantly during stimulation as compared to pre-stimulation (mean ± sem: 3.36 ± 1.25 vs. 9.8 ± 2.27; t = 3.44, df= 4, P = .03) and subsequently retumed to pre-stimulation levels (9.28 ± 2.56) within one to two minutes. Of the 'off cells labeled with Nb in the current study, the majority were immunopositive for GAD (517), as was also the case in our previous study. The Nb+/GAD+ 'off cells identified here were alliocated in the MCPO (n = 5; Fig. 1). All ofthese Nb+/GAD+ 'off cells (Fig. 2C and D) appeared to be labe1ed for the Œ2AAR by the presence ofpunctate staining over the cell body and cytoplasm surrounding the nucleus (Fig. 2C' and D'). 36 Figure 3 Nb+/GAD+/a2A+ Off cell A. EEG Somalie Stimulation B. Unit Discharge Rate 5 o o 1 Il 1 , Il 1 60, - - _ 20 sec , 260 120 C.EEG ~ D. Unit Discharge W 2 sec ~11mv 1111111111111111 37 Figure 3. Unit activity together with simultaneously recorded EEG from prefrontal cortex showing a typical decrease or cessation offiring in association with stimulation-evoked cortical activation and recovery offiringfollowing stimulation in a urethane-anesthetized rat. This cortical activation 'off' cell was labeled with Nb and immunopositive for GAD and the a2.AAR (Fig. 2, C and C 'J. The compressed EEG recording (A) is expanded (C) and shown together with the unit recording (D) below. The unit discharge rate plot (spikes per second in B) shows the decrease and cessation in firing that occurs as the EEG changes from irregular slow wave activity to fast activity during stimulation and the return to pre-stimulation rates that occurs as the EEG resumes irregular slow wave activity following stimulation. 38 2.6 Discussion This study documents by immunohistochemistry combined with electrophysiology that the majority (59%) ofbasal forebrain GABAergic neurons possess u2AR and that the GABAergic neurons that fire maximally with cortical slow wave activity bear these receptors. In our previous recording studies of GABAergic neurons in urethane-anesthetized rats (Manns et al., 2000a), the majority (57%) ofNb+/GAD+ neurons through the MCPO-SI were found to discharge at a higher rate with cortical slow wave activity than with cortical activation. No Nb+/ChAT+ cells did so (Manns et al., 2000b), and only a minority (18%) ofNb+/GAD- cells did so (Manns et al., 2000a). These results suggested that a substantial group of GABAergic basal forebrain neurons would be most active in association with cortical slow wave activity during SWS and thus correspond in large part to previously identified sleep-active neurons in this region (Szymusiak and McGinty, 1986b). These neurons could correspond to the major proportion (59%) of GAD+ neurons that bear U2AAR documented here. The u2AR is responsible for hyperpolarization of the membrane through opening ofK+ channels, as shown on preoptic area neurons as on locus coeruleus neurons (Williams et al., 1985, Bai and Renaud, 1998). Although other subtypes of u2AR have been shown to be present in the basal forebrain (Rosin et al., 1996), the U2A was previously shown to be distributed densely therein (Talley et al., 1996) and using the same antibody as that in the present study, to be present upon locus coeruleus neurons (Hou et al., 2002). It cannot be 39 excluded in the present study performed using epifluorescence microscopy that u2AR labeling could also be present on presynaptic terminaIs of the noradrenergic fibers as documented in other areas by electron microscopy (Glass et al., 2001) or of other fibers as indicated in the preoptic area by electrophysiological evidence (Matsuo et al., 2003). However, the u2AR labeling over the cell body and proximal dendrites of GAD+ cells here was predominantly associated with the postsynaptic cells since it often appeared to be located within the cytoplasm surrounding the cell nucleus, as also described in other neuronal perikarya (Talley et al., 1996). Examination for triple labeling by Nb, GAD and U2AAR of physiologically characterized cells showed here that cortical activation 'off GABAergic cells do bear U2AR. These GAD+/u2 AAR+ neurons could be inhibited byNA in association with cortical activation and waking when LC neurons discharge at their highest rate (AstonJones and Bloom, 1981). Reciprocally they would be disinhibited during cortical slow wave activity and SWS when LC neurons decrease firing. They could also be active during paradoxical sleep (PS) when LC neurons are silent and many sleep-active neurons in the forebrain continue to discharge (Szymusiak and McGinty, 1986a, Koyama and Hayaishi, 1994, Szymusiak et al., 1998). However, in view of the present results, they are more likely to be silent in association with the cortical activation that occurs during PS as well as waking. Silence by these cells during PS could be effected by acetylcholine (ACh), which also inhibits the non-cholinergic cells inhibited by NA (Fort et al., 1998, Gallopin et al., 2000) and which is released by neighboring cholinergic cells during both PS and waking (Vazquez and Baghdoyan, 2001). The GABAergic u2AR-bearing neurons identified here thus most likely correspond to sleep-active cells that are 40 maximally active during SWS. Given the evidence from lesion studies that destruction of cells in the basal forebrain leads to a loss of sleep (Szymusiak and McGinty, 1986b), these sleep-active neurons must also function importantly as sleep-promoting neurons. They may do so via projections to and inhibitory effects on local cholinergie neurons, the cortex (Gritti et al., 1997) or the posterior hypothalamus (Gritti et al., 1994). In this role, they may function in synergy with GABAergic neurons of the preoptic region, inc1uding the VLPO (Sherin et al., 1996, Szymusiak et al., 1998), that project in paralle1 to the posterior hypothalamus (Gritti et al., 1994, Sherin et al., 1998) and are similarly inhibited by NA (Fort et al., 1998, Gallopin et al., 2000). 41 2.7 Acknowledgements This research was supported by grants from the Canadian Institutes ofHealth Research (CIHR, 13458) and United States National Institute of Mental Health (NIMH, ROI MH60119":OIAI). Y.P. Hou was a Visiting Scientist from the Lanzhou Medical College, Lanzhou, China. I.D. Manns held a graduate student fellowship from the Canadian Natural Science and Engineering Research Council (NSERC). Lynda Mainville is greatly appreciated for her technical assistance. 42 2.8 Preface to chapter 2 The first chapter revealed that the majority of identified GABAergic neurons decreased their firing in association with evoked cortical activation in urethaneanesthetized rats. It moreover showed that GABAergic cortical activation "off' neurons that discharged maximally in association with cortical SWA, were labeled for the o,2AAR. These GABAergic neurons could thus be distinguished from other cortical activation "on" GABAergic cells by their differential response to NA. Accordingly, the GABAergic "off' neurons that also bear o,2AAR would be inhibited by NA during waking whereas they would be removed from inhibition during sleep when release of NA is lowest and thus could take part in sleep promotion and maintenance. Based on these results, we proposed that O,2AAR-bearing GABAergic basal forebrain neurons constitute sleep-active and sleep-promoting cells. In the next chapter in order to verify our results in naturally sleeping animaIs, we designed a paradigm of sleep deprivation and sleep recovery in rats and by applying immunohistochemistry for GAD, the enzyme needed for GABA synthesis, ChAT and cFos, as an indicator for cell activity, we test the possibility ofwhether 1) the basal forebrain and adjacent preoptic area are comprised of GABAergic sleep-active cells and 2) these sleep-active cells are not cholinergic. Finally by applying triple labeling for cFos, GAD and O,2AAR across different conditions, we examined whether GABAergic sleep-active cells bear in common O,2AARs and thus could be distinguished from other GABAergic cells of the region. In addition, the study in the next chapter provided us the 43 opportunity to study simultaneously the activity of populations of cells across the widespread regions of the basal forebrain and the preoptic area. 44 3. Chapter 2: GABAergic Neurons with Alpha 2- Adrenergic Receptors in Basal Forebrain and Preoptic Area Express c-Fos during Sleep2 Mandana Modirrousta, Lynda Mainville and Barbara E. Jones 2 Neuroscience.2004;129(3):803-10 45 3.1 Abbreviations AR, adrenergic receptor BF, basal forebrain ChAT, choline acetyltransferase DBB, diagonal band of Broca nuclei GAD, glutamic acid decarboxylase LPO, lateral preoptic area MCPO, magnocellular preoptic area MnPO median preoptic nucleus MPO medial preoptic area NA, noradrenaline NDS, normal donkey serum non-REMS, non-rapid eye movement sleep POA, preoptic area; REMS, rapid eye movement sleep SC, sleep control SD, sleep deprivation SIa, substantia innominata, anterior part SIp, substantia innominata, posterior part SR, sleep recovery SWS, slow wave sleep; VLPO, ventrolateral preoptic area 46 3.2 Abstract The basal forebrain (BF) contains cholinergie neurons that stimulate cortical activation during waking. In addition, both the BF and adjacent preoptic area (POA) contain neurons that promote sleep. We examined c-Fos expression in cholinergie and GABAergic neurons in the BF and POA to determine whether they are differentially active following sleep deprivation versus recovery and whether the GABAergic neurons are active during sleep. Whereas the numbers of c-Fos+ cells and proportions of c-Fos+ cells that were cholinergie were decreased, the proportions that were GABAergic were increased following sleep recovery across BF and POA nuclei. Moreover, the sleepactive GABAergic neurons were immunostained for a2A-adrenergic receptors. We conclude that GABAergic neurons that commonly bear a2-adrenergic receptors comprise sleep-active cells of the BF and POA. These GABAergic cells would be inhibited by noradrenaline (NA) released from locus coeruleus neurons during waking; they would be disinhibited through diminished NA release during drowsiness and thus become active to promote sleep by inhibiting in tum wake-promoting neurons. 47 3.3 Introduction Since early studies, the BF has been known to play an important role in cortical activation as the ventral extrathalamic re1ay to the cerebral cortex from the brainstem ascending activating system (Starzl et al., 1951, Jones, 2000). Yet, e1ectrical stimulation in the region of the BF and adjacent POA also elicited cortical slow wave activity with slow wave sleep (SWS) (Sterman and Clemente, 1962b, Sterman and Clemente, 1962a), and lesions therein produced insomnia (McGinty and Sterman, 1968). From single unit recording studies, it became apparent that whereas many neurons in the BF and POA increase their firing with cortical activation or waking, others increase their firing with cortical slow wave activity or SWS (Detari et al., 1984, Szymusiak and McGinty, 1986a, Koyama and Hayaishi, 1994, Alam et al., 1996). More recentlyby examining c-Fos expression as a reflection of neural activity, a restricted collection of neurons was identified within the ventrolateral preoptic area (VLPO) that was active during sleep and thus proposed to be the principal sleep-promoting cell group of the POA and forebrain (Sherin et al., 1996). In immunohistochemical studies, many cells in the VLPO, LPO and BF, inc1uding projection neurons, were shown to contain glutamic acid decarboxylase (GAD), the synthetic enzyme for GABA (Gritti et al., 1993, Gritti et al., 1994, Sherin et al., 1998). The possibility was thus raised that GABAergic neurons in both POA and BF could comprise sleep-active and promoting cells. Indeed, in studies employing juxtacellular labeling of recorded neurons in urethane-anesthetized rats, a majority of immunohistochemically identified GABAergic BF neurons were found to fire maximally 48 with cortical slow wave activity, whereas all cholinergie neurons fired maximally with stimulation-induced cortical activation (Manns et al., 2000b, Manns et al., 2000a). Wake-active versus sleep-active neurons most likely respond differently to the major neurotransmitters of the ascending arousal systems, most notably noradrenaline (NA) contained in locus coeruleus neurons projecting to the BF and POA (Jones and Moore, 1977, Jones and Cuello, 1989, Chou et al., 2002). Indeed, it was first found in vivo that NA excited wake-active neurons, whereas it inhibited sleep-active neurons through u2_adrenergic receptors (u2AR) in the POA and adjacent BF (Osaka and Matsumura, 1995). It was also found in vitro that whereas all cholinergie neurons were excited by NA in the BF, sorne co-distributed non-cholinergie neurons (~15%) were inhibited by NA (Fort et al., 1995, Fort et al., 1998). It was subsequently discovered that the majority of neurons in the VLPO (~60%) were inhibited by NA and also contained mRNA for GAD (Gallopin et al., 2000). Most recently, we found that immunohistochemically identified GABAergic BF neurons which fired maximally with slow wave activity in urethane-anesthetized rats were immunostained for U2AAR (Manns et al., 2003). These results suggested that GABA and u2AR may reflect a common phenotype as well as mechanism for a population of sleep-promoting neurons distributed across the BF and POA. In the present study, c-Fos expression was examined in sleep deprived versus sleep recovery groups of rats to test the hypotheses that sleep-promoting neurons 1) are distributed across nuc1ei ofthe BF and POA, 2) are not comprised of cholinergie neurons and 3) are comprised of GABAergic neurons that are inhibited by NA and thus bear 49 a2AR. A portion of these results was presented in abstract form (Modirrousta et al., 2003, Modirrousta et al., 2004a). 50 3.4 Experimental procedures Male Wistar rats (Charles River Canada, St. Constant, Quebec, Canada) weighing between 200-250 g were housed individualIy in cages with free access to food and water. Three different groups of four rats per group were treated in their home cages for: 1) total sleep deprivation (SD) by gentle touching for 3 hours (1200 to 1500 h), 2) total sleep deprivation for 3 hours (900 to 1200 h) folIowed by sleep recovery (SR) for 3 hours (1200 to 1500 h), or 3) undisturbed sleep and waking as sleep control (SC) for 3 hours (1200 to 1500 h). Visual observation was used to record the behavioral state every 20 sec as wake, non-rapid eye movement sleep (non-REMS) or REMS, whereby nonREMS was scored when the animal was recumbent with eyes closed and showing little or no movement and REMS when the animal was recumbent with eyes closed and showing rapid movements or twitches ofthe eyes, whiskers, ears or paws (as previously determined to correspond to polygraphicalIy scored sleep states (Maloney et al., 1997». AnimaIs were prevented from sleeping during SD by gentle touching (with a small soft paint brush inserted through the bars of the cage top) upon eye closure. The deprivation procedure resulted in the total absence of sleep during 3 hours for the SD and SR groups and was folIowed in the SR group by an increase in total sleep relative to both the SD group and SC group. At the end of the experimental period (1500 h), the rats were immediately killed under pentobarbital anesthesia (100 mglkg, i.p.) by intra-aortic perfusion with a fixative solution of3% paraformaldehyde. AlI procedures were approved by the McGill University Animal Care Committee and the Canadian Council on Animal Care. 51 Following immersion in a 30% sucrose solution, the brains were frozen and stored at -80 0 C. They were cut in coronal sections at 20 Jlm, which was determined to be the greatest thickness that would allow full penetration and staining with the antibodies employed. Adjacent series of sections were collected and processed for double immunohistochemical staining using peroxidase-antiperoxidase (P AP) for 1) c-Fos (rabbit antiserum,1:10,000, Ab-5, PC38, Oncogene Research Products, La Jolla, CA) with DAB-Ni as chromogen and 2) a) at 400 Jlm intervals, choline acetyltransferase (ChAT, mouse antibody, 1:2000, MAB5270, Chemicon International, Temecula, CA) or b) at 200 Jlm intervals, GAD (mouse antibody, detecting both GAD65 and GAD67 isoforms, 1: 100, MSA-225, Stressgen Biotechnologies, Victoria, BC) with pink, alphanaphthol pyronin B. Other series taken at 800 Jlm intervals were processed for triple immunostaining for 1) c-Fos in the first position using DAB-Ni, 2) U2AAR (goat purified antiserum, 1:50, sc-1478, Santa Cruz Biotechnology, Santa Cruz, CA) in the second position using red Cy3-conjugated donkey antigoat antiserum (Jackson ImmunoResearch Laboratories, West Grove, PA), and 3) GAD (rabbit antiserum, 1:1000, AB5992; Chemicon) using green Cy2-conjugated donkey antirabbit antiserum (Jackson) in the third position. Incubations with primary antibodies were performed at room temperature overnight for c-Fos, ChAT and GAD antibodies and three nights at 4 oC for u2AAR antibody using a Tris-saline solution (0.1 M) containing 1% normal donkey serum (NDS) (following initial blocking with 3% NDS). Sections were viewed by light and fluorescence microscopy (Leica DMLB microscope equipped with an x/ylz movement-sensitive stage and video camera attached to a computer). Cells immunopositive (+) for single c-Fos+, double c-Fos+/ChAT+ or c- 52 Fos+/GAD+ and triple c-Fos+/GAD+/u2AAR+ were counted by applying stereology using the Optical Fractionator program of Stereo Investigator (2003, MicroBrightFie1d, Williston, VT). Cells were counted within the contours of 8 nuclei in the BF and POA, respectively: SIa, SIp, MCPO, DBB and LPO, VLPO, MPO, MnPO. The nuclei were delineated in a computer resident atlas (modified from (Gritti et al., 1993» that was placed in register with each section. For each nucleus, labe1ed cells were counted bilaterally in at least three sections (3 - 14, depending upon the length of each nucleus) at 400 /lm intervals for the c-Fos/ChAT-immunostained series through the BF region nuclei or 200 /lm for the c-Fos/GAD-immunostained series through the BF and POA regions. Through all nuclei, cells were counted under a 63x oïl objective (with 1.4 numerical aperture) within a counting frame of 125 x 125 /lffi. In most nuclei, cells were counted through 25% of the area at each level by setting the grid size (within which the counting frame is automatically inserted) to 250 x 250 /lm. For the smaller nuclei (VLPO and MnPO), cells were counted through the entire area of the nucleus by setting the grid size to 125 x 125 /lm. Counts oftriple-Iabeled cells immunostained for c-Fos/GAD/u2AAR were combined across nuclei for estimates of total cell numbers within the BF and POA regions. By counting nuclei that came into focus beneath the surface of the section, cFos+ nuclei were counted within a counting block of 8 /lm in depth (in the dehydrated, delipidated, mounted and coverslipped sections that were on average 10 /lm thick). Sleep and cell counts were analyzed between groups and across nuclei or regions using one and two way ANOVA in Systat (v10.2, Richmond, CA). All main effects were confirmed by nonparametric rank order tests (K.ruskal-Wallis, p < .05) to insure that the distribution of variance in groups (containing zeros as a result of the experimental 53 condition) did not distort the parametric statistics. Figures were composed using Adobe Photoshop 6 and Illustrator 9 (Adobe, San Jose, CA). 54 3.5 Results Rats submitted to sleep deprivation (SD) by gentle touching did not sleep during the 3 hours prior to sacrifice at 1500 h, whereas those permitted sleep recovery (SR) during the same period after 3 hours deprivation in the moming (900 to 1200 h) slept ~91 % of the time. The amount oftotal sleep differed significantly across conditions of SD, SR and sleep control (SC, Table 1). Recovery and control sleep were comprised predominantly ofbehaviorally quiet, non-REMS (non-REMS: 77.23 ± 2.94% in SR and 68.68 ± 0.10% in SC with REMS: 13.58 ± 1.30% in SR and 7.10 ± 0.66% in SC, mean ± S.E.M. of total time). 55 Table 1. Average Percent Sleep and c-Fos expression across nuc1ei (Nuc) of the basal forebrain (BF) and preoptic area (POA) under conditions (Cond) ofsleep control (SC), sleep deprivation (SD) and sleep recovery (SR) in three groups ofratsa sc Variable % Sleep 75.75 ± 0.61 650.53 ± 86.29 Total Number c-Fos+ Cells Percent c-Fos+/ChAT+ Percent c-Fos+/GAD+ 3.57 ± 1.61 - 44.00 ± 3.66 SD F (Cond) SR F (Nuc) 1095.40 *** 2462.70 ± 341.40 (SC,SR) 575.80 ± 95.02 (SD) 70.72 *** 0.00 ± 0.00 1 (SC,SR) 90.80 ± 2.47 (SC, SD) 1 16.67 F (CondxNuc) *** 11.68 ± 2.64 (SC, SR) 0.00 ± 0.00 (SD) 8.95 *** 0.33 ns 17.16 _:±: ~.7L_ ~C,SR) 41.16 ± 4.04 (SD) 25.86 *** 6.53 56 *** 4.77 *** 0.16 ns 1.35 ns Table 1. aMean ± S.E.M values from 4 rats per group. % Sleep was calculated as % of3 br observation period prior to killing and was examined across groups by a one-way ANOVA. For number or percent of c-Fos expressing cens, two-way ANOVAs were performed with Cond (3) and Nuc (8) as factors. For each variable, there was a significant main effect of condition. Post-hoc paired comparisons with Bonferroni correction were performed to examine differences between conditions (indicated in parentheses, p < .05). For % Sleep, SD and SR both differed from SC and from each other; for c-Fos+ cen variables, SD differed from SC and SR but SR did not differ from Sc. For total number of c-Fos+ cens (ca1culated as c-Fos+ plus c-Fos+/GAD+ cens in the c-Fos/GAD dual-immunostained series), there was a significant difference across nuclei and a significant interaction of condition with nuclei. Post-hoc comparisons between conditions in each nucleus revealed a main effect of condition in every nucleus except the MnPO. For percent of c-Fos+ cens (as c-Fos+/ChAT+ or c-Fos+/GAD+ of the total), there was a significant difference for GAD+ cens between nuc1ei, but there was no significant interaction between condition and nuclei for GAD+ or ChAT+ cells, thus contraindicating post-hoc comparisons for condition in each nucleus. (*** indicates Fratio withp < .001; ns means not significant.) 57 In SR and/or SD rats, c-Fos was expressed in ChAT-immunopositive (+) and GAD+ cens as wen as in ChAT-immunonegative (-) and GAD- cens within the BF and POA (Fig. lA-D). The numbers of c-Fos+ cens varied significantly across conditions, differing between SR or SC and SD but not between SR and SC (Table 1), According to a two-way ANOVA with condition and nuc1ei as factors, c-Fos+ cens were significantly decreased in SR (like SC) as compared to SD (see Figs. 2A-B and 3B, Table 1). Given a significant interaction between condition and nuc1ei, post-hoc comparisons were performed to determine if the decrease was significant in an nuc1ei (Table 1). The difference was found to be significant in an nuc1ei, except the MnPO. The proportions of c-Fos+ cens that were ChAT+ were also significantly decreased in SR (like SC) as compared to SD (Figs. 2C-D and 3C, Table 1). In this case, there was no significant interaction between condition and nuc1ei, indicating that the effect of condition was not different across individual nuc1ei ofthe BF. In contrast to those being ChAT+, the proportions of c-Fos+ cens that were GAD+ were significantly increased in SR (like SC) as compared to SD (Figs. 2E-F and 3D; Table 1). In this case, there was also no significant interaction between condition and nuc1ei, indicating that the main effect of condition did not differ across the nuc1ei of the BF and POA. 58 Figure 1 59 Figure 1. Labeled cells in the nuc/ei of the BF or POA. A. Single-Iabeled cFos+ cell (stained black with DAB-Ni, black arrowhead), single-Iabeled ChAT+ cell (stained pink with alpha-naphthol pyronin B, white arrowhead) and double-Iabeled cFos+/ChAT+ cell (double arrowhead) in the MCPo. B-D. Single-Iabeled c-Fos+ cells (stained black, black arrowheads), single-Iabeled GAD+ cells (stained pink, white arrowheads) and double-Iabeled c-Fos+/GAD+ cells (double arrowheads) in the MCPO (B), MPO (C) and VLPO (D). E and F. A triple-Iabeled c-Fos+/GAD+/a2AAR+ cell shown in images of c-Fos staining (stained black with DAB-Ni, black arrowhead in E) and a2AAR (stained by redfluorescence with Cy3) together with GAD staining (stained by green fluorescence with Cy2, white arrowhead in F). The a2AAR labeling is evident over the GAD+ cell body (in orange or yellow). Magnification bars = 20 j.lm (for A-D shown in D and E-F shown in F). 60 Figure 2 E .t. c.-Fos+/GAD+ SD SR 5D 61 SR Figure 2. Distribution ofc-Fos+ cells in one SD and one SR representative brain through the BF and POA (at ~ A8.6 in A, C and E and ~A9.0 in B, D and F, (Gritti et al., 1993)). A-B. c-Fos+ cells (black dots) within BF and POA nuclei, C-D. Double-labeled c-Fos+/ChAT+ cells (red circles) distinguishedfrom single-labeled c-Fos+ cells (black dots) within the BF, and E-F. Double-labeled c-Fos+/GAD+ cells (blue triangles) distinguishedfrom single-labeled c-Fos+ cells (black dots) within the BF and POA nuclei. Atlas images show cells plotted in one 20 pm thick section per level (~A 8.6 on left and ~A 9.0 on right) from adjacent series (separated by 200 f1Jn) dual-immunostained for c-Fos/ChAT (C-D) or c-Fos/GAD (A-B and E-F). Abbreviations: SIa, substantia innominata, anterior part; (SIp, substantia innominata, posterior part, not shown); MCPO, magnocellular preoptic area; DBB, diagonal band ofBroca nuclei; LPO, lateral preoptic area; VLPO, ventrolateral preoptic area; MPO, medial preoptic area; MnPO, median preoptic nucleus. (Note that all nuclei examined are contained within the sections illustrated in Fig. 2, except SIp, which is located in the same relative position as SIa at more caudallevels.). 62 Figure 3 A B Sla ~ ~û '5 1 ~ ~ ~ Ù '5 1iE g ~ 1 C ... 8 DBB Sla ~ l 6 ". SR D SR ~ ô ~ ~ >fi SO SR Condition SO SR Condition 63 Figure 3. Bar charts showing the percentage ofsleep (in A) and the total numbers of c-Fos+ cells (in B) or proportions of c-Fos+ cells that were cholinergic or GABAergic (in Cor D) within each nucleus of the BF and/or POA in SD and SR groups. A. The % sleep (representing % total sleep of 3 hour period prior to sacrifice) was significantly increased in SR as compared to SD. B. The total numbers of c-Fos+ cells (obtainedfrom c-Fos/GAD dual-immunostained series as the total of c-Fos+ and cFos+/GAD+ cells) were significantly decreased across nuclei of the BF and POA in SR as compared to SD (with a significant difference across nuclei and a significant interaction between condition and nuclei, due to a significant decrease in ail nuclei except MnPO, Table 1). C. The percentages of c-Fos+ cells that were ChAT+ (obtained from the c-Fos/ChAT dual-immunostained series as the % of c-Fos+ plus c-Fos+/ChAT+ cells that were c-Fos+/ChAT+) were significantly decreased across nuclei of the BF in SR as compared to SD (with no significant difference across nuclei or interaction between condition and nuclei, Table 1). D. The percentages of c-Fos+ cells that were GAD+ (calculatedfrom total numbers shown in B) were significantly increased in SR as compared to SD (with a significant difference across nuclei but no significant interaction between condition and nuclei, contraindicating post-hoc comparisons in individual nuclei, Table 1). For each group (n = 4), mean rS.E.M are presented. For abbreviations see Fig. 2. 64 The incidence of Œ2AAR labeling was subsequently examined on c-Fos+/GAD+ cells in the SD and SR groups by triple-immunostaining (Fig. lE and F). The proportions of c-Fos+/GAD+ cells that were Œ2AAR+ were small in the SD group and significantly higher in the SR group across the BF and POA regions (with no significant difference across the regions or interaction of condition with region, Fig. 4). On average across regions, 3.88 ± 1.73% of c-Fos+/GAD+ cells were Œ2AAR+ in the SD group and 77.70 ± 22.07% in the SR group. 65 Figure 4 BF fi) A ~ + ~ .. ~ + Cl ~ !!l u.. +" <) '* SO SR Condition POA fi) B ~ + et: .. <f .... ~ + ~'" 0 ~ '* SO SR Condition 66 Figure. 4. Bar charts showing the proportions of c-Fos+/GAD+ cells that bear a2AAR in the BF (A) and POA (B) in SD and SR groups. There was a significant increase in c-Fos+/GAD+/a2AAR+ cells in SR as compared to SD (ANOVAfor main effect of condition, F = 7.168, p < 0.05 with no significant difference across the two regions and no significant interaction between condition and region, p > 0.05). 67 3.6 Discussion The present results indicate that across the BF and POA fewer neurons are active during sleep than during waking, and that of those active during sleep, none are cholinergic and a substantial proportion are GABAergic. Moreover, the majority of the sleep-active GABAergic neurons appear to bear <X2AAR. In aIl nuclei of the BF and aIl but the MnPO of the POA, more neurons accumulated c-Fos protein following 3 hours ofwaking produced by gentle deprivation of sleep than following 3 hours of sleep either during recovery from deprivation or control condition. These results suggest that the majority of neurons in the BF and POA are more active during waking than during sleep. Similar conclusions had been reached by other studies employing c-Fos immunohistochemistry or in situ hybridization in the past (Pompeiano et al., 1992, Cirelli et al., 1993, Ledoux et al., 1996, Sastre et al., 2000). Only recently was it reported tirst for the VLPO and then for the MnPO in the POA, that more neurons expressed c-Fos during sleep than during waking (Sherin et al., 1996, Gong et al., 2000). It is possible that the particular experimental conditions in those studies, such as the housing, surgery or method of sleep deprivation, which can be associated with certain degrees of stress and associated c-Fos expression at particular times (Dragunow and Faull, 1989, Cullinan et al., 1995, Maloney et al., 1999), resulted in relatively higher c-Fos expression in those nuclei during sleep than in the present study. Here, we employed conditions that would minimize stress prior to and during the experimental period by studying the animaIs in their home cages, using behavioral observation to record sleep so as to avoid prior surgery and applying gentle touching with a soft brush 68 for preventing sleep during a re1ative1y short (3 hour) period. Vnder these conditions, an increase in the total number ofneurons expressing c-Fos was not observed in any nucleus ofBF or POA during recovery or control sleep as compared to enforced waking. Indeed, the number ofneurons active in SR was on average <25% ofthat active in SD. The proportions did differ however across nuclei with the highest proportions being in the MnPO (~75%) (~20%), and VLPO (~50%) and lower proportions in the BF and LPO nuclei suggesting different concentrations of sleep- versus wake-active neurons with the highest concentrations in MnPO and VLPO. These results are supported by both in vivo and in vitro e1ectrophysiological studies showing re1atively high concentrations of putative sleep-promoting neurons in these POA nuclei (Szymusiak et al., 1998, Ga1lopin et al., 2000, Suntsova et al., 2002). Single unit recording studies have also found relative1y small proportions of neurons that are maximally active specifically during non-REMS or SWS in BF and POA nuclei (Detari et al., 1984, Szymusiak and McGinty, 1986a, Koyama and Hayaishi, 1994, Szymusiak et al., 2000, Suntsova et al., 2002, Lee et al., 2004). On the other hand in the same studies, a large and varying proportion of neurons has been characterized through these regions as active during REMS, sorne as non-REMS/REMS-active, sorne se1ective1y REMS-active and others as Wake/REMS-active. In a study examining c-Fos in pharmacologically produced predominant REMS versus predominant non-REMS conditions, it was found that REMS was associated with increased c-Fos expression in many forebrain regions (Sastre et al., 2000). We assume that in the present study the major percent oftime spent in non-REMS time spent in REMS (~14%) (~77%) as compared to the small percent of in the 3 hours preceding sacrifice served as the major determinant in c-Fos expression and accumulation during recovery as well as control 69 conditions. We thus conclude that the c-Fos+ neurons seen following sleep recovery or controllargely represent non-REMS-active neurons (which would include nonREMS/REMS-active but not selective REMS-active or Wake/REMS-active neurons) that are distributed across BF and POA as a small population though with relatively greater representation in certain nuclei of the POA. In parallel with the total number ofneurons expressing c-Fos, the proportions of those neurons that were cholinergie across BF nuclei were diminished during sleep. In fact, no c-Fos+/ChAT+ neurons were detected in the sleep recovery condition. These results confirm electrophysiological studies indicating that presumed cholinergie neurons are silent during SWS (Jones, 2004, Lee et al., 2004). They also confirm that cholinergie neurons are active during waking. According to release of acetylcholine and firing of presumed cholinergie BF neurons, however, they should also be active during REMS (Marrosu et al., 1995, Jones, 2004). As for aIl c-Fos+ cells (above), we assume that the lack of c-Fos immunostaining in cholinergie neurons in the recovery condition, as weIl as control, is due to the predominance of non-REMS in the 3 hours prior to sacrifice and the small amount oftime spent in REMS (14%) as weIl as waking (~9%) that would be inadequate for accumulation of the Fos protein during that period (Dragunow and FauIl, 1989). In contrast to the proportion of cholinergie neurons, the proportion of GABAergic neurons expressing c-Fos increased significantly with SR as compared to SD across BF and POA nuclei. These results indicate that many GABAergic neurons distributed across these regions remain or become active in association with sleep following waking. They support claims that neurons in the BF and POA including the VLPO that project to the posterior hypothalamus and contain GAD may be sleep-active and promoting neurons 70 and may act by inhibiting wake-promoting neurons ofthat region (Gritti et al., 1994, Sherin et al., 1998). They also support the hypothesis that identified GABAergic BF neurons that discharge in association with cortical slow wave activity in urethaneanesthetized rats and project either to posterior hypothalamus or cortex, if not locally, as determined by antidromic activation, could be sleep-active and promoting as well (Manns et al., 2000a). Finally, they confirm similar results most recently reported by others for cFos expression in GAD+ neurons of the MnPO and VLPO (Gong et al., 2004). GABAergic neurons of different BF and POA nuc1ei could thus exert an inhibitory influence upon wake-active and promoting systems in the posterior hypothalamus, cortex and/or basal forebrain. However, in single unit recording studies, it was also found that another physiologically distinct group of GABAergic neurons discharged maximally in association with cortical activation (Manns et al., 2000a). Therefore, different groups of GABAergic neurons in the BF and POA are likely active in association with cortical activation of waking and others in association with SWS. The major proportion (~78%) ofGAD+ neurons that expressed c-Fos with SR in the present study were immunostained for the a2AAR, whereas only a minimal percentage (~4%) ofthose that did so with SD were so labe1ed. These results confirm the hypothesis that GABAergic neurons in the BF and POA that are active during sleep bear a2AR and would accordingly be inhibited by NA (Osaka and Matsumura, 1995, Bai and Renaud, 1998, Manns et al., 2003, Saint-MIeux et al., 2004). This particular group of GABAergic neurons would be held under inhibition during waking by re1ease of NA from afferent locus coeruleus neurons (Jones and Cuello, 1989) that fire maximally during active waking, decrease firing during quiet waking, and further diminish firing 71 during SWS to cease firing during REMS (McCarley and Hobson, 1975, Aston-Jones and Bloom, 1981). With decremental re1ease of NA during drowsiness, these GABAergic neurons would be disinhibited to become active and promote sleep by inhibiting in tum the noradrenergic neurons together with other neurons of the brainstem and forebrain activating systems (Gritti et al., 1994, Luppi et al., 1995, Sherin et al., 1998, Steininger et al., 2001, Chou et al., 2002). 3.7 Acknowledgments The research was supported by a grant from the Canadian Institutes of Health Research (13458). 72 3.8 Preface to chapter 3 The study in the second chapter revealed that across the BF and POA, higher numbers of cens s expressed c-Fos during sleep deprivation than they did during sleep recovery. Similarly, the proportions of c-Fos expressing cens that were cholinergic were higher after SD than they were after SR. In contrast, the proportions of c-Fos expressing cens that were GABAergic were higher after SR as compared with the SD condition. Moreover, the majority of sleep c-Fos expressing GABAergic cens bore a2AAR, whereas few wake c-Fos expressing bore a2AAR. These results revealed that the BF and POA are comprised of many GABAergic cens that are active during sleep and thus might be important in sleep promotion and maintenance. We showed that these sleep-active GABAergic cens are distributed across the BF and POA yet they are more concentrated in sorne nuc1ei inc1uding the VLPO and the MnPO. Our study also explained one potential mechanism being able to explain how sleep promoting cens become active in a cyc1ical manner. These particular GABAergic cens are under NA inhibition through a2AAR during waking when the release of NA is highest. They subsequently become disinhibited during sleep, when the release of NA is lowest, to become active to promote and perhaps maintain sleep. These studies chemicany identified the neurons of the BF and the anterior hypothalamic POA that are involved in sleep-wake regulation. As has been long known, in addition to the importance of the forebrain, the posterior hypothalamus plays a critical role in the regulation ofbehavioral states, particularly arousal and waking. With the goal of identifying the chemical transmitters of neurons in the posterior hypothalamus that 73 play roI es in the sleep-waking cycle, the next study was devoted to investigate the potential roles of the two hypothalamic peptides, Orx and MeR. We hypothesized that Orx neurons are active during waking whereas MeR cens are active during sleep. Using the same paradigm of sleep deprivation and sleep recovery in rats and by applying immunohistochemistry for antibodies against Orx and MeR and by studying c-Fos expression, the activity of these two cen populations across behavioral conditions were studied. Furthermore in order to understand the mechanisms of cyclical regulation of Orx vs. MeR cens, the incidence of alAAR vs. a2AAR on these cens were studied to test the hypothesis that during waking Orx cens would be excited by NA and therefore might bear alAAR whereas MeR cens would be inhibited by NA during waking and thus might beara2AAR. 74 4. Chapter 3: Orexin and MCR Neurons Express c-Fos Differently After Sleep Deprivation vs. Recovery and Bear Different Adrenergic Receptors 3 Mandana Modirrousta, Lynda Manville and Barbara E. Jones 3 Eur J Neurosci. 2005 May;21(10):2807-16 75 4.1 Abbreviations AR, adrenergic receptor DMH, dorsomedial hypothalamus LH, lateral hypothalamus MCH, melanin concentrating hormone NA, noradrenaline non-REMS, non-rapid eye movement sleep Orx,orexin PF, perifomical area REMS, rapid eye movement sleep SC, sleep control SD, sleep deprivation SR, sleep recovery ZI, zona incerta 76 4.2 Abstract Though overlapping in distribution within the posterior hypothalamus, neurons containing orexin (Orx) and melanin concentrating hormone (MeR) may play different roi es in the regulation ofbehavioral state. In the present study in rats, we tested whether they express c-Fos differently after total sleep deprivation (SD) vs. sleep recovery (SR). Whereas c-Fos expression was increased in Orx neurons after SD, it was increased in MeR neurons after SR. We reasoned that Orx and MeR neurons could be differently modulated by noradrenaline (NA) and accordingly bear different adrenergic receptors (ARs). Of aIl Orx neurons (estimated at ~6700), substantial numbers were immunostained for the 0, 1AAR, including ceIls expressing c-Fos after SD. Yet, substantial numbers were also immunostained for the o,2AAR, also including ceIls expressing c-Fos after SD. Of aIl MeR neurons (estimated at ~ 12,300), rare neurons were immunostained for the 0, 1AAR, whereas significant numbers were immunostained for the o,2AAR, including cells expressing c-Fos after SR. We conclude that Orx neurons may act to sustain waking during sleep deprivation, whereas MeR neurons may act to promote sleep foIlowing sustained waking. Sorne Orx neurons would participate in the maintenance ofwaking during deprivation when excited by NA through o,lARs, whereas MeR neurons would participate in sleep recovery after deprivation when released from inhibition by NA through o,2ARs. On the other hand under certain conditions, Orx neurons may also be submitted to an inhibitory influence by NA through o,2ARs. 77 4.3 Introduction The posterior hypothalamus has long been known to play a critical role in the maintenance ofwakefulness (Jones, 2000). Recently two peptides, orexin (Orx, also ca11ed hypocretin) and melanin concentrating hormone (MCR) have been found to be contained in distinct populations of magnocellular neurons that partially overlap in their distribution through the mid to posterior hypothalamus and commonly give rise to widespread projections through the central nervous system (Bittencourt et al., 1992, Broberger et al., 1998, de Lecea et al., 1998, Peyron et al., 1998). They were both initially found to have a stimulatory influence upon eating and thus to be considered as contingents of the hypothalamic 'orexigenic' systems (Williams et al., 2004). Yet, as became evident in knock out experiments e1iminating the peptides or their receptors, their normal roles appeared to extend beyond influences upon eating and to differ with regard to energy homeostasis. Most notably, mice lacking the gene for orexin or dogs lacking the gene for its receptor manifested a syndrome ofnarcolepsy (Chemelli et al., 1999, Lin et al., 1999) and humans with narcolepsy were found to lack the gene, peptide or neurons containing Orx (Peyron et al., 2000, Thannickal et al., 2000). In addition, Orx knock out mice became obese despite being hypophagic, attributed to a decrease in activity and basal metabolic rate. As evidenced by c-Fos expression and Orx re1ease, Orx cells also appeared to be most active during the night, when animaIs were awake and engaged in motor activity (Estabrooke et al., 2001, Yoshida et al., 2001). Orx thus appeared to stimulate arousal with motor activity and increased energy expenditure. In contrast, 78 although MeR knoek out miee were also hypophagie, they manifested a decrease in body weight attributed to an increase in aetivity and basal metabolic rate (Shimada et al., 1998, Marsh et al., 2002). MeR thus appeared to decrease arousal and energyexpenditure. Aecording to these results, Orx and MeR neurons may perform opposite roles in wake vs. sleep promotion and/or maintenance. Rere, we examined whether Orx and MeR cells respond differently to total sleep deprivation (SD) vs. sleep recovery (SR) by using c-Fos expression as an indieator of neuronal aetivity. Different activity profiles eould be due to different responses of the Orx and MeR neurons to the major neurotransmitters of the ascending arousal systems inc1uding most importantly noradrenaline (NA). In the basal forebrain and preoptic areas, cholinergie wake-aetive neurons are exeited by NA through al adrenergic reeeptors (aIARs), whereas GABAergic putative sleep-aetive neurons are inhibited by NA through a2ARs (Fort et al., 1995, Fort et al., 1998, Gallopin et al., 2000, Manns et al., 2003, Modirrousta et al., 2004b). We thus examined whether the Orx and MeR neurons bear different adrenergic receptors while possibly expressing c-Fos under different conditions of sustained waking vs. reeovery sleep. 79 4.4 Material and methods AlI procedures were approved by the McGill University Animal Care Committee and the Canadian Council on Animal Care, whose standards meet those of the Association for Assessment and Accreditation of Animal Care International. Male Wistar rats (200-250 g) were used in minimal numbers to make up three test groups. The rats were housed individually with free access to food and water at all times and maintained on a 12:12lightldark schedule (with lights on from 700 to 1900 hours). As previously described (Modirrousta et al., 2004b), the experiment was designed to deprive rats of sleep under non-stressful conditions and was thus performed in the home cages. The three different groups (n = 4 per group) were submitted to 1) total sleep deprivation (SD) for 3 hours (1200 to 1500 h), 2) total sleep deprivation for 3 hours (900 to 1200 h) followed by sleep recovery (SR) for 3 hours (1200 to 1500 h), or 3) undisturbed sleep and waking as sleep control (SC) for 3 hours (1200 to 1500 h). The experimenter (MM) brought each rat in its cage (in groups of 3) to an experimental room in the animal facility and habituated each rat to her presence and to a paint brush inserted through the cage top for 3 days prior to the experimental day. During the experiment for the SD and SR groups, she deprived each rat of sleep by gently touching it with the brush when it c10sed its eyes. She deprived and/or observed one rat at a time (on one day) and scored by visual observation its behavioral state every 20 sec. as comprised in the majority by wake, non-rapid eye movement sleep (non-REMS) or REMS. The behavioral scoring was based upon previous studies in the current lab (Maloney et al., 1997) together with 80 demonstrations in other labs (Bergmann et al., 1987, Espana et al., 2003), showing that behavior can be used in nonnal rats to reliably score the major states ofW, non-REMS and REMS. Thus, as previously detennined to correspond to polygraphically scored sleep states in our lab (Maloney et al., 1997), non-REMS was scored when the animal was recumbent with eyes closed and showing little or no movement and REMS when the animal was recumbent with eyes closed and showing rapid movements or twitches of the eyes, whiskers, muzzle, ears or paws. The deprivation procedure resulted in the total absence of sleep during 3 hours for the SD and SR groups and was followed in the SR group by an increase in total sleep relative to both the SD and SC groups. At the end of the experimental period (1500 h), the rats were immediately killed under pentobarbital anesthesia (100 mg/kg, i.p.) by intra-aortic perfusion with a fixative solution of3% parafonnaldehyde. Following immersion in a 30% sucrose solution, brains were frozen and stored at -80 0 C. They were cut in corona! sections at 20 /Jm, which was detennined to be the greatest thickness that would allow full penetration and staining with the antibodies employed. Adjacent series of sections were collected at 400 /Jm intervals and processed for double immunohistochemical staining using peroxidase-antiperoxidase (P AP) for 1) c-Fos (rabbit antiserum, 1:10,000, Ab-5, PC38, Oncogene Research Products, La Jolla, CA) with DAB-Ni as chromogen and 2) Orexin (Orexin-A (C-19), goat antibody, 1:500, sc-8070, R230, Santa Cruz Biotechnology, Santa Cruz, CA) or MCR (MCR (E-16) goat antibody, 1:500, sc-14507, L281, Santa Cruz Biotechnology) with pink, alpha-naphthol pyronin B. Other series were processed for triple immunostaining for 1) c-Fos in the first position using DAB-Ni, 2) a 1AAR (goat purified antiserum, 1:50, sc-14 75, R31 0, Santa 81 Cruz Biotechnology) or U2AAR (goat purified antiserum, 1:50, sc-1478, K0702, Santa Cruz Biotechnology) in the second position using Cy3-conjugated donkey anti-goat antiserum (Jackson ImmunoResearch Laboratories, West Grove, PA), and 3) Orx (Orexin A, rabbit antiserum, 1:2000, R261-3, Phoenix Pharmaceuticals, Belmont, CA) or MCR (rabbit antiserum, 1:5000, R177-6, Phoenix Pharmaceuticals) in the third position using Cy2-conjugated donkey anti-rabbit antiserum (Jackson). Incubations with primary antibodies wereperformed at room temperature ovemight for c-Fos, Orx and MCR antibodies and two nights at 4° C for UIAAR and U2AAR antibodies using a Tris-saline solution (0.1 M) containing 1% normal donkey serum (NDS, fonowing initial blocking with 3% NDS). Sections were viewed by light and fluorescence microscopy with a Leica DMLB microscope equipped with an x/y/z movement-sensitive stage and video camera attached to a computer. Single-, double- and triple-immunostaining were evaluated for c-Fos, Orx or MCR and UIAAR or U2AAR. Single-, double- and triple-Iabeled cens were counted by applying stereology using the Optical Fractionator program of Stereo Investigator (2003, MicroBrightField, Williston, VT). cens were sampled and counted within delineated contours at appropriate levels of a computer resident atlas (expanded from (Gritti et al., 1993)). On each side, a large contour was employed to inc1ude the nuc1ei in which the Orx neurons were located (lateral hypothalamus (LR), perifomical area (PF), and dorsomedial hypothalamus (DMR)) or those in which the MCR neurons were located (LR, PF, DMR and zona incerta (ZI)). Partially overlapping through the hypothalamus, the Orx and MCR cens were distributed within these contours and nuc1ei over approximately 1600-1800 !lm from anterior to posterior. Accordingly, cens single-, 82 double- or triple-Iabe1ed for c-Fos and/or peptide (Orx or MCR) and/or receptor (a,lAAR or a,2AAR) were sampled and counted bilaterally within the contours in three sectionslevels at 400 ~m intervals from anterior to posterior. Within the stereology program, the sizes of the counting frame, within which cells are counted, and the grid, within which the counting frames are automatically placed, were optimized for each single-, double- or triple-Iabeled series so that a suitable number of cells were counted per level in the deprived or recovery condition. Accordingly, the counting frame (125 x 125 grid size (300 x 300 ~m or 350 x 3 50 ~m) ~m) and were set to sample ~ 13 or 17% of the area for single-Iabe1ed c-Fos, Orx and MCR cell counts; the counting frame (140 x 140 ~m) and grid size (200 x 200 ~m) were set to sample 49% of the area for counting double-Iabeled c-Fos/Orx or c-Fos/MCR celIs; and the counting frame and grid size (both at 140 x 140 /lm) were set to sample 100% ofthe area for double-Iabe1ed Orx or MCR and a,lAAR or a,2AAR cell counts as well as triple-Iabe1ed profiles with c-Fos. The cells were counted under a 63x oil objective (with 1.4 numerical aperture). Nuc1ei or cells that came into focus beneath the surface of the section were counted within a counting block of 8 /lm in depth (in the dehydrated, de1ipidated, mounted and coverslipped sections that were on average 10 /lm thick). Sleep and cell counts were analyzed between groups and across nuc1ei or regions using one and two way ANOVAs in Systat (v10.2, Richmond, CA). AlI main effects were confirmed by nonparametric rank order tests (Kruskal-Wallis, p < .05) to insure that the distribution of variance in groups (containing zeros as a result of the experimental condition) did not distort the parametric statistics. Figures were composed using Adobe Photoshop Creative Suite (CS) and Adobe Illustrator CS (Adobe, San Jose, CA). 83 4.5 Results Sleep deprivation and recovery Rats in the SD group did not sleep during the 3 h in the aftemoon prior to sacrifice (at 1500 h), whereas rats in the SR group that were allowed to recover sleep in the aftemoon after 3 h of sleep deprivation in the moming (900-1200 h) slept ~91 % of the time prior to sacrifice (at 1500 h). The amount of sleep differed significantly across conditions of SD, SR and SC (Table 1). The major proportion of time for the SR and SC groups was spent in non-REMS (77.23 ± 2.94% in SR and 68.68 ± 0.10% in SC) and a minor proportion in REMS (13.58 ± 1.30% in SR and 7.10 ± 0.66% in SC, mean ± S.E.M. of total time). c-Fos expression in Orx and MCH neurons after sleep deprivation and recovery c-Fos was expressed in neurons ofthe hypothalamus inc1uding Orx, MCH, nonOrx and non-MCH cells (Fig. lA, B). According to ANOVA with condition (and peptide series) as factors, total numbers of c-Fos-immunopositive (+) cells varied significantly across conditions (independent of peptide series) being highest in the SD condition (Table 1). Total numbers of Orx- and MCH-immunopositive neurons did not vary significantlyas a function of condition. The Orx+ neurons, which are distributed across the LH, PF and DMH (Figs. 1C and 2A) numbered on average ~6700 (Table 1). MCH neurons, which are distributed across LH, PF, DMH and ZI (Figs. ID and 2D) numbered 84 on average ~ 12,300 and were significantly more numerous than the Orx cells (Table 1). The numbers of c-Fos+/Orx+ and c-Fos+/MCH+ cells varied significantly across conditions but also varied significantly as a function of an interaction between condition and peptide (Table 1). Numbers of c-Fos+/Orx+ cells were significantly decreased in SR as compared to SD (Figs. 2B, C and 3C; Table 1), whereas numbers of c-Fos+/MCH+ cells were significantly increased in SR as compared to SD (Figs. 2E, F and 3F; Table 1). Numbers of c-Fos+/MCH+ cells were also significantly higher in SR as compared to SC (Table 1). Across the SD and SR groups, the numbers of c-Fos+/Orx+ cells were negatively correlated with the numbers of c-Fos+/MCH+ cells (correlation coefficient for pairwise values, r = -0.82, n = 8, p = 0.01). Relative to all c-Fos+ cells in SD, the cFos+/Orx+ cells represented a small proportion (~6%, Figs. 2B and 3B, C). Theyalso represented a small proportion of the Orx cell population (~9%, Figs. 2A, B and 3A, C). Similarly, relative to ail c-Fos+ cells in SR, the c-Fos+/MCH+ cells represented a small proportion (~6%, Figs. 2F and 3E, F). They also represented a small proportion of the MCH cell population (3%, Figs. 2D, F and 3D, F). 85 Table 1. Average percent sleep and numbers of Orx and MCR cells expressing c-Fos under conditions ofSleep Control (SC), Sleep Deprivation (SD) and Sleep Recovery (SR) Variable SC % Sleep 75.75 ± 0.61 0.00 ± 0.00 .(sC, SR) c-Fos+ 2535.67 ± 424.00 11924.38 ± 1382.56 (SC,SR) Orx+ 4888.75 ± 536.98 8042.00 ± 1421.88 25.50 ± 25.50 675.75 ± 123.84 10906.67 ± 1120.81 12711.75 ± 1430.25 25.50 ± 14.72 38.25 ± 12.75 c-Fos+/Orx+ MCH+ c-Fos+IMCH+ F (Coud) SR SD 86 (SC, SR) 90.80 ± 2.47 (SC, SD 1095.40 5187.88 ± 1112.65 (SD) 19.57 *** 7227.25 ± 397.04 3.26 us 20.54 *** 1.21 us 30.28 *** 142.50 ± 39.54 (SD) 13212.00 ± 592.98 (SR) *** 382.50 ± 60.70 (SC,_~I> Table 1. Values represent Mean ± S.E.M. for 3 groups of 4 rats (n = 12). Cell numbers were obtained from stereological estimates for total numbers on left and right sides. Values across conditions (Cond) were compared by one or two-way ANOVAs (with condition and peptide series as factors), followed by one-way ANOVA for condition (with p < 0.001 indicated by *** in table) and post hoc pair-wise comparisons between conditions (using Fisher's LSD with p < 0.05 indicated for SD and SR with respect to SC, SR or SD). The estimated total numbers of c-Fos+ cells differed across conditions in both the Orx and MCH series and did not differ between (or in interaction with) the peptide series (df = 2, 1, 2, 18) and are thus presented as the mean values for the two peptide series (n = 24). The numbers of Orx and MCH cells did not differ across (or in interaction with) conditions but did differ between each other, Orx cells (grand mean ± S.E.M., 6719 ± 622) being significantly fewer than MCH cells (12,277 ± 649; F = 18.26, df= 1,2,9; P = 0.002). In a two-way ANOVA, the numbers of c-Fos+/Orx+ and cFos+/MCH+ neurons differed significantly as a function of condition (F = 18.3; df= 2, 9; p < 0.001), differed between the two peptide series (F = 6.56; df= 1,9; p = 0.03) and differed as a function of an interaction between condition x peptide (F = 25.62; df = 2, 9; p < 0.001). Given the significant interaction, one-way ANOVAs were performed for each series and presented in the table along with post hoc pair-wise comparisons. 87 Figure 1 88 Figure 1. c-Fos expression in Orx and MCH neurons. A. Single-Iabeled c-Fos+ cell (stained black with DAB-Ni, black arrowhead), single-Iabeled Orx+ cell (stained pink with alpha-naphthol pyronin B, white arrowhead) and double-Iabeled c-Fos+/Orx+ cells (double arrowheads). B. Single-Iabeled c-Fos+ cell (stained black, black arrowhead), single-Iabeled MCH+ cells (stained pink, white arrowheads) and doublelabeled c-Fos+/MCH+ cell (double arrowhead). C Orx cell distribution in the LH, PF and DMH (stained brown with DAB). D. MCH cell distribution in the LH, PF, DMH and ZI (stained brown). Abbreviations: f, fornix. Magnification bars and 1 mm in C, D. 89 = 20 pm in A, B Figure 2 SD SR . .~~~~-------,--. . ~~~~--~------,.~~~~~~-------,--~  Orx+ • c-Fos+ .. c-Fos+/Orx+ ~~~~~----~--~~~~~~------.,--~~~r-~----~~~o MCH+ • c-Fos+ • c-Fos+/MCH+ MCH 90 Figure2: Distribution of Orx and MCH neurons expressing c-Fos after SD or SR. A. Orx ce/l distribution in the LH, PF and DMH (open triangles). B, C. Double-Iabeled c-Fos+/Orx+ ce/ls (fi/led triangles) distinguishedfrom single-Iabeled c-Fos+ ce/ls (black dots) in one SD (B) and one SR (C) representative brain. D. MCH ce/l distribution in the LH, PF, DMH and Z1 (open squares). E, F. Double-Iabeled c-Fos+/MCH+ ce/ls (fi/led squares) distinguishedfrom single-Iabeled c-Fos+ ce/ls (black dots) in one SD (E) and one SR (F) representative brain. Abbreviations: f, fornix; DMH, dorsomedial hypothalamus; LH, lateral hypothalamus; PF, perifornical area; ZL zona incerta. Magnification bar = 1 mm. 91 Figure 3 A B § t.. D 1 ~ 1 ~ <f ~ '1 (,) D ~ E 111 B fi, t ,.,.â :t 0 .. i! & .:. ~ ~6 MCH 92 Figure 3. Numbers of Orx and MCH neurons expressing c-Fos after SD or SR. A. Total numbers of Orx + ceUs counted bilateraUy and estimated by stereological analysis in the hypothalamus including LH, PF and DMH B. Total numbers ofc-Fos+ ceUs (obtainedfrom c-Fos/Orx dual-immunostained series as the total ofsingle c-Fos+ and double c-Fos+/Orx+ ceUs) were significantly decreased in SR as compared to SD (Table 1). C Total numbers ofc-Fos+/Orx+ ceUs were significantly decreased in SR as compared to SD (Table 1). D. Total numbers ofMCH+ ceUs counted and estimated by stereological analysis in the hypothalamus including LH, PF, DMH and ZL E. Total numbers ofc-Fos+ ceUs (obtainedfrom c-Fos/MCH dual-immunostained series as the total ofsingle c-Fos+ and double c-Fos+/MCH+ ceUs) were significantly decreased in SR as compared to SD (Table 1). F. Total numbers of c-Fos+/MCH+ ceUs were significantly increased in SR as compared to SD (Table 1). Note same scale in A, B, D and E (0 - 20,000) for single-labeled ceUs and smaUer scale in C and F (0 - 1000) for double-labeled ceUs. 93 Adrenergic receptors on Orx and MeR, including c-Fos expressing, neurons after sleep deprivation and recovery Immunostaining for adrenergic receptors (UIAAR or U2AAR), peptide (Orx or MCR) and c-Fos was performed to examine the incidence of UIAAR or U2AAR on Orx and MCR cens and also on Orx and MCR cens expressing c-Fos in the different conditions. Orx cens were immunostained for both types of adrenergic receptors, ~ 1100 for UIAAR (Fig. 4A) and ~1400 for U2 AAR (Fig. 4B; Table 2). Orx cens bearing the different ARs were distributed across the LR, PF and DMR with no ostensible segregation (Fig. 5A, B). Very few MCR neurons were immunostained for UIAAR (~160 having minimal positive staining, Fig. 4C), whereas a large number were immunostained for U2AAR (~1800, Table 2; Fig. 4D). MCR cens immunopositive for U2AAR were distributed across the LR, PF, DMR and ZI (Fig. 5D). The numbers of Orx and MCR AR-immunopositive cens differed significantly between the two peptide cen types (Table 2). There was a significantly higher prevalence of Orx+/UIAAR+ cens than MCR+/UIAAR+ cens. Moreover, whereas there was no significant difference between the numbers of UIAAR+ vs. U2AAR+ Orx cens, there was a significant difference between the numbers OfUIAAR+ vs. U2AAR+ MCR cens (Table 2). Indeed, ~16% of Orx cens bore UIAAR and ~21 % bore U2AAR, whereas only ~1.5% ofMCR cells bore UIAAR and ~15% bore U2AAR. The numbers of AR-immunopositive Orx and MCR cells did not vary significantly as a function of condition (Table 2). Analysis of the tripleirnrnunostaining revealed sorne c-Fos expressing Orx cells in SD which bore UIAAR and 94 sorne which bore U2AAR (Fig. 4E, F). No c-Fos expressing MeR neurons in SR were found to be immunopositive for UIAAR, whereas sorne were found to be irnrnunopositive for U2AAR (Fig. 40). 95 Table 2. Average number of Orx or MCH cells that were immunostained for UIAAR or U2AAR under conditions of SC, SD and SR. Variable SC SD Orx+/alAR+ 1102.75 ± 122.74 1058.33 ± 456.97 1325.00 ± 450.69 1084.56 ± 179.41 Orx+/a2AR+ 1531.75 ± 297.58 1133.33 ± 475.29 1735.00 ± 761.07 1406.33 ± 290.04 MCH+/alAR+ 213.25 ± 48.48 166.67 ± 87.00 141.67 ± 117.56 MCH+/a2AR+ 1479.25 ± 170.93 2300.00 ± 478.93 1933.33 ± 147.43 Mean SR F (Cond) F (AR) F (CondxAR) 0.29 ns 1.95ns 0.27 ns 2.4 ns 81.64** 1.86 ns 161.44 ± 44.38 1818.56 ± 196.29 1 Values represent Mean ± S.E.M. for 3 groups of 3 or 4 rats (n = 10). Cell numbers were obtained from stereological estimates for total numbers on left and right sides. Values of each peptide series (Orx and MCH) were analyzed by two-way ANOVA for condition (Cond) and adrenergic receptor subtype (AR). Total numbers of Orx and MCH neurons with UIAAR or U2AAR did not vary significantlyas a function of condition (ns, not significant). Whereas the number of neurons immunostained for UIAAR vs. U2AAR did not differ for the Orx neurons, it did differ for the MCH neurons (**, p < 0.01). 96 Figure 4 97 Figure 4. Adrenergic receptors (AR) on Orx and MCH neurons including those expressing c-Fos. A. Cell immunostainedfor Orx (Al) and aJ~R (Al and in superimposed image inA3). B. Cell immunostainedfor Orx (Bl) and a2~R B3). C Cell immunostainedfor MCH (Cl) that was minimally stainedfor (Bl and aJ~R (Cl and C3). D. Cells immunostainedfor MCH (Dl) ofwhich one was also stainedfor a2~R (Dl and D3). E. Labeled Orx+ cell (El) that was (E3). F. Labeled Orx+ cell (Fl) that was MCH+ cell (Gl) that was a2~R+ a2~R+ aJ~R+ (El) and c-Fos+ (Fl) and c-Fos+ (F3). G. Labeled (Gl) and c-Fos+ (G3). Peptides were stained by green fluorescence with Cy2; receptors were stained by redfluorescence with Cy3 and cFos by black DAR-Ni. Magnification bar = 20 !lm. 98 Figure 5 <dAR r~~'-"",--~=""'-""'~~""'-T;;:';';:;';"-~-- * Orx+/aIAR+ o Orx+/a2AR+ * MCH+/alAR+ o MCH+/a2AR+ 99 Figure 5. Distribution of al vs. a2AR immunopositive Orx and MCH neurons. A. Distribution of al~R+/Orx+ ceUs (stars) and B. a2~R+/Orx+ cells (circ/es) across LH, PF and DMH. C Distribution of al~R+/MCH+ ceUs (stars) and D. a2~R+/MCH+ ceUs (circ/es) across LH, PF, DMH and ZI. For abbreviations see Fig. 2. Magnification bar = 1 mm. 100 4.6 Discussion Paralle1 with a large population of neurons in the posterior hypothalamus, more Orx cells express c-Fos after sleep deprivation than after sleep recovery, whereas more MCH cells express c-Fos after sleep recovery. This reciprocal pattern of activity could be explained in part by different adrenergic receptors and thus response to NA in the Orx and MCH neurons. c-Fos differentially expressed in Orx and MeR neurons as a function of sleep A large population of neurons in the posterior hypothalamus expressed c-Fos following 3 hours oftotal sleep deprivation. As a reflection ofneural activity, the c-Fos expression thus confirms unit recordings showing the maximal discharge rate by the vast majority ofneurons in this region to be during waking as compared to non-REMS (Steininger et al., 1999, Alam et al., 2002, Koyama et al., 2003). In paralle1 with the major population ofhypothalamic neurons, more Orx neurons also expressed c-Fos following sustained waking than following recovery or control sleep. These results confirm microdialysis studies showing that Orx re1ease is high in association with waking and low in association with sleep (Fujiki et al., 2001, Yoshida et al., 2001, Zeitzer et al., 2003). However, the percentage of c-Fos+ cells that were Orx+ in the present study was very small (~5%) indicating that the Orx cells were the not major contributors in this region to the maintenance of the waking state. Moreover, the percentage ofOrx+ cells that were c-Fos+ was also very small (~10%), indicating that the 101 Orx cells were not maximally activated for the maintenance of the waking state under the present conditions. In fact, the experimental conditions were intended to deprive rats of sleep without stress and accordingly employed behavioral observations for sleep-wake state scoring to avoid the surgery and tethering needed for polygraphic recording, maintained the animaIs in their home cages with food and water ad libitum to avoid any alimentary deprivation and used gentle touching with a soft brush when necessary to avoid continuous or stressful stimulation and evoked activity during the day. The results suggest that although other neurons in the posterior hypothalamus are active in association with quiet waking and thus potentia11y involved in maintaining cortical activation of the waking state, the Orx neurons may only be active with more aroused or stressful waking. In another study, it was found that Orx neurons expressed c-Fos in large numbers during the day only in association with high arousal and stressful waking induced by continuous auditory stimulation (Espana et al., 2003). Selective deprivation of REM sleep was also found to be associated with increased c-Fos expression in Orx neurons (Verret et al., 2003), perhaps due to stress that may be associated with that procedure. Orx re1ease has also been found to be maximal in association with motor activity (Kiyashchenko et al., 2002, Zeitzer et al., 2003). Effects of intracerebroventricular (leV) administration of Orx and loss offunction in Orx knock out mice moreover indicate that Orx can stimulate energy metabolism along with motor activity by positive influences upon the sympathetic nervous system (stimulating increased heart rate, blood pressure, temperature and thermogenesis), the hypothalamopituitary thyroid axis (HPT, stimulating increased basal metabolic rate) and the hypothalamo-pituitary adrenal axis (HP A, stimulating increased corticosteroid levels) (Lubkin and Stricker-Krongrad, 1998, Shirasaka et al., 1999, Ida et al., 2000, Hara et al., 102 2001, Espana et al., 2002, Monda et al., 2003, Yamanaka et al., 2003). An integral role in stimulating arousal, activity and metabolism can be mediated by excitatory influences of Orx upon multiple central arousal systems, inc1uding noradrenergic locus coeruleus neurons and cholinergic brainstem and basal forebrain neurons (Horvath et al., 1999, Bayer et al., 2001, Eggermann et al., 2001, BurIet et al., 2002), upon central motor and sympathetic systems (Peyron et al., 1998, van den Pol, 1999, Krout et al., 2003, Yamuy et al., 2004) and upon hypothalamic neurons (van den Pol et al., 1998, Ferguson and Samson, 2003). In contrast to the Orx cells, MCH cells showed more c-Fos activation after sleep recovery than after sleep deprivation. c-Fos expression was specifica1ly associated with recovery sleep, when animaIs slept ~90% of the time and not with normal sleep in the control condition, when animaIs slept ~75% of the time. These results suggest that the cFos activation is associated with the recovery pro cess and not simply with natural sleep. Nonetheless, the percentage ofMCH neurons that expressed c-Fos during recovery following 3 hours of gentle sleep deprivation was very small «5%). In another study examining c-Fos expression following 72 hours ofparadoxical sleep (PS) deprivation upon inverted flower pots, it was found that a large proportion ofMCH neurons (>50%) expressed c-Fos during recovery, when animaIs slept 77% ofthe time, comprised by 33% SWS and 44% PS (Verret et al., 2003). In our paradigm, the recovery sleep was comprised in greatest proportion by non-REMS sleep (estimated at ~ 77% as compared to 14% REMS), and the percentages ofboth non-REMS and REMS were significantly increased above control (estimated at ~69% and 7% respective1y). Accordingly, the increased c-Fos expression in MCH cells could be explained by the predominance of non-REMS in SR or by a recovery process afforded by total sleep that inc1udes 103 unimpeded non-REMS and REMS along with the loss ofpostural muscle tone. MCR cells may thus become activated only when there is an increased need as well as possibility for the behavioral inactivity and rest that is permitted by non-REMS and REMS sleep. Based upon evidence ofMCR administration or MCR knock out, it has been shown that MCR decreases energy metabolism along with activity through a negative influence upon the sympathetic nervous system, the RPT axis and the RP A axis (Shimada et al., 1998, Kennedy et al., 2001, Marsh et al., 2002, Ito et al., 2003, Shearman et al., 2003, Zhou et al., 2005). MCR neurons could thus play a role in opposition to Orx neurons by decreasing activity and energy metabolism in association with sleep through inhibition of transmission in the hypothalamus and other arousal systems (Bittencourt et al., 1992, van den Pol et al., 1998, Gao and van den Pol, 2001). Adrenergic receptors differentially distributed on Orx and MCH including c-Fos expressing neurons C-Fos expression was inverse1y re1ated in Orx and MCR neurons across the deprived and recovery conditions in the present study suggesting a reciprocal re1ationship in their activity and roles. Orx and MCR cells are partially overlapping in their distribution within the hypothalamus and have reciprocal synaptic re1ationships, which could mediate reciprocal profiles of activity (Bayer et al., 2002b, Guan et al., 2002). It is also possible that they would be under reciprocal influences by other activating systems involved in behavioral state and energy regulation, such as the noradrenergic systems, including importantly the locus coeruleus neurons (Jones and Yang, 1985). It couid thus be expected that Orx neurons would bear alAR, associated with depolarizing responses to NA as present upon cholinergie basal forebrain neurons, whereas MCR neurons would 104 bear a2AR, associated with hyperpolarizing responses to NA as present upon GABAergic sleep-active basal forebrain neurons (Fort et al., 1995, Fort et al., 1998, Modirrousta et al., 2004b). Many Orx neurons were found to be immunostained for the alAAR, although unexpectedly many were also immunostained for the a2AAR. In contrast, MCH cens were almost exclusive1y immunostained for the a2AAR. The difference in the incidence of alAAR on Orx vs. MCH cens explains one mechanism by which Orx neurons may be stimulated, whereas MCH neurons would be inhibited during waking when noradrenergic locus coeruleus neurons are active (Aston-Jones and Bloom, 1981). The MCH neurons may become active when released from inhibition by noradrenergic input with sleep onset. These conclusions were recently substantiated in electrophysiological studies using slices from rat brain, in which NA was found to have an excitatory effect upon Neurobiotin-Iabeled Orx cens and to have an inhibitory effect upon Neurobiotin-Iabe1ed MCH cens (Bayer et al., 2005). On the other hand, the present finding that sorne Orx neurons bear a2AARs suggests that sorne Orx cens may be inhibited by NA. In fact, in transgenic mice expressing GFP in Orx neurons, NA was found to have a hyperpolarizing effect upon the GFP-Iabeled Orx cens in vitro (Li et al., 2002). And most recently it was discovered that the excitatory effect of NA on Orx cens in the rat slice was transformed into a hyperpolarizing or biphasic effect after two hours of sleep deprivation (Grivel et al., 2004). It is thus possible that under certain circumstances in mi ce and rats, Orx cens may mobilize or express a2ARs. In the present experiment, the numbers of cens bearing alAARs or a2AARs did not vary as a function of sleep-wake condition, thus indicating that mild sleep deprivation or recovery in adult rats does not appear to alter the 105 expression of these receptors. On the other hand, sleep deprivation in younger rats (~15 - 20 days), as employed in the in vitro studies, would be associated with stress, as indicated by e1evated corticosterone (Hairston et al., 2004), and stress or corticosteroids have been shown to be associated with changes in the expression of Œ2ARs (JhanwarUniyal et al., 1986, Flugge et al., 2003). Thus, the influence ofnoradrenergic input from locus coeruleus or other brainstem cell groups may under normal conditions be predominantly excitatory upon Orx neurons through ŒIARs, however change following stress to become predominantly inhibitory upon them through increased expression or mobilization of Œ2ARs. This dual potential may allow adaptive changes under conditions of stress, when sleep along with energy conservation, instead of sustained waking along with energy expenditure, could enhance survival. In summary, the present results show that Orx and MCH neurons are active in a reciprocal manner during sleep deprivation and sleep recovery such as to suggest that they play opposing roles in regulating sleep-wake states along with activity and energy metabolism. These opposing roles may in part be mediated by reciprocal relationships or by differential responses to afferent inputs inc1uding importantly noradrenergic inputs. 4.7 Acknowledgements The research was supported by a grant from the Canadian Institutes ofHealth Research (13458). 106 4.8 Preface to chapter 4 The study in the third chapter revealed that within the posterior hypothalamus, higher numbers of cens expressed c-Fos during sleep deprivation than they did during sleep recovery. Similarly, it showed that the numbers of c-Fos expressing Orx cens were higher after SD as compared with the SR condition. In contrast, the numbers of c-Fos expressing MeR cens were higher after SR as compared with the SD condition. In addition, many Orx cens were immunostained with UIAAR including those that expressed c-Fos during SD while few MeR cens were immunostained with UIAAR. Many MeR cens on the other hand, were found to bear U2AAR including those that expressed c-Fos during sleep recovery. Sorne Orx neurons were also immunostained with U2AAR. This study substantiates the role of Orx as one of the posterior hypothalamic peptides important in waking regulation. The presence of UIAAR on Orx cens could moreover explain how Orx cens are under further excitation during waking when the release of NA is highest. These results also identified MeR cens as a population of neurons that play roI es in opposition to Orx neurons. Under certain situations ofhigh pressure for sleep, they become active during sleep to facilitate rest behaviors. The presence of U2AAR on MeR cens could explain how these cens might be inhibited during waking when release of the NA is highest and how they would be removed from inhibition during sleep when NA release is at its lowest. The fact that sorne Orx cens also bear u2AAR brings the possibility that sorne Orx neurons might be inhibited by NA. It moreover suggests the possibility that under certain circumstances, such as prolonged sleep deprivation or stress, 107 Orx neurons might express or mobilize U2AAR to their surface to dampen their own activity. In the first, second and third chapter, we showed that sleep vs. wake promoting neurons might respond differentially to NA as one of the main neurotransmitters of waking and therefore might bear different adrenergic receptors. This could be one mechanism to explain how cells are modulated during sleep vs. waking in a cyclical manner. Another possible mechanism is that sleep or wake promoting neurons show different amount of receptor expression or mobilization on their membranes. As shown in the first and second chapters, GABAergic cell activity and therefore GABA release is important for sleep promotion and maintenance. We moreover showed, in the second chapter, that BF cholinergie cells are active during waking but not during sleep. With the goal ofunderstanding the interaction of sleep promoting GABAergic cells with the wake promoting cholinergie neurons, we designed our last experiment to test the hypothesis that cholinergie cells differentially respond to GABA across behavioral states by changes in the availability of GABAARs on their membrane. In a paradigm of sleep deprivation and sleep recovery in rats we studied possible changes that might occur both in the presence and/or intensity of GABAARs on cholinergie cell membranes across different conditions. 108 5. Chapter 4: GABAA Receptor Modifications on Basal Forebrain Cholinergie Cells across the Sleep-Waking Cycle4 Mandana Modirrousta, Lynda Mainville and Barbara E. Jones 4 In preparation 109 5.1 Abbreviations ACh, acetylcholine ChAT, choline acetyl transfrase MCPO, magnocellular preoptic nucleus mIPSCs, miniature Inhibitory Post Synaptic Currents NDS, normal donkey serum non-REMS, non-rapid eye movement sleep REMS, rapid eye movement sleep Rs, receptors SC, sleep control SD, sleep deprivation SR, sleep recovery SWA, slow wave activity SWS, slow wave sleep 110 5.2 Abstract The basal forebrain cholinergie cells play a critical role in cortical activation and arousai. These cells discharge in bursts during waking with cortical activation and become silent during slow wave sleep (SWS) with cortical slow wave activity. We hypothesized that the activity of cholinergie cells across the sleep-waking cycle could be homeostatically regulated by changes in GABAA receptors (Rs). In a paradigm of sleep deprivation (SD) and sleep recovery (SR) in rats, we tirst counted the numbers of cholinergie cells in the magnocellular preoptic nucleus of the BF that were immunostained for GABAAR (P2-3 chain). We subsequently measured in fluorescent microscopy the luminance of GABAAR over the cell membrane. We found that both the numbers of cholinergie cells that were positively labe1ed for the GABAAR and the intensity of the GABAAR fluorescence labeling over the cell membrane were signiticantly increased after SD as compared to SR and sleep control (SC) conditions. We conclude that the expression or mobilization of GABAARs on cholinergie cell membranes increases after sustained waking. This increase in membrane GABAAR could be associated with higher susceptibility to inhibition of the cholinergie neurons by GABA and consequent decrease in cortical activation with a greater propensity for SWS. These results suggest a mechanism involving homeostatic regulation of GABAARs and GABA inhibition of cholinergie neurons that could underlie the sleep-waking cycle. 111 5.3 Introduction The cholinergie cells of the basal forebrain (BF) play an important role in cortical activation as the ventral extra thalamic relay from the brainstem arousal systems to the cerebral cortex (Starzl et al., 1951, Jones, 2000). Recently, extracellularrecording and juxtacellular labeling in the unanesthetized head fixed rats revealed that identified BF cholinergie neurons dis charge in bursts with cortical activation (Lee et al., 2005). In parallel, microdialysis studies have shown that acetylcholine (ACh) release in the cortex is highest with cortical activation and attentive behaviors (Marrosu et al., 1995, Himmelheber et al., 2000). Similarly, pharmacological block of ACh receptors diminishes cortical activation (Vanderwolf, 1975). We have recently shown that cholinergie BF neurons express c-Fos during sleep deprivation (SD) and not sleep recovery (SR) during the day (Modirrousta et al., 2004b). All together these studies demonstrate that the cholinergie BF neurons are active in association with cortical activation during waking and inactive in association with slow wave activity (SWA) during SWS. Their relative silence during SWS is likely imposed by GABAergic inhibitory input (Khateb et al., 1998). We considered the possibility that the effect of GABA might be under homeostatic regulation across the sleep-waking cycle. Accordingly, cholinergie neurons would be regulated by synaptic homeostasis such that after a period of activity, they would decrease their activity in order to preserve stability and survive (Turrigiano, 1999). It has been shown that the amount of inhibitory vs. excitatory synaptic activity varies depending upon prior activity (Shatz, 1990). Application ofTTX on pyramidal neurons decreased miniature Inhibitory Post Synaptic 112 Currents (mIPSCs), the number of open GABAA receptor (GABAAR) channe1s and the intensity of GABAARs immunostained at synaptic sites (Kilman et al., 2002). Moreover, application ofbicuculline in rat hippocampal slices increased the density and size of GABAAR (a. subunits) clusters (Marty et al., 2004). These studies suggest that prolonged activity elicits more inhibition on neurons, whereas activity blockade elicits more excitation. Whether the same process occurs during the sleep-waking cycle remains to be investigated. We thus hypothesized that after a prolonged waking period, those cells which are active like the cholinergie BF cells would undergo certain synaptic or other changes to reduce their activity in association with SWA and SWS. This process could occur through decreasing the excitatory/inhibitory balance by an increase in GABAARs on the active cholinergie cells. Here, by applying a paradigm of SD and SR in rats, we investigated the incidence of cholinergie cells of the magnocellular preoptic nucleus (MCPO) that appeared to be immunostained for the GABAAR in association with c-Fos under the different conditions. We then analyzed the intensity ofGABAAR (132-3 chain subunits) on cholinergie cell membranes across conditions. 113 5.4 Materials and Methods AlI procedures were approved by the McGill University Animal Care Committee and the Canadian Council on Animal Care. These procedures also conform to those of the Association for Assessment and Accreditation of Animal Care International. Sleep deprivation. Male Wistar rats (200-250 g) were housed individually with free access to food and water at aIl times with a 12:12 lightldark schedule (lights on from 700 to 1900 hours). As previously described (Modirrousta et al., 2004b), the rats were deprived of sleep in home cages. Therefore, the experimenter (MM) deprived and/or observed one rat at a time (on one day) and scored its behavioral state every 20 sec by visual observation as comprised ofwake, non-rapid eye movement sleep (non-REMS) or rapid eye movement sleep (REMS). In this manner, the stress of surgery and cage displacement necessary for recording which can affect c-Fos expression were avoided (Maloneyet al., 1999). Non-REMS was scored when the animal was recumbent with eyes closed, showing little or no movement and REMS when the animal was recumbent with eyes closed, showing rapid movements or twitches of the whiskers, ears or paws. Rats were submitted to 1) total sleep deprivation for 3 hours (1200 to 1500 h, n=3) as the SD group, 2) total sleep deprivation for 3 hours (900 to 1200 h,) followed by sleep recovery for 3 hours (1200 to 1500 h, n=3) as the SR group, or 3) undisturbed sleep and waking as sleep control (SC) for 3 hours (1200 to 1500 h, n=4). Rats were habituated to the experimenter's presence and to a paint brush (for sleep deprivation) inserted through the cage top for 3 days prior to the experimental day. During the experiment, the deprivation for the SD and SR groups was done by gentle touching using the brush upon 114 dosure of the eyes. Sleep deprivation resulted in the total absence of sleep during 3 hours for the SD and SR groups which was followed by an increase in total sleep in the SR group relative to both the SD and SC group. At the end of the experiment (1500 h), rats were immediate1y killed under pentobarbital anesthesia (100 mglkg, i.p.) by intraaortic perfusion with a fixative solution of 3% paraformaldehyde. Immunohistochemistry. Following immersion in a 30% sucrose solution, brains were frozen and stored at -80° C. They were cut in coronal sections at 20 Jlm, the maximal allowable thickness that was determined to allow full penetration and staining with the antibodies employed. Adjacent series of sections were collected at 800 Jlm intervals and processed for triple or double immunohistochemical staining for c-Fos (rabbit antiserum, 1:10,000, Ab-5, PC38, Oncogene Research Products, La Jolla, CA) using DAB-Ni, choline acetyl transfrase (ChAT) (rabbit antiserum, 1:1000, AB143, Chemicon International, Temecula, CA, USA) using Cy2-conjugated anti-rabbit antiserum (Jackson ImmunoResearch Laboratories, West Grove, PA and GABAAR (mouse antiserum, 1: 100, MAB341, Chemicon) using Cy3-conjugated anti-mouse antiserum (Jackson). In order to examine whether GABAARs were present upon cholinergic neurons that expressed c-Fos following SD, SR or SC, triple immunostaining was performed for c-Fos in the first position incubated over night at room temperature and ChAT and GABAAR in the second position co-incubated for three nights at 4° C. Subsequently, other series were processed for double immunostaining of ChAT and GABAAR co-incubated for three nights at 4° C. Incubations with antibodies were performed using a Tris-saline solution (0.1 M) containing 1% normal donkey serum (NDS), following initial blocking with 3% NDS. 115 Microscopie analysis. Sections were viewed by light and fluorescence microscopy with a Nikon Eclipse E800 microscope equipped with an x/ylz movementsensitive stage and video camera (Optronics, S99808) attached to a computer. Cell counts and image acquisition were performed by systematic random sampling using Stereo Investigator (2003, MicroBrightField). Luminance measurement on acquired images was performed by using Neurolucida software (2003, MicroBrightField, Williston, VT). Cell counts. In triple labeled series, single-, double- and triple-immunostained cells were evaluated in the MCPO for ChAT, ChAT/GABAAR and cFos/ChAT/GABAAR immunostained cells respectively. Single-, double- and triplelabeled cells were counted by applying stereology using the Optical Fractionator program of Stereo Investigator. Cells were sampled and counted within a delineated contour of the left side for the M CPO at appropriate levels of a computer resident atlas (expanded from (Gritti et al., 1993)). Labeled cells were sampled and counted in three sections at 800 !lm intervals from anterior to posterior. Within the stereology pro gram, the size of the counting frame, within which cells were counted, was set at 125 x 125 !lm and the grid, within which the counting frames were automatically randomly placed, was set at 250 x 250 !lm. Accordingly, 25% of the MCPO area at each level was sampled. The cells were counted under a 60x oil objective (with 1.4 numerical aperture, NA). Cells were counted within a counting block of 8 !lm in depth (in the dehydrated, delipidated, mounted and coverslipped sections that were on average 10 !lm thick) when the cell bodies came into focus beneath the surface of each section 116 Luminance measurement. In double labeled series stained for ChAT and GABAARs, images were sampled and acquired by using random sampling through Stereo Investigator. In each sampling site, images were acquired using at 60x oil objective (1.4 NA) and filters for Cy2 and Cy3. For images acquired with both filters and across aIl sections, the exposure time was consistently set at 100 ms with a gain of 4 and target intensity of 70%. Within Stereo Investigator, the size of the counting frame used to sample cells was set at 50 x 50~m and the grid at 250 x 250 ~m. In each frame, the Cy2- labeled ChAT+ cell which was the closest to the centre of the counting frame was selected for acquisition and brightness measurement of the membrane Cy3-labeled GABAAR. For luminance measures, a measurement box was employed with a length of 5 ~m and a width of 1 ~m, which was determined to be the maximal width observed for membrane GABAAR staining. For each selected ceIl, boxes were positioned over the nucleus and the portions of the cell membrane that had the minimal distance from the nucleus and were most paralIel in orientation on each si de of the cell. One other box was placed over the cytoplasm adjacent to the box over the upper membrane. Luminance information was colIected as the average luminance of pixels, based on luminance values ofNeurolucida software within each box (Fig. lB). The overalI membrane luminance in each condition was obtained by averaging the values per animal and then per condition. Statistical analysis. Sleep, celI counts and luminance data from celIs were analyzed between groups using one way ANOVAs in Systat (vI 0.2, Richmond, CA). When there was a significant main effect, Fisher's LSD post-hoc paired comparisons were applied between groups. AlI main effects were confirmed by nonparametric rank order tests (Kruskal-WalIis, p < .05) to insure that the distribution of variance in groups 117 (containing zeros in sorne cases as a result of the experirnental condition) did not distort the parametric statistics. Figures were cornposed using Adobe Photoshop Creative Suite (CS) and Adobe Illustrator CS (Adobe, San Jose, CA). 118 5.5 Results Sleep amount in the three experimental groups Rats in the sn group did not sleep during the 3 hours in the afternoon prior to sacrifice (at 1500 h), whereas rats in the SR group that were allowed to recover sleep in the afternoon after 3 hours of sleep deprivation in the morning (900-1200 h) slept -91 % ofthe time prior to sacrifice (at 1500 h). Similarly, rats in SC that had undisturbed sleep or waking for 3 hours before sacrifice spent the majority oftime, -75%, in sleep prior to sacrifice (1200-1500 h). The amount of sleep differed significantly across conditions of SC, sn and SR (Table 1). spent in non-REMS (77.23 The major proportion of time for the SR and SC groups was ± 2.94% in SR and 68.68 ± 0.10% in SC) and a minor proportion in REMS (13.58 ± 1.30% in SR and 7.10 ± 0.66% in SC, mean ± S.E.M. of total time). Cholinergie eeUs immunostained with GABAAR According to one way ANOVA with condition as the main factor, the total number of cholinergie cells (ChAT+) in the MCPO did not differ across conditions (Table 1), whereas the proportion of the cholinergie cells that were immunostained for GABAAR (ChAT+/GABAAR+) significantly varied across conditions (F=7.49, df=2, df=7, p<O.05) with higher proportions in sn as compared with both SC and SR (p=0.006 and p=0.05 respectively) and with no significant difference between SC and SR 119 conditions (Fig. 2, 3A, Table 1). In addition, analysis ofthe triple labe1ed cells revealed that in the SD group sorne ChAT+/GABAAR+ cells expressed c-Fos whereas no c-Fos was expressed in the ChAT+/GABAAR+ cells in either SC or SR groups (Fig. lA). Table 1. Average percent sleep and numbers ofChAT+ and ChAT+/GABAAR+ cells under conditions of Sleep Control (SC), Sleep Deprivation (SD) and Sleep Recovery (SR) Variable SD' SC % Sleep 75.75 ± ChAT+ 12750 ± ChAT+/GABAAR+ 2350 ± Membrane GABAAR luminance 2.23 ± 9:!?J~~,~~,QA~~:,:~ 2.32 ± F (Cond) SR ± 0 90.8 ± 2.47 1975.29*** 2418.50 15000 ± 3500.48 14400 ± 871.78 0.228 ns 298.608 78°ot ± 1833 4666.67 ± 1387.2 5.437 * ± 0.91 4.25tt 0.79 14.102***,.,. 0.38 0.604 0.61 0.35 0.27 ott 7.~lt t 2.91 ~-~'"' ± ± ~~mm 0.41 3.1 ± luminance Values represent Mean ± S.E.M. for 3 groups ofrats. Cell numbers were obtained from stereological estimates for total numbers on the left side. Values across conditions (Cond) were compared by one-way ANOVAs (with condition as the main factor), and post hoc pair-wise comparisons between conditions (using Fisher's LSD). The estimated total numbers ofChAT+ cells did not differ across conditions (df= 2; dferror=7). Total numbers ofChAT+/GABAAR+ cells increased significantly across conditions (df=2; df error=7). Average membrane GABAAR luminance significantly differed (df=2; df error=222) while average cytoplasmic GABAAR luminance did not differ across conditions (df=2; df error=223) (ns indicates non-significant; *, p<0.05; ***, p<O.OOl; 120 t, significant differences between SD and SC; t between SD and SR and tt between SR and SC). Luminance of GABAAR membrane staining across conditions In each brain, 19-27 ChAT+ cells in the MCPO across three sections were selected for luminance measures of GABAAR immunofluorescent staining by systematic random sampling using Stereo Investigator. The luminance value of membrane staining for each cell was calculated as the average between the two boxes located on two sides over the membrane and minus that of the nucleus which was considered to represent the background staining (Fig. lB). According to one way ANOVA with condition as the main factor, the luminance ofGABAAR membrane labeling (-nucleus) significantly differed across conditions (F=14.10, df=2, dferror=222, p<O.OOl) (Table 1). Post-hoc paired comparisons with Fisher's LSD correction revealed that membrane labeling was significantly higher in SD as compared with both SC and SR (p<O.OOl and p<0.05 respectively) and it was significantly higher in SR as compared to the SC condition (p< 0.05) (Figs. 1C-E and 3B) (Table1). In contrast, the luminance over the cytoplasm (nucleus) did not differ across conditions (F=1.45; df=2; df error=223; p>0.05) (Table 1). 121 Figure 1 122 Figure 1. c-Fos expression and GABAAR on cholinergie cells of the BF. A. A triple labeled cell for c-Fos (Al) and ChAT( stained green with cy2) bearing GABAAR (stained red with cy3) (A2). B. A sampIe cholinergie cell in green (B1) stainedfor GABAAR in red (B2) and the location ofboxes (in yellow) that were usedfor luminance measurements of the two membrane sides, cytoplasm and nucleus. C Cell immunostainedfor ChAT (Cl) and GABAAR (C2) in one SC representative brain. D. Cel! immunostainedfor ChAT (Dl) and GABAAR (D2) in one SD representative brain. E. Cel! immunostainedfor ChAT (El) and GABAAR (E2) in one SR representative brain. Arrow heads indicate the locations ofboxes on each membrane sides. Magnification bar =20J.ll1t 123 Figure 2 sc SD SR Fig.2 Figure 2. Distribution ofChAT+ neurons that were (filled circ/es) or were not (open circ/es) positively immunostainedfor GABAAR in SC (A), SD (B) and SR (C) representative brains. Magnification bar = 1 mm. 124 Figure 3 0.6 J!l 0.5 f3 0:::: 0.4 <l ~ 0.3 ~ 0.2 Ü '#. 0.1 0.0 <IJ v c: SC SD SR SD SR 10 <IJ u ~ 0 ::1 q: 0:: < CX) «~ <IJ 5 C ~ .0 E 111 :2 >- .'!.: VI C ~ .E 0 SC Cordtion 125 Figure 3. A. Percent ChAT+ ceUs that were also GABAAR+ in the MCPO afier SC, SD and SR conditions. B. Luminance of GABAAR membrane immunostaining on ChAT+ ceUs in the MCPO afier Sc, SD and SR conditions. Membrane luminance represents the mean values per animal per condition. For each ceU, the membrane luminance was obtained by averaging over two membranes minus that over the nue/eus of each ceU. (* indicates comparisons with the SD group;*, p= <O. 05; **, p<O.Ol; ***, p<O.OOl. t indicates the comparison between the SC and SR groups with p<0.05). 126 5.6 Discussion We show that the proportion of cholinergic cells with detectable GABAARs increased at the end of SD, when the animaIs were awake continuously, relative to the proportions after SC and SR conditions when the animaIs slept the vast majority of time. We also show that the immunostaining of membrane GABAARs was higher after SD as compared with both SC and SR conditions. It is then possible that a period ofwaking enhances expression or mobilization of GABAAR to the cell surface, whereas sleep reduces the expression or mobilization ofthe GABAAR. From our c-Fos study, we recently found that sleep active neurons of the BF and the preoptic area are comprised of GABAergic cells. These GABAergic cells by their local or descending projections (Sherin et al., 1998, Henny and Jones, 2003) can inhibit neurons of the arousal systems and promote sleep. Here we show that the numbers or intensity of membrane GABAARs was altered as a function ofbehavioral state. From pharmacological studies, it has been known that many anesthetics and hypnotic drugs act through GABAARs(Lancel and Steiger, 1999). Although sedatives that induce sleep or anesthesia are physiologically distinct from naturally occurring sleep, certain EEG and behavioral similarities do exist. Studies in rats showed that after 24 hours of sleep deprivation with a 6 hour period of either ad libitum sleep or propofol anesthesia, recovery for sleep was similar in timing and persisted not more than 12 hours in both groups. This study suggests that recovery pro cesses that occur during natural sleep also take place during anesthesia and suggest that sleep and anesthesia share common neuroregulatory mechanisms (Tung et al., 2004). Moreover, both circadian 127 rhythmicity and sleep deprivation affect anesthetic actions implying a link: between factors which regulate natural sleep and drug induced sedation, hypnosis or anesthesia (Munson et al., 1970, Tung et al., 2002). It is likely then that increases in GABAAR availability on neurons of the arousal systems including the BF cholinergic cens would enhance inhibitory responses and consequently sleep promotion. The profile of GABAAR changes on cholinergic BF cens is comparable to the homeostatic changes of SWA during sleep. It has been known that SWA is directly correlated with the duration of prior waking and that SWA is highest during the first hours and lowest during the last hours of sleep (Borbely et al., 1984). Interestingly, application of GABA agonists including muscimol and TRIP, enhance SWA (Lancel, 1997, Lancel et al., 1997, Vyazovskiy et al., 2005). It is assurnable thus that increased numbers of GABAARs on cholinergic cens and likelyon other wake promoting neurons lead to higher SWA in the beginning of sleep and as sleep pressure reduces, the GABAAR expression attenuates leading to disinhibition of arousal systems and preparation of animaIs for the fonowing waking period. Although there was not any significant difference in the nurnber of cholinergic cens irnrnunostained for GABAAR between SC and SR groups, the intensity of membrane GABAAR staining was significantly higher in SR when compared with the SC condition. This difference could be explained by the prior waking during deprivation of the SR group in contrast to undisturbed sleep of the SC group during the same moming period (900-1200 h). The difference in the amount of time spent in sleep, ~ 91 % and ~ 75% in SR and SC respectively, indicates that SR animaIs needed to sleep more and recovered from the sleep deficit which would explain the somewhat enhanced GABAAR on the cholinergic cens. It is thus possible that during 3 hours of sleep recovery, the 128 GABAAR membrane staining appears to decrease progressively retuming to control levels, although not entirely given the slightly higher staining. We show that after 3 hours of sustained waking, BF cholinergic cells show increased immunofluorecent staining for GABAAR. Although previous experiments showed a time course of days for the increases or decreases of GABAAR as a result of inactivity (TTX) or activity (bicuculline) in cultured visual or hippocampal cortex respectively(Kilman et al., 2002, Marty et al., 2004), other studies have demonstrated a shorter time scale for trafficking of membrane GABAARs. Application of insulin caused a rapid increase within minutes the expression of GABAARs on the postsynaptic and dendritic membranes through translocation of intracellular GABAARs to the plasma membrane (Wan et al., 1997). Similarly, brief stimulation of rat hippocampal cultured neurons by a dopamine agonist significantly enhanced protein synthesis and surface expression of GluI receptor within 60 minutes (Smith et al., 2005). In our paradigm therefore, it appears that 3 hours of waking could be enough for either rapid translocation ofGABAARs to the post-synaptic domain or translational protein synthesis. Consequently, enhancement of membrane GABAAR immunofluorecence after the SD condition could be due to more expression, more translation of GABAAR or it could be due to more recruitment of subsynaptic GABAAR to surface synaptic zones. The differential expression or mobilization of GABAARs to cell membranes can be attributed to several plausible mechanisms. Many studies have shown that higher neural activity leads to increase in GABA transmission (Marty et al., 2000, Marty et al., 2004). In parallel, after activity deprivation in cultured rat visual cortex for 2 days, the number of synaptic sites that expressed detectable levels of GABAARs was decreased by approximately 50% and the amplitude of mIPSCs similarly diminished (Kilman et al., 129 2002). AIl together, these studies suggest that in general, stimulation leads to enhancement of inhibitory synapses. In our study, cholinergie ceIls as shown previously by c-Fos expression (Modirrousta et al., 2003) are active during waking and inactive during sleep that represented mainly non-REMS either in SC or SR groups with minimal amount of REMS. Although unit recording has shown that the activity of cholinergie ceIls is highest during both arousal and REMS (Lee et al., 2005), the lack of c-Fos expression in cholinergie ceIls during SC or SR conditions could reflect minimal amount of REMS to be able to influence on sustained cholinergie activity. The increase in both numbers and density of GABAAR on the cholinergie ceIls after SD condition and significant decrease after SC or SR condition could thus be related to their higher activity during waking and lower activity during sleep. The trafficking of GABAARs could be regulated by various mechanisms. It has been shown that application ofbrain derived neurotrophic factor (BDNF), a member of the neurotrophin family of peptides, in rat visual cortex induced a rapid increase in the total number of ceIl surface GABAARs, through the activation ofTrk B receptor tyrosine kinases. BDNF also rapidly induced a sustained potentiation of GABAAR-mediated currents and se1ective1y increased the mean mIPSC current (Mizoguchi et al., 2003). In another study, BDNF application prevented the decrease in GABA-mediated inhibition onto pyramidal neurons that was induced by activity blockade of cultured rat visual cortex (Rutherford et al., 1997). Interestingly, the expression ofBDNF is regulated by neuronal activity (Castren et al., 1992, Tabuchi et al., 2000). BDNF is in addition one of the genes of which expression is upregulated by waking independent of the time of day (Cirelli et al., 2004). Considering that BDNF mRNA is expressed in the BF (Conner et al., 1997), it is possible that neuronal activity of cholinergie ceIls during waking, drives 130 expression of more GABAAR on cholinergie cells and this change could be partly regulated by release of BDNF from active, likely, glutamatergic neurons. In summary, these results suggest a novel mechanism through which cholinergie cells, as part of arousal systems, are self regulated in a homeostatic manner during waking and thus by accumulation of more inhibitory receptors as waking continues, they will be prepared to be inhibited to allow sleep occurrence and maintenance. Whether or not the same mechanism occurs in other neurons of the arousal systems is a subject for future study. 5.7 Acknowledgements The research was supported by a grant from the Canadian Institutes ofHealth Research (13458). 131 6. General Discussion Experiment one The results of the first chapter revealed that in anesthetized rats, a substantial proportion of the BF GABAergic cens (57%) discharged at higher rates during cortical SWA and their firing rates decreased during evoked cortical activation. This finding suggested that those identified GABAergic cens in the BF that are maximany active during cortical SWA are putative sleep promoting cens. In the next step, by dual immunohistochemical staining for GAD and a2AAR, we first found that approximately 60% ofGABAergic cens distributed through the MCPO and SI were immunostained for a2AAR. Second in urethane anesthetized rats, we found that an Nb-Iabe1ed GAD+ cells that were maximally active during cortical SWA bore a2AAR. These cells can correspond in a large part to previously identified sleep-active neurons in this region (Szymusiak and McGinty, 1986a). Moreover, this study proposed that these neurons could be differentiated from other GABAergic SWA-off cells perhaps by their inhibitory response to NA through a2AAR. The a2AR, through opening ofK+ channe1s, causes hyperpolarization of the membrane as shown on the neurons of the POA and locus coeruleus (Williams et al., 1985, Bai and Renaud, 1998). Consequently sleep promoting GABAergic cells could be inhibited by NA during waking when LC neurons discharge at their highest rate (Aston-Jones and Bloom, 1981). Reciproca1ly, they would be disinhibited during cortical SWA and SWS when LC neurons decrease their firing rate. These results indicated a captivating possibility that particular BF GABAergic cells 132 might be important for sleep promotion and maintenance. In order to confirm these results in natural sleep and waking behaviors we designed our next experiment that is presented in chapter two. Experiment two The study in chapter two looked at the pattern of c-Fos activation, as an indicator for neuronal activity in the cholinergie and GABAergic cells of the BF and/or POA after different conditions. Consistent with our results in the first chapter and in anesthetized rats, these results for the first time revealed that substantial proportions of neurons that were active during sleep across both the BF and POA were GABAergic and none were cholinergie. Furthermore, our results for the first time showed that GABAergic sleepactive cells are not confined to only one or two nuclei but are distributed across these two regions and are intermingled among other wake-active cells. These GABAergic neurons by their ascending projections to the cortex (Szymusiak et al., 1989, Gritti et al., 1997), descending projections to the other arousing systems such as the posterior hypothalamus (Gritti et al., 1994, Gong et al., 2004) or also likely by their local projections to the BF cholinergie cells (Manns et al., 2000a) could exert an inhibitory influence and thus lead to cortical dampening and inhibition of arousing systems in the posterior hypothalamus and BF (Fig. 1). In addition, our results showed that across both the BF and POA more neurons, being either GABAergic or non-GABAergic, expressed c-Fos during waking than during sleep. This finding is consistent with previous c-Fos and in situ hybridization studies (Pompeiano et al., 1992, Cirelli et al., 1993, Ledoux et al., 1996, Sastre et al., 133 2000). However, it is in opposition with recent reports demonstrating that in the VLPO and MnPO more cells expressed c-Fos during sleep than during waking. This apparent discrepancy could result from different experimental conditions, such as housing, surgery and method of sleep deprivation which could be associated with more stress and therefore more c-Fos expression. Furthermore, in most previous studies, animaIs were allowed to recover from sleep deprivation or spontaneously sleep not more than two hours. Taking into consideration that almost 90 minutes are required for Fos protein synthesis and considering the Fos halflife of ~2-3 hours, it is possible that those results reporting higher numbers of c-Fos in the VLPO during sleep, in fact mostly reflected activity from the prior waking state. In the current study by keeping the animaIs in their home cages during the experiment, by using behavioral observation instead of electrode implantation for sleep-waking scoring and finally by applying gentle touching when needed for sleep deprivation, the amount of imposed stress was minimized. It is thus considered that 3 hours of sleep deprivation is short enough for minimization of sleep deprivation induced stress and long enough for accumulation of Fos protein (Sheng and Greenberg, 1990, Morgan and Curran, 1991). Vnder these conditions, an increase in the total number of neurons expressing c-Fos was not observed in any nucleus ofBF or POA during the recovery or control sleep as compared with sleep deprivation. In parallel with increased c-Fos expression during waking, higher numbers of cholinergic cells expressed c-Fos after SD as compared with both SR and SC conditions. Indeed, no c-Fos was observed in cholinergic cells after SC or SR conditions. Although it has been known that in addition to arousal, the cholinergic cells discharge during REMS (Lee et al., 2004), we assume that small proportion of REMS in either SC or SR conditions, 14% vs. 9% respectively, was inadequate to allow accumulation of c-Fos in these groups. 134 In addition to those GABAergic cens that expressed c-Fos during sleep, we showed that across the BF and POA, many GABAergic cens expressed c-Fos also after sleep deprivation. This finding is in accordance with unit recording that showed physiologicany distinct population of GABAergic cens discharged at higher rate during cortical activation (Manns et al., 2000a). In the first chapter, we showed that an SWA-on GABAergic cells bore <l2AAR. Similarly in the second chapter, we showed that on average ~80% vs. ~4% of c-Fos expressing GABAergic cells were immunostained for <l2AAR after sleep recovery vs. sleep deprivation respective1y. It confirms the hypothesis that particular GABAergic neurons of the BF and POA that are active during sleep have the common phenotype ofbearing <l2ARs and differ from those GABAergic cells that are active during waking. In paralle1, it has been shown that GABAergic neurons of the VLPO were inhibited by application of NA (Gallopin et al., 2000). Consistent with adrenergic receptor results, evidence from autoradiographie and retro grade tracing studies have suggested projections to the BF from the locus coeruleus noradrenergic neurons that could be a source of NA to act on <l2AARs (Jones and Moore, 1977, Semba et al., 1988, Jones and Cuello, 1989). A recent e1ectron microscopie study has shown that an individual LC axon can establish synaptic contacts with both cholinergie and noncholinergie neuronal e1ements (Hajszan and Zaborszky, 2002). The <l2AAR bearing GABAergic cens of the BF and the POA thus appear to be under inhibition during waking by NA. Accordingly, when re1ease of NA is maximal during waking, particular sleep-active GABAergic cells would be inhibited through <l2AAR and with decremental re1ease of NA during sleep, they would be disinhibited. They in tum by inhibiting the monaminergic neurons of the brainstem and cholinergie neurons of the BF could initiate 135 sleep (Gritti et al., 1994, Luppi et al., 1995, Sherin et al., 1998, Steininger et al., 2001, Chou et al., 2002). Experiment three In the third chapter, in order to chemically identify neurons of the posterior hypothalamus that exert roles in sleep vs. wake promotion, we looked at c-Fos expression in Orx and MCR cells after conditions of sleep deprivation vs. sleep recovery. The results indicated that in paralle1 with neurons of the BF and POA, more cells in the posterior hypothalamus expressed c-Fos after sustained waking than after recovery sleep. These findings reflect and confirm the participation of the posterior hypothalamus in the maintenance ofwaking (Fig. 1). They also confirm electrophysiological studies showing that in the posterior hypothalamus the majority of cells fire in association with waking (Steininger et al., 1999, Alam et al., 2002, Koyama et al., 2003). Similarly, higher numbers of Orx cens expressed c-Fos after sleep deprivation than after sleep recovery. This result verifies microdialysis studies showing that re1ease of Orx is high during waking and low during sleep (Fujiki et al., 2001, Yoshida et al., 2001, Zeitzer et al., 2003). Yet, only 5% of total c-Fos expressing cens during waking in the posterior hypothalamus were Orx+ meaning that under quiet waking states, other posterior hypothalamic cens might be more important than Orx cens in the maintenance ofwaking. The role of Orx cens, on the other hand, might be more prominent when animaIs are in transition from sleep to waking state (Lee and Jones, 2004) or when they have more locomotor activity or need more energy expenditure and thus increased food seeking (Sakurai et al., 1998, Wu et al., 2002). Orx neurons may be particularly important to 136 maintain a continuous, stable wakefulness and to avoid rapid fluctuations in behavioral state (Mieda et al., 2004). Orx cells project to multiple neuronal systems including the cholinergie neurons in the BF, laterodorsal tegmental (LDTg) and pedunculopontine tegmental (PPTg) nuclei, monoaminergic neurons including histaminergic in tuberomammillary nucleus, noradrenergic neurons in locus coeruleus, serotoninergic neurons in raphe nuclei and dopaminergic neurons in the ventral tegmental area, as well as other neurons in the brainstem and the spinal cord regions (Peyron et al., 1998, Horvath et al., 1999, Marcus et al., 2001, Beuckmann and Yanagisawa, 2002, Taheri et al., 2002). The widespread projections of Orx neurons to the two main components of the ascending arousal system in the brainstem and in the BF suggest a central role of Orx in promoting wakefulness. It is possible that activation of Orx cells during sleep deprivation or waking could drive more excitation of other arousing systems for further maintenance of an aroused state. The orexinergic system together with other monoaminergic and cholinergie arousing systems may thus work in concert to regulate sleep and wakefulness. In addition, the link between the sleep-wake regulatory role of the hypothalamus and other hypothalamic functions such as metabolism, hormone release, regulation of food and water intake and temperature is now becoming more evident(Gong et al., 2000, Rechtschaffen et al., 2002). Interestingly, it is shown that Orx neurons, in addition to their important role in arousal, stimulate energy metabolism through the hypothalamo-pituitary thyroid axis (HPT) (Lubkin and Stricker-Krongrad, 1998) and the hypothalamo-pituitary adrenal axis (HP A, stimulating increased corticosteroid leve1s) (Ida et al., 2000). They are also sensitive to metabolic eues such as glucose, leptin and ghrelin (Beuckmann and Yanagisawa, 2002, Taheri et al., 2002) and 137 have a positive influence on the sympathetic nervous system by increasing heart rate, blood pressure and temperature (Shirasaka et al., 1999). It is therefore more likely that Orx cells play an integrative role to enhance arousal whenever there is a higher demand for increasing basal metabolism, energy expenditure or locomotion by their excitatory influence upon other arousing systems (Rorvath et al., 1999, Bayer et al., 2001, Eggermann et al., 2001, Burlet et al., 2002) or upon other hypothalamic neurons (van den Pol et al., 1998, Ferguson and Samson, 2003). Furthermore, the direct projections from the circadian pacemaker, SeN, to Orx and MeR containing cells (Abrahamson et al., 2001) suggests the interaction between sleep control in the posterior hypothalamus and circadian rhythm. In contrast to Orx cells, MeR cells that are distributed in almost the same area as the Orx cells, expressed more c-Fos after recovery for sleep than after sleep deprivation. Increase of c-Fos expression in MeR cells was only observed in association with recovery for sleep when the animaIs slept more than 90% of the 3 hours and it was not seen after the control natural sleep condition in which the animaIs slept -75% ofthe total time. This means that in naturally sleeping animaIs and under normal conditions, hypothalamic MeR cells might not participate in sleep promotion and/or maintenance but only during a recovery process when there is a high demand for sleep, these cells could become active perhaps to take part in sleep facilitation and associated recovery processes. It has been shown that MeR decreases energy metabolism and activity through a negative influence on the sympathetic nervous system, the RPT and the RP A axes (Shimada et al., 1998, Kennedy et al., 2001, Marsh et al., 2002, Ito et al., 2003, Shearman et al., 2003, Zhou et al., 2005). In this contex, MeR neurons could play an 138 opposite role to that of Orx neurons by decreasing activity and energy metabolism in association with sleep through inhibition of transmission in the hypothalamus and other arousal systems (Fig. 1) (Bittencourt et al., 1992, van den Pol et al., 1998, Gao and van den Pol, 2001) We found that many Orx neurons were immunostained for u lAARs, although some were also immunostained for u2AARs. In contrast, MeR cens were almost exclusively immunostained for u2AARs. The incidence of UIAARs on Orx cens that induces depolarization could explain one mechanism by which Orx neurons would be stimulated by locus coeruleus noradrenergic neurons during waking when the release of NA is high (Aston-Jones and Bloom, 1981). In fact it is known that noradrenergic locus coeruleus neurons send ascending projections to the posterior hypothalamus (Jones and Moore, 1977, Jones and Yang, 1985). Although it is not known whethernoradrenergic cens from the Le make synapses on Orx neurons, in vitro studies have shown that NA can directly depolarize and excite Orx cells (Bayer et al., 2005). Orx cells in turn by their excitatory projections to the locus coeruleus are able to further enhance or maintain release of NA. On the other hand, the MeR neurons would not be excited by NA during waking rather they would be inhibited through u2AAR. Rowever, they may become active when released from the inhibitory influence of NA with sleep ons et and decreasing NA release. The finding that some Orx neurons bear u2AARs suggests that populations of Orx cens are likely inhibited by NA. It is possible that under certain circumstances such as stress, or prolonged excitation by NA, Orx cells mobilize or express u2AARs on their plasma membrane to be inhibited and consequently permit sleep initiation. In fact, in vitro studies on neonatal rat slices have shown that the depolarizing effect of NA on 139 Orx cens changes into a hyperpolarizing response after 2 hours of sleep deprivation (Grivel et al., 2005). In other studies, it is shown that stress or corticosteroids are associated with changes in expression of u2ARs (Jhanwar-Uniyal et al., 1986, Flugge et al., 2003). Knowing that sleep deprivation in neonatal rats increases corticosterone levels and is thus stress fui (Hairston et al., 2004), we assume that in response to the corticosterone and stress, Orx cens could potentiany express U2AAR. Here, we did not find any significant difference on the numbers of Orx cens bearing UIAAR vs. U2AAR across conditions, and since the receptors were not judged as being in cytoplasm or on the membrane, we could not judge whether those receptors were functional. However, changes in receptor expression or mobilization leading to changes in their functions could be partly responsible for excitation vs. inhibition of different sleep vs. wake-active cens across behavioral states. This phenomenon might be another potential mechanism for sleep-wake regulation. To test this possibility we designed our last experiment to study receptor plasticity under different conditions of sleep deprivati~n vs. sleep recovery. Experiment four In the fourth and last chapter, we analyzed the numbers and the intensity of membrane GABAARs on BF cholinergie cens. We found that the proportion of cholinergie cens that were immunostained for GABAARs was increased after sleep deprivation as compared with either control or recovery sleep. Moreover, we showed that fluorescence GABAAR immunostaining was increased after sustained waking. These results showed that a period of sleep vs. a period of sleep deprivation have contrary 140 effects on the expression or mobilization of the GABAARs on cholinergic ceIls. During waking, the BF cholinergic cells discharge in bursts and in the meantime accumulate more membrane GABAARs. It is known that the strength of inhibitory synaptic currents is directly corre1ated with the number of synaptic GABAARs (Otis et al., 1994) (Nusser et al., 1997, Nusser et al., 1998). Therefore, this build up ofGABAARs on cholinergic ceIl surfaces would make them more susceptible to GABA release that could occur with sleep onset. Consequently, after a prolonged waking period along with an increase in sleep pressure, the cholinergic cells may respond to even small amounts of GABA because of their enhanced membrane GABAARs. This in tum would dampen cholinergic activity and facilitate sleep promotion by decreasing cortical activation. Moreover in the tirst and second chapters, we showed that the GABAergic sleep-active ceIls are intermingled with the BF cholinergic neurons and other ceIls in the adjacent POA. These GABAergic ceIls, in paraIle1 with the attenuation of arousal and cortical activation, would be removed from inhibition and begin their activity which then by local projections to the cholinergic cells and acting through expressed or mobilized GABAARs lead to stronger inhibition of cholinergic neurons. The fact that many anesthetics and hypnotic drugs, inc1uding barbiturates and benzodiazepines, act through GABAARs suggests the importance of this receptor in sleep. Although the EEG during anesthesia is distinct from naturaIly occurring sleep, recent studies suggested that there are behavioral and perhaps neurophysiological similarities between sleep and anesthesia. Studies in rats revealed that recovery from sleep deprivation can occur during propofol anesthesia (Tung et al., 2004). 141 We showed that the immunofluorecent intensity of GABAARs was higher after sleep deprivation relative to sleep recovery and control conditions and it was higher after recovery for sleep relative to control conditions. It appears thus that during 3 hours of sleep recovery the membrane GABAARs staining decreases to return to controllevels, although not entirely and still would be higher than after 3 hours of control sleep. This difference could possibly be related to the condition prior to the experiment. Whereas the animaIs in sleep recovery group were deprived of sleep for 3 hours in the morning and before the experiment, the animaIs in the control group were aIlowed to sleep at the same time. In other words, after sleep recovery condition the animaIs were probably still under more pressure for sleep than after sleep control condition. The significantly higher GABAAR intensity after sleep recovery, could explain higher demand for maintaining sleep in those animaIs. Several studies have shown that neuronal activity leads to enhancement of membrane GABAAR c1usters. Activity blockade of cultured visual cortex by TTX after 2 days reduced the intensity of GABAAR labeling (Kilman et al., 2002). Similarly, the activity driven by the application ofbicuculline on cultured hippocampal cells after 13 days increased the density of GABAARs (Marty et al., 2004). These results suggest that cellular activity for a period of time is followed by synaptic changes toward a direction that leads to cellular inactivity or inhibition. In reverse, when neurons are under inhibition for a certain period, their synaptic changes prepare them for subsequent robust excitation. These findings are aIso consistent with the theory of synaptic homeostasis stating that in complex systems homeostatic plasticity has to take place to prevent destabilization during various physiological processes (Turrigiano, 1999, Marder and 142 Prinz, 2002, Mody, 2005). During waking, many cells fire continuously in order to sustain wakefulness. Yet, no one investigated whether the same phenomenon happens during waking on the arousing activating neurons. Here, for the first time we showed that the numbers and the intensity of GABAARs on cholinergie cells as part of the arousing systems were increased after 3 hours of sleep deprivation. As we showed in the second chapter, the BF cholinergie neurons are active during sleep deprivation represented by c-Fos expression. A recent study in the CUITent lab moreover revealed that identified BF cholinergie cells discharge in bursts during cortical activation and arousal in unanesthetized head-fixed rats (Lee et al., 2003). The enhanced membrane GABAARs on the cholinergie neurons at the end of the waking period could be thus the consequence of their dis charge activity during waking suggesting a homeostatic mechanism for the activity regulation of cholinergie cells as a part of the arousing systems. We showed that 3 hours of sleep deprivation was enough to induce significant changes in the amount and the intensity of fluorescence GABAARs staining. Other studies indeed have shown that insulin could provoke rapid translocation of intracellular GABAARs to the membrane (Wan et al., 1997). In paralle1, the application of dopamine agonists on cultured hippocampal cells induced glutamate receptor 1 (GluR1) synthesis within 60 minutes (Smith et al., 2005). This evidence thus shows that even for protein synthesis, changes could occur in a short time scale. Here, we did not assess the mRNA for GABAARs and therefore what we recorded as the increase in numbers or intensity of detectable membrane GABAARs could be either due to recruitment of intracellular/subsynaptic pools or it could reflect more receptor synthesis. 143 The insertion of GABAARs into cell surface membrane appears to be regulated by diverse signaling pathways. Application ofBDNF, a neurotrophin factor, on rat visual cortex, through activation ofTrkB receptors resulted in increased mIPSC amplitude in pyramidal cell neurons (Mizoguchi et al., 2003). In paraIlel, BDNF prevented the decrease in GABA-mediated inhibitory currents that was induced by activity blockade in rat visual cortex cultures (Rutherford et al., 1997). Besides, both neuronal activity and wakefulness are known to increase the expression ofBDNF (Castren et al., 1992, Cirelli et al., 2004). One potential signaling mechanism of regulating the cell surface GABAARs thus could be expression ofBDNF (Conner et al., 1997)that might occur following the activity of cholinergie cells and therefore through activating TrkB receptors and intracellular Ca++ cascades (Mizoguchi et al., 2003) would be able to recruit more GABAARs to the plasma membrane. 144 Summary In summary, multiple brain regions and neurotransmitters play roi es to regulate the sleep-waking cycle. During waking, beside the glutamatergic, monoaminergic and cholinergic arousing systems in the brainstem, we showed that the cholinergic BF neurons and the Orx cens of the posterior hypothalamus take part in cortical activation and behavioral arousal (Fig. 1). These findings show that different aspects ofwaking are regulated by activation of different neurons. Accordingly, the BF cholinergic cens by their cortical projections stimulate cortical activation. Orx neurons on the other hand, by their widespread excitatory projections to the cortex, the spinal cord as wen as to the multiple arousing systems including the BF cholinergic cens and the brainstem monoaminergic and cholinergic neurons would maintain continuous waking. They are in addition, of paramount importance during those arousal periods that are associated with increased motor activity, high demand for metabolism, energy expenditure and increased sympathetic drive. While exciting arousing systems, these cens receive reciprocal excitatory inputs from other elements ofthe arousal systems including most importantly from the Le noradrenergic fibres. As shown for Orx cens, theyare endowed by ŒIAAR and would be further excited during waking when release of NA is highest. Similar to waking states, several neuronal systems act in coordination to facilitate sleep promotion and maintenance. We showed that particular GABAergic cens of the BF and the adjacent POA become active during sleep and thus might be important for sleep generation and maintenance (Fig. 1). These particular GABAergic cens through their 145 long cortical projections (Gritti et al., 1997) might be involved in cortical dampening and SWA (Szymusiak et al., 1989, Manns et al., 2000a). In addition, locally projecting GABAergic neurons could inhibit the BF cholinergie cells during SWA (Manns et al., 2000a, Szymusiak et al., 2000) and finally by descending projections to the posterior hypothalamus (Gritti et al., 1994, Henny and Jones, 2003), they could inhibit the activity of Orx neurons. These findings c1arify how the arousal systems inc1uding the BF cholinergie cells or the posterior hypothalamic Orx neurons could become and remain inactive during sleep. Consistently, it has been shown that the GABA release is highest during SWS in the posterior hypothalamus (Nitz and Siegel, 1996). Due to the intrinsic depolarization state of Orx neurons (Eggermann et al., 2003), by which wakefulness may be maintained over long periods, their inhibition through GABAergic inputs from the BF and POA is crucial for sleep initiation. The inhibition of Orx cells in turn attenuates the sympathetic nervous system drive, motor activity, energy metabolism and feeding behaviors and thus facilitates a restful behavioral quiescence and sleep. Our results could provide a physiological basis for the hypnotic and sedative effects of many drugs that act through enhancement of GABA transmission. We found that the sleep-active GABAergic cells are distributed across the BF and the POA yet are more concentrated in sorne nuc1ei inc1uding the VLPO and the MnPO. These particular GABAergic cells bear U2AAR and thus could be distinguished from other wake-active GABAergic neuron across the BF and POA. These findings show how sleep-active cells are reciprocally influenced by arousal systems such as noradrenergic LC neurons. Consequently, they are under inhibition through U2AAR during waking when the release of NA is greatest whereas with decremental release of NA that occurs 146 during sleep, they become activated to dampen arousal systems and bring on sleep. In addition to the activity of GABAergic neurons, GABA transmission could be moreover enhanced postsynaptically through increases in expression or mobilization of GABAARs on arousal systems including the BF cholinergic cells at the end of waking phase to promote sleep. During waking, the BF cholinergic cells are active to maintain cortical activation. As a consequence of their activity, we showed that the cholinergic cells accumulate more GABAAR on their surface. The accumulated membrane GABAARs in tum leads to robust inhibition of cholinergic cells to allow sleep occurrence. These results explain a novel and intriguing mechanism for homeostatic inactivation or activation of the BF cholinergic cells across the sleep-waking cycle. We found that under certain situations such as high pressure for sleep, the hypothalamic MCH neurons in paralle1 with the GABAergic cells of the BF and POA become active so that by their widespread projections and their negative influence on energy metabolism, activity and sympathetic nervous systems, in opposition to Orx cells, facilitate recovery for sleep (Fig. 1). We showed that similar to the waking state, sleep is regulated by different cell types. Depending on the prior state and whether it imposes high demand of sleep or energy compensation, additional systems such as MCH neurons become active to aid the GABAergic sleep promoting cells. We found that MCH neurons bear uzAAR. Accordingly during waking, MCH cells are inhibited by NA through uzAAR, whereas during sleep recovery, they would be disinhibited and thus could exert a role in the recovery process. These studies e1ucidated different chemically identified neurons that play roles in sleep-waking cycle regulation. Moreover, they explained in part how sleep and wake 147 promoting neurons interact with each other to create a cycle and to modulate their activity. They also revealed that as heing part of complex systems, sleep or wake promoting neurons could show dynamic plasticity of their receptors to regulate their own activity in a homeostatic manner. The results have enriched our understanding of the sleep-waking cycle that could he important for the understanding and treatment of sleep or waking disorders. 148 Figure 1 Fast EEG (W & PS): !~liW!"il~ Slow EEG (SWS): 1\V1'1\j ,J\,Vt\VrI\\ Fast EEG active (Gamma+/Delta-; W-PSl .. ACh A GABA Slow EEG active (Gamma-/Delta+; SWSl À GABA (a2-ARJ Behavioral wake active (EMG+;W) o NA 1iji(- Orx Behavioral sleep active ŒMG-; SWS-PS) .... GABA (IÛ-AR) ,. MCH 149 Figure 1. Sleep vs. wake promoting systems. Schematic sagittal view ofrat brain (modifiedfrom (Jones, 2005)) showing the major neuronal systems and their major pathways (red arrows) and transmitters (red symbols) involved in waking. The major ascending pathway emerges from the brainstem reticular formation (RF) to ascend along a dorsal pathway into the thalamus (Th) from where the second thalamo-cortical projection fibres in turn reach the cerebral cortex (Cx) in a widespread manner. They also through a ventral pathway project to the hypothalamus and then up to the basal forebrain (BF) neurons where they terminate and the second basal-cortical fibres again in a wide spread manner project to the cerebral cortex. Noradrenergic neurons of the locus coeruleus (LC) send axons along the major ascending arousal systems to project in a diffuse manner to the cerebral cortex, hypothalamus and the BF. The basal forebrain cholinergie neurons which contain acetylcholine (ACh) in addition to laterodorsal and pedunculopontine tegmental (LDTg and PPTg) nuclei of the brainstem are located in the BF from where they project to the cortex. Orexinergic neurons (Orx) in the mid- and posterior hypothalamus (PH) project diffusely through the forebrain, brainstem and spinal cord to exert an excitatory influence at multiple levels. Some specifie GABAergic neurons may be on during waking perhaps to project to cortical interneurons and induce an indirect excitation ofpyramidal cells. The major sleep promoting systems and their major pathways (blue arrows) together with their chemical transmitters (blue symbols) are also shown. Particular cortical/y projecting GABAergic cel/s of the BF and preopticanterior hypothalamic areas (POAH) may have the capacity to dampen cortical activation during sleep. Local/y projecting GABAergic cel/s may inhibit the BF cholinergie cel/s. Other GABAergic neurons of the BF and POA project caudally to the PH where they may inhibit multiple arousing systems including the hypothalamic Orx 150 neurons. MCH cells with their widespread inhibitory projections to the en tire brain can influence multiple systems. Along with sleep-on GABAergic neurons they could potentially send projections to the cortex to inhibit cortical activation. They may also inhibit their neighbouring Orx cells by local projections or even by proximal cell to cell contacts. Abbreviations: 7g, genu 7th nerve; ac, anterior commissure; CPu, caudate putamen; Cx, cortex; EEG, electroencephalogram; EMG, electromyogram; Gi, gigantocellular RF; GiA, gigantocellular, a part RF; GiV, gigantocellular, ventral part RF; GP, globus pallidus; Hi, hippocampus; ic, internaI capsule; LDTg, laterodorsal tegmental nucleus; Mes RF, mesencephalic RF; opt, optic tract; PH, posterior hypothalamus; PnC, pontine, caudal part RF; PnO, pontine, oral part RF; POA, preoptic area; PPTg, pedunculopontine tegmental nucleus; Rt, reticularis nucleus of the thalamus; s, solitary tract; sep, superior cerebellar peduncle; SL substantia innominata; SN, substantia nigra; Sol, solitary tract nucleus; Th, thalamus; TM, tuberomammillary nuclei; VTA, ventral tegmental area. 151 7. Appendix 152 7.1 Ethics approval '. Vo/WN.mcgiltcafrgolanfmaU Prolocol 1#: Approval end date: ~M.u.\I., ~(, ~O()<O RENEWAL Faellity Commluee: \-t}..) 1.. of Animal Use Protocol Researeh l8J Teaehing 0 For: RenewalN: p~oject Principallnvestigalor: Dr. Barbara E. Jones Prolocol Tille: Neuml BnsÎs orthe Slcee·Wakins Cycle Mhl NcurolollY & Neurosurgery, Montreal Neurologieal Institllte. 3801 University St" #896. Montreal, Quebec HlA 284 Unit, Depl. & Address: Emoil: t~ li '1 MeGiIl University Animal Care Committee barbnra.joncs!Cllmcgîll.ca Slart of Funding: Prolocoll# Level: April 1. 20DI ' '1. Phone: loi 1943·0 --~~-----------. . ::;39:..::8:.. . :. :19:.:.1;::.3_______ . .::c39:..::8:..;.S::.;:8:..:.7.:.1_ _ _ _ __ Fax: Funding source: 0 ~ CIHR q ')..1\.<.- End ol Fundlng: _r.:.:;I"'nr:.::c:.;.h.:;.3.;.,1.c.:;2:..:.0.:;.06=--_ _ _ _ _ _ _ _ _ __ Emergency contact III + phone Ils Dr. Barbara E. Jones· 514·937·5550 Emergcncy COntact 1#2 + phone 115 -=Lyn=d=a..:.:r.:::la:::in.:..:v..:.:iI:.:le:....•...:4.::.S0;::.-4:.:.5~8:....•.:. :IS:;.: 8:.:.4_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ 1. Personnel and ualifications List the bames of the Principal Investlgalor and orau individunis who will bc ln Clmlaet wlth anlmab in this sludy and thclr emptoymenl classification (investigator, technicÎnn, research assistant, undergradualeJ graduale student, fellow). Ir an undergraduale student!s involved, the role of the sludent and the supervision received must be described. Training Is mandalory for ail personnellisted here. Reler to www.lln;malca.e.mcglll.cl/for detalls. ElIch person l!sled ln thls seelion must 51 n. (S lice will .... and as needetl. Oceupntional Helllth Name Classification Animal Related Signature ~ lias rClld Ih, I1rlglnal Training Information Program • 1411 rolol:ol" Dr. Barbara E. Jones PI Lynda Mainville Technician Initial training with > 30 years experience Theory course + rat workshop no Mandana Modirroust.'1 PhO Student Theory course + tat workshop no Frédéric Brischoux POF. Theory course + rat workshop no no 3. Summary (in language that will be understood by members of the general public) AIMS AND BENEFITS: Oescrlbe. in a short paragraph, the overallnim oC the study and Ils potentinl bene lit 10 humnnlllnimnl henUb or to Ihe ndvtmtemellt of sclenlilit knowlcdlte (Iflas sectloll 5a 1/1 maill protoc:ol). Understand howan attentive wakeful stale is maintaincd and how normal sleep occurs 50 as to know why such O.~FEV. 200~ 153 MONTREAL NEUROLOGICAL INSTITUTE AND HOSPITAL INSTITUT ET HÔPITAL NEUROLOGIQUES DE MONTRÉAL MeCi/! University Université McCil/ Barabara E. Jones, Ph..D. Professor Departmmt ofNeuro/ogy and Neurosurg"y Oecember 7, 2005 To Whom It May Concern, This letter authorizes Mandana Modirrousta to reprint a manuscript in her Ph.D. thesis of which we are co-authors. The manuscript is: Modirrousta M, Mainville L, Jones, BE. GABAp, Receptor Modifications on Basal Forebrain Cholinergie CeUs across the Sleep-Waking Cycle, in preparation. Thank you. Sincerely, ~~~ Lyn Mainville Research Technician Int 3801 University Street, #896 Montreal, Quebee Canada H3A 2B4 Telephone: (514) 3981913 Fax: (514) 398 5871 E-mail: barbarajones@mcgill. 154 -~--- Original Message ----- Ft:qm: eilams"Zôe'; :"'" To: Chalifour Kristin; [email protected] Sent: Monday, July 04,20058:10 AM Subject: RE: Permission for publishing my pa pers in my Ph.D. thesis Dear Mandana Thank you for yOuf email request. Permission is granted for you to use the mate rial below for yOuf thesis subject to the usual acknowledgements and on the understanding that you will reapply for permission ifyou wish to distribute or publish yOuf thesis commercially. Goodluck! Best wishes, Zoë Ellams (Miss) Permissions Co-ordinator Blackwell Publishing 9600 Garsington Road Oxford 0X42DQ Tel: 00441865476149 Fax: 00441865471149 [email protected] AlI future permission requests should be sent to mailto:[email protected] From: Mandana Modirrousta [mailto:[email protected]] Sent: Thursday, June 30, 2005 10:49 AM To: Customer Service Requests - Oxford; Customer Service Requests - USA Subject: Permission for publishing my papers in my Ph.D. thesis 155 Eur J Neurosci. 2005 May;21(10):2807-16. Orexin and MCH neurons express c-Fos differently aCter sleep deprivation vs. recovery and bear different adrenergic receptors. Modirrousta M, Mainville L, Jones BE. Eur J Neurosci. 2003 Aug;18(3):723-7. Alpha 2 adrenergic receptors on GABAergic, putative sleep-promoting basal forebrain neurons. Manns ID, Lee MG, Modirrousta M, Hou YP, Jones BE. 156 TRADITION j EXCEUENCE , fO!·the~{ of the ~CQmpmyin 18;80. Our ref: HG/smc/July 2005.j1238 22 July 2005 Dr Mandana Modirrousta [email protected] Dear Dr Modirrousta NEUROSCIENCE, Vol 129, No 3, 2004, Pages 803-10, 'Gabaergic neurons with .. .' As per your letter dated 30 June 2005, we hereby grant you permission to reprint the aforementioned material at no charge in your thesis subject to the following conditions: 1. If any part of the material to be used (for example, figures) has appeared in our publication with credit or acknowledgement to another source, permission must also be sought from that source. If such permission is not obtained then that material may not be included in your publication/copies. 2. 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Helen Gainford Rights Manager Your future requests will be handled more quickly if you complete the online form at www.elsevier.com/locate/permissions 158 7. References 159 Abrahamson, E. E., Leak, R. K. and Moore, R. Y., 2001. The suprachiasmatic nucleus projects to posterior hypothalamic arousal systems. Neuroreport. 12,435440. Alam, M. N., Gong, H., Alam, T., Jaganath, R., McGinty, D. and Szymusiak, R., 2002. Sleep-waking discharge patterns of neurons recorded in the rat perifornical lateral hypothalamic area. J Physiol. 538,619-631. Alam, M. N., McGinty, D., Bashir, T., Kumar, S., Imeri, L., Opp, M. R. and Szymusiak, R., 2004. Interleukin-lbeta modulates state-dependent discharge activity of preoptic area and basal forebrain neurons: role in sleep regulation. Eur J Neurosci. 20,207-216. Alam, M. N., McGinty, D. and Szymusiak, R., 1996. Preoptic/anterior hypothalamic neurons: thermosensitivity in wakefulness and non rapid eye movement sleep. Brain Res. 718, 76-82. 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Manns, Maan Gee Lee, Mandana Modirrousta, Yiping P. Hou* and Barbara E. Jones Department of Neurology and Neurosurgery, McGill University, Montreal Neurologicallnstitute Montreal, Quebec, Canada H3A 284 Keywords: cholinergie, cortical activation, EEG, noradrenergic, rat Abstract The basal forebrain plays an important role in the modulation of cortical activity and sleep-wake states. Vet its role must be multivalent as lesions reportedly diminish cortical fast activity and also cortical slow activity along with slow wave sleep (SWS). Basal forebrain cholinergie vs. GABAergic cell groups could differentially influence these processes. By labelling recorded neurons with Neurobiotin (Nb) using the juxtacellular technique and identitying them by immunostaining, we previously found that whereas ail cholinergie cells increased their firing, the majority of GABAergic neurons decreased their firing in association with evoked cortical activation in urethane-anaesthetized rats. Here, we examined the possibility that such GABAergic, cortical activation 'off' cells might bear alpha 2 adrenergic receptors (u2AR) through which noradrenaline (NA) could inhibit them during cortical activation. First using simple dualimmunostaining for glutamic acid decarboxylase (GAD) and the u2AAR, we found that the majority (~60%) of GAD-immunopositive (GAD+) neurons through the magnocellular preoptic nucleus (MCPO) and substantia innominata (SI) were labelled for the u2AAR. Second, in urethane-anaesthetized rats, we examined whether Nb-Iabelled, GAD+ cortical activation 'off' neurons that discharged maximally in association with cortical slow wave activity, were immunopositive for u2AAR. We found that ail the Nb+/GAD+ 'off' cells were labelled for the u2AAR. Such cells could be inhibited in association with cortical activation and waking when noradrenergic locus coeruleus (LC) neurons discharge and be disinhibited with cortical slow waves and SWS when these neurons become inactive. We thus propose that u2AR-bearing GABAergic basal forebrain neurons constitute sleep-active and sleep-promoting neurons. Introduction The basal forebrain plays an important role in modulating cortical activity and sleep-wake states (Jones, 2000). Lesions or inactivation of the basal forebrain reportedly diminish cortical fast activity during waking (Stewart et al., 1984; Cape & Jones, 2000) but also diminish cortical slow wave activity and slow wave sleep (SWS) (Szymusiak & McGinty, 1986a). The basal forebrain may thus be comprised of different cell groups that promote different cortical activities and states. In addition to cholinergic neurons that are known to stimulate cortical activation, the basal forebrain contains large numbers of GABAergic neurons (Gritti et al., 1993). By employing juxtaceUular labelling and immunohistochemical identification of recorded neurons in urethane-anaesthetized rats, we recently discovered that GABAergic ceUs behave very differently as a group than cholinergic cells. Whereas aU cholinergic neurons increased their firing with evoked cortical activation, the majority of GABAergic neurons decreased their firing in association with cortical activation and discharged at their highest rate in association with cortical slow wave activity (Manns et al., 2000a). We proposed that such GABAergic ceUs could be sleep-active neurons. Correspondence: Dr Barbara E. Jones, as above. E-mail: [email protected] .1 'Present address: Department of Anatomy, Lanzhou Medical College, Lanzhou, P. R. China 730000. Received 21 March 2003, revised 15 May 2003, accepted 20 May 2003 doi: \ 0.\ O46Ij.\460-9568.2003.02788.x Neurons that discharge at their highest rate during natural SWS have indeed been recorded in the basal forebrain and in the adjacent preoptic area of naturally sleeping-waking rats and cats (Szymusiak & McGinty, 1986b; Koyarna & Hayaishi, 1994; Szymusiak et al., 1998). Such SWS-active neurons were inhibited by stimulation of the locus coeruleus (Le) or iontophoretic application of noradrenaline (NA) in vivo (Osaka & Matsumura, 1994, 1995). In vitro studies also identified noncholinergic neurons in the basal forebrain and GABAergic neurons in the ventrolateral preoptic area (VLPO) that are commonly hyperpolarized and inhibited by NA (Fort et al., 1998; GaUopin et al., 2000). Such inhibitory effects of NA are mediated by alpha 2 adrenergic receptors (U2AR) (Osaka & Matsumura, 1995; Bai & Renaud, 1998). The presence of U2AR on basal forebrain neurons might thus identify them as sleep-active cells. The aim of the present study was to determine immunohistochemicaUy whether GABAergic and potentially sleep-promoting, basal forebrain neurons possess U2AR. In a preliminary investigation, we employed dual-immunostaining for glutamic acid decarboxylase (GAD) and the U2AAR to assess if a significant proportion of GAD-immunopositive (GAD+) neurons in the magnoceUular preoptic nucleus (MCPO) or overlying substantia innominata (SI) of the rat were labelled for the u2AAR. We then examined using urethane-anaesthetized rats, whether neurons that fire maximaUy in association with cortical slow wave activity and are GAD+ are labeUed for the u2AAR. Recorded within the MCPO or overlying SI, such characterized units were labelled with Neurobiotin 724 1. D. Manns et al. (Nb) using the juxtacellular technique and subsequently triple-stained Results for Nb, GAD and the a2AAR. Materials and methods For the immunohistochemical study of a2AAR on GAD-immunoreactive neurons, three male Wistar rats (weighing 200-250 g) were employed for simple perfusion-fixation of the brain under barbiturate anaesthesia (Somnotol, 120 mglkg, Lp.). For the study of the receptors on GAD-immunoreactive physiologically characterized cells, seven male Long Evans rats (weighing 200-250 g) were employed for acute electrophysiological recording prior to perfusion-fixation of the brain. Held in a stereotaxic frame, they were anaesthetized with urethane for the duration of the recording (1.4 glkg initial dose followed by boosters of 0.14 glkg as needed, Lp.) and for the subsequent perfusion-fixation (with an additional booster if needed). Ail procedures were approved by the McGill University Animal Care Committee and the Canadian Council on Animal Care. ln the electrophysiological experiments, units were recorded in the MCPO or SI using glass micropipettes and characterized in association with electroencephalogram (BEG) or field potentials recorded with surface or deep electrodes from retrosplenial or prefrontal cortex and olfactory bulb, as described in previous publications (Manns et al., 2000b, 2000a). Following isolation of a single unit, its discharge was characterized during baseline cortical irregular slow wave activity and during stimulated cortical activation evoked by tail pinch. Cells that decreased their rate of discharge with cortical activation, previously described as 'off' ceIls, were selected. One such cell per animal was then labelled with Neurobiotin (Nb, Vector Laboratories, Burlingame, CA) by applying the 'juxtacellular' technique. As established in our previous publications, modulating the discharge of one recorded unit using 200 ms current pulses over a period of 5-30 min resulted in labelling of only one neuron. Brains were fixed by intra-aortic perfusion of a paraformaldehyde solution (4% in 0.1 M phosphate buffer, PB, pH7.4, ~500mL) preceded by saline (0.9% NaCI, ~50 mL). After removal, all brains were placed in a sucrose solution (30% in PB at 4 oC) for ~2 days and then frozen. Sections were cut at 25-J.lID thickness in the coronal plane on a freezing microtome. For dual-immunostaining of GAD and a2AAR. sections were collected at 400 ~m intervals through the basal forebrain. They were incubated for three nights at 4 oC with an affinity-purified goat polyclonaI antibody for the a2AAR (C-19, sc-1478, Santa Cruz Biotechnology, Santa Cruz, CA) as employed previously (Hou et al., 2002), together with a rabbit polyclonal antibody for GAD67 (1: 3000, Chemicon International, Temecula, CA). The a2AAR was revealed using Cy3-conjugated donkey antigoat antiserum and GAD revealed simultaneously using Cy2-conjugated donkey antirabbit antiserum (Jackson Immuno Research, West Grove, PA). GAD-immunoreactive neurons in the MCPO and overlying SI were exarnined by fluorescence microscopy for the presence of a2AAR. For triple staining of recorded cells for Nb, GAD and a2AAR, aIl sections were collected through the basal forebrain for staining of the Nb-Iabelled cell using AMCA-conjugated streptavidin (Jackson ImmunoResearch Laboratories). After locating the Nb-Iabelled cell, the section containing it was subsequently incubated with the a2AAR and GAD antibodies for three nights and revealed with Cy2-conjugated and Cy3-conjugated secondary antibodies as described above. Nb-Iabelled cells that were also GAD+ were exarnined by fluorescence microscopy for the presence of a2AAR. To provide images of double labelling, they were superimposed using Adobe Photoshop (v9, Adobe Systems, San Jose, CA). In dual-immunostained series, GAD+ cells within the MCPO and overlying SI were often immunopositive for the a2AAR (GAD+/ a2AAR+, triangles in Fig. 1; filled arrowheads in Fig.2A' and B'). In these cells, the receptor labelling was punctate and distributed over the cell body and proximal dendrites. In many cases, it appeared to be within the cytoplasm, as it surrounded the nucleus of the cell. Punctate or diffuse receptor labelling was also present over blood vessels in the region (bv, Fig. 2A' and B'). In the same area, sorne GAD+ cells were immunonegative for the a2AAR (GAD+/a2AAR-, empty arrowheads, Fig.2A' and B'). The GAD+/a2AAR+ and GAD+/a2AAR- cells were present in the same vicinity (Fig. 2A and A') and sometimes situated immediately adjacent to one other (Fig. 2B and B'). In a quantitative sampling, a majority of the GAD+ neurons in the MCPO (average of 66%) and a substantial proportion of those in the. SI (average of 38%) or an overall majority in the MCPO-SI (average of 59% in three brains) were judged positively labelled for a2AAR. In recording experiments performed under urethane anaesthesia, cells were selected that decreased their firing with stimulationevoked cortical activation for labelling with Nb (Manns et al., 2000a). Generally discharging in a tonic irregular manner in association with the baseline irregular cortical slow wave activity, these 'off' cells decreased or ceased firing in association with cortical activation (Fig. 3). They subsequently increased their discharge following cessation of the stimulation as cortical slow wave activity returned to prestimulation levels (Fig. 3). Across cells, the average 1. GAD+ cells that were labelled for the <X2AAR (grey triangles) are plotted in the BF cholinergie cell ares, including the magnocellular preoptic nucleus (MCPO) and substantia innominata (SI) (Gritti et al., 1993). In this same area, units were recorded and selected for juxtacellular labelling and ·triple-staining for Nb, GAD, and <X2AAR. Ali Nb+/GAD+ cortical activation 'off' cells were located in the MCPO and were positively labelled for <X2AAR (black stars). Scale bar, 1 mm. Other abbreviations: ac, anterior commissure; CPU, caudate putamen; GP, globus pallidus; LPO, lateral preoptic area; MPO, medial preoptic area; oc, optic chiasm, OTu, olfactory tubercle. FIG. © 2003 Federation of European Neuroscience Societies, European Journal of Neuroscience, 18,723-727 Alpha 2 adrenergic receptors on GABAergic neurons 725 FIG.2. Photomicrographs showing GAD irnrnunostaining withCy2 by green fluorescence emission (A, B, C and D) and a2AAR irnrnunostaining with Cy3 by red fluorescence of the same view (A', B', C'! and D') ..In the left panels, dual-irnrnunostaining reveals that some GAD+ cells (filled arrowhead, A and B) are labelled for the azAAR+ (filled arrowheads, A' and B'), and others are not (open arrowheads, ft( and B'). Blood vessels (bv) are also irnrnunostained for the a2AAR+. In the right panels, triple-staining reveals that the GAD+ cells (filled arrowheads, C and D) that were physiologically characterized as cortical activation 'off' cells and labelled with Nb in blue fluorescence by AMCA-conjugated streptavidin (C' and D') are also irnrnunopositive for a2AAR (solid arrows, C' and D'). Photomicrographs show a simple image with single green fluorescence emission for GAD irnrnunostaining (left) and superimposed images with blue and red fluorescence for Nb and azAAR irnrnunostaining (right, C' and D'). Ali Nb+/GAD+/a2AAR+ neurons were located in the MCPO (Fig. 1). Scale bars, 20 Iffil. discharge rate decreased significantly during stimulation as compared to prestimulation (mean ± SEM: 3.36 ± 1.25 vs. 9.8 ± 2.27; t = 3.44, d.f. = 4, P = 0.03) and subsequently retumed to prestimulation levels (9.28 ± 2.56) within one to two minutes. Of the 'off' ceUs labeUed with Nb in the current study, the majority were immunopositive for GAD (5n), as was also the case in our previous study. The Nb+/GAD+ 'off' ceUs identified here were aU located in the MCPO (n = 5; Fig. 1). AU of these Nb+/GAD+ 'off' ceUs (Fig.2C and D) appeared to he lahelled for the a2AAR by the presence of punctate staining over the celI body and cytoplasm surrounding the nucleus (Fig. 2C' and D'). Discussion This study documents by immunohistochemistry combined with electrophysiology that the majority (59%) of basal forebrain GABAergic neurons possess <X2AR and that the GABAergic neurons that fire maximally with cortical slow wave activity bear these receptors. © 2003 Federation of European Neuroscience Societies, European Journal of Neuroscience, 18, 723-727 726 1. D. Manns et al. Nb+/GAD+/a2A+ Off œil A. EEG Somalie Stimulation ~", , B. Unit Discharge Rate 5 1 Il 1 1 Il 1 1 Il 1 1 lit 60, 20 sec 120 160 1/ • 200 ': , , , , , , '260 C.EEG ~ D. Unit Discharge - 2 sec -..-.I,mv IIIIIIIIIIIIII~ FIG. 3. Unit activity together with simultaneously recorded EEG from prefrontal cortex 'showing a typical decrease or cessation of firing in association with stimulation-evoked cortical activation and recovery of firing following stimulation in a urethane-anaesthetized rat This cortical activation 'off' œIl was labelled with Nb and inununopositive for GAD and the <l2AAR (Fig. 2C and C'). The compressed EEG recording (A) is expanded (C) and shown together with the unit recording (D) below. The unit discharge rate plot (spikes per second in B) shows the decrease and cessation in firing that occurs as the EEG changes from irregular slow wave activity to fast activity during stimulation and the retum to prestimulation rates that occurs as the EEG resumes irregular slow wave activity following stimulation. In our previous recording studies of GABAergic neurons in urethane-anaesthetized rats (Manns et al., 2000a), the majority (57%) of Nb+/GAD+ neurons through the MCPO-SI were found to discharge at a higher rate with cortical slow wave activity than with cortical activation. No Nb+/ChAT+ cells did so (Manns et al., 2000b), and only a minority (18%) of Nb+/GAD- cells did so (Manns et al., 2000a). These results suggested that a substantial group of GABAergic basal forebrain neurons would he most active in association with cortical slow wave activity during SWS and thus correspond in a large part to previously identified sleep-active neurons in this region (Szymusiak & McGinty, 1986a). These neurons could correspond to the major proportion (59%) of GAD+ neurons that bear <lzAAR documented here. The <lzAR is responsible for hyperpolarization of the membrane through opening of K+ channels, as shown on preoptic area neurons as on locus coeruleus neurons (Williams et al., 1985; Bai & Renaud, 1998). Although other subtypes of <lzAR have been shown to be present in the basal forebrain (Rosin et al., 1996), the <lZA was previously shown to he distributed densely therein (Talley et al., 1996) and using the same antibody as that used in the present study, found to he present upon locus coeruleus neurons (Hou et al., 2(02). In the present study, performed using epifluorescence microscopy, it cannot be excluded that <lzAR labelling could also be present on presynaptic terminaIs of the noradrenergic fibers as documented in other areas by electron rnicroscopy (Glass et al., 2001) or of other fibers as indicated in the preoptic area by electrophysiological evidence (Matsuo et al., 2(03). However, the <l2AR labelling over the cell body and proximal dendrites of GAD+ cells here, was predominantly associated with the postsynaptic cells as it often appeared to he located within the cytoplasm surrounding the cell nucleus, as also described in other neuronal perikarya (Talley et al., 1996). Examination for triple labelling by Nb, GAD and <lzAAR of physiologically characterized cells showed here that cortical activation 'off' GABAergic cells do hear <l2AR. These GAD+/<l2AAR+ neurons could be inhibited by NA in association with cortical activation and waking when LC neurons discharge at their highest rate (Aston-Jones & Bloom, 1981). Reciprocally they would be disinhibited during cortical slow wave activity and SWS when LC neurons decrease firing. They could also he active during paradoxical sleep (PS) when LC neurons are silent and many sleep-active neurons in the forebrain continue to discharge (Szymusiak & McGinty, 1986b; Koyama & Hayaishi, 1994; Szymusiak et al., 1998). However, in view of the present results, they are more likely to be silent in association with the cortical activation that occurs during PS as weIl as waking. Silence by these cells during PS could he effected by acetylcholine, which also inhibits the noncholinergic cells inhibited by NA (Fort et al., 1998; Gallopin et al., 2(00) and which is released by neighbouring choli. nergic cells during both PS and waking (Vazquez & Baghdoyan, 2(01). The GABAergic <lzAR-hearing neurons identified here, thus, most likely correspond to sleep-active cells that are maximally active during SWS. Given the evidence from lesion studies that destruction of cells in the basal forebrain leads to a loss of sleep (Szymusiak & McGinty, 1986a), these sleep-active neurons must also function irnportantly as sleep-promoting neurons. They may do so via projections to and inhibitory effects on local cholinergic neurons, the cortex (Gritti et al., 1997) or the posterior hypothalamus (Gritti et al., 1994). In this role, they may function in synergy with GABAergic neurons of the preoptic region, including the VLPO (Sherin et al., 1996; Szymusiak et al., 1998), that project in parallel to the posterior hypothalamus (Gritti et al., 1994; Sherin et al., 1998) and are similarly inhibited by NA (Fort et al., 1998; Gallopin et al., 2000). Acknowledgements This research was supported by grants from the Canadian Institutes of Health Research (CIHR, 13458) and United States National Institute of Mental Health (NIMH, ROl MH60119-01Al). Y.P. Hou was a Visiting Scientist from the Lanzhou Medical College, Lanzhou, China. 1.0. Manns held a graduate student fellowship from the Canadian Natural Science and Engineering Research Council (NSERC). Lynda Mainville is greatly appreciated for her technical assistance. Abbreviations <l2AR, alpha2 adrenergic receptor; GAD, glutarnic acid decarboxylase; LC, locus coeruleus; MCPO, magnocellular preoptic nucleus; NA, noradrenaline; Nb, Neurobiotin; PS, paradoxical sleep; SI, substantia innominata; SWS, slow wave sleep; VLPO, ventrolateral preoptic region. References Aston-Jones, G. & Bloom, F.E. (1981) Activity of norepinephrine-containing locus coeruleus neuronsin behaving rats anticipates fluctuations in the sleepwaking cycle. J. Neurosci., 1, 87~886. Bai, D. & Renaud, L.P. (1998) Median preoptic nucleus neurons: an in vitro patch-clamp analysis of their intrinsic properties and noradrenergic receptors in the rat. Neuroscience, 83, 905-916. Cape, E.G. & Jones, B.E. (2000) Effects of glutamate agonist versus procaine microinjections into the basal forebrain cholinergic œil area upon gamma and theta EEG activity and sleep-wake state. Eur. 1. Neurosci., 12, 216~2184. Fort, P., Khateb, A., Serafin, M., Muhlethaler, M. & Jones, B.E. (1998) Pharmacological characterization and differentiation of non-cholinergie nucleus basalis neurons in vitro. Neuroreport, 9, 1-5. © 2003 Federation of European Neuroscienee Societies, European Journal of Neuroscience, 18, 723-727 Alpha 2 adrenergic receptors on GABAergic neurons Gallopin, T., Fort, P., Eggennann, E., Cauli, B., Luppi, P.H., Rossier, J., Audinat, E., Muhlethaler, M. & Serafin, M. (2000) Identification of sleeppromoting neurons in vitro. Nature, 404, 992-995. Glass, M.J., Huang, J., Aicher, S.A., Milner, T.A. & Pickel, V.M. (2001) Subcellular localization of alpha-2A-adrenergie receptors in the rat medial nucleus tractus solitarius: regional targeting and relationship with catecholamine neurons. J. Comp. Neurol., 433, 193-207. Gritti, L, Mainville, L. & Jones, B.E. (1993) Codistribution of GABA- with acety\choline-synthesizing neurons in the basal forebrain of the rat. J. Comp. Neurol., 329, 438-457. Gritti, 1., Mainville, L. & Jones, B.E. (1994) Projections of GABAergie and cholinergie basal forebrain and GABAergie preoptic-anterior hypothalamic neurons to the posterior lateral hypothalamus of the rat. J. Comp. Neurol., 339,251-268. Gritti, L, Mainville, L., Mancia, M. & Jones, B.E. (1997) GABAergic and other non-cholinergie basal forebrain neurons project together with cholinergic neurons to meso- and iso-cortex in the rat. J. Comp. Neurol., 383,163-177. Hou, Y.P., Manns, I.D. & Jones, B.E. (2002) Immunostaining of cholinergie pontomesencephalic neurons for alphal versus alpha 2 adrenergie receptors suggests different sleep-wake state activities and roles. Neuroscience, 114, 517-52\. Jones, B.E. (2000) Basie Mechanisms ofSleep-Wake States. In Kryger, M.H., Roth, T., & Dement, W.C. (Eds), Princip/es and Practice of Sleep Medicine. Saunders, Philadelphia, pp. 134-154. Koyama, Y. & Hayaishi, O. (1994) Firing of neurons in the preopticJanterior hypothalamic areas in rat: its possible involvement in slow wave sleep and paradoxical sleep. Neurosci. Res., 19, 31-38. Manns, 1.0., Alonso, A. & Jones, B.E. (2000a) Discharge profiles of juxtacellularly labeled and immunohistochemically identified GABAergic basal forebrain neurons recorded in association with the electroencephalogram in anesthetized rats. 1. Neurosci., 20, 9252-9263. Manns, 1.0., Alonso, A. & Jones, B.E. (2000b) Discharge properties of juxtacellularly labeled and immunohistochemically identified cholinergie basal forebrain neurons recorded in association with the electroencephalogram in anesthetized rats. J. Neurosci., 20, 1505---1518. Matsuo, S., Jang, I.S., Nabekura, J. & Akaike, N. (2003) alpha 2-adrenoceptormediated presynaptic modulation of GABAergic transmission in mechani- 727 cally dissociated rat ventrolateral preoptic neurons. J. Neurophysiol., 89, 1640---1648. Osaka, T. & Matsumura, H. (1994) Noradrenergie inputs to sleep-related neurons in the preoptic area from the locus coeruleus and the ventrolateral medulla in the rat. Neurosci. Res., 19,39-50. Osaka, T. & Matsumura, H. (1995) Noradrenaline inhibits preoptic sleep-active neurons through Clrreceptors in the rat. Neurosci. Res., 21, 323-330. Rosin, D.L., Talley, E.M., Lee, A., Stometta, R.L., Gaylinn, B.D., Guyenet, P.G. & Lynch, K.R. (1996) Distribution of alpha 2C-adrenergic receptor-like immunoreactivity in the rat central nervous system. J. Comp. Neurol., 372, 135---165. Sherin, J.E., Elmquist, J.K., Torrealba, F. & Saper, C.B. (1998) Innervation of histaminergic tuberomammillary neurons by GABAergic and galaninergic neurons in the ventrolateral preoptic nucleus of the rat. J. Neurosci., 18, 4705-472\. Sherin, J.E., Shiromani, P.J., McCarley, R.W. & Saper, C.B. (1996) Activation of ventrolateral preoptic neurons during sleep. Science, 271, 216-219. Stewart, D.1., MacFabe, D.F. & Vanderwolf, C.H. (1984) Cholinergie activation of. the electrocorticogram: Role of the substantia innominata and effects of atropine and quinuclidinyl benzilate. Brain Res., 322, 219-232. Szymusiak, R, Alam, N., Steininger, T.L. & McGinty, D. (1998) Sleep-waking discharge patterns of ventrolateral preopticJanterior hypothalamic neurons in rats. Brain Res., 803, 178-188. Szymusiak, R. & McGinty, D. (1986a) Sleep suppression following kainic acid-induced lesions of the basal forebrain. Exp. Neurol., 94, 598-614. Szymusiak, R & McGinty, D. (l986b) Sleep-related neuronal discharge in the basal forebrain of cats. Brain Res., 370, 82-92. Talley, E.M., Rosin, D.L., Lee, A., Guyenet, P.G. & Lynch, K.R (1996) Distribution of alpha 2A-adrenergic receptor-like immunoreactivity in the rat central nervous system. 1. Comp. Neurol., 372,111-134. Vazquez, J. & Baghdoyan, H.A. (2001) Basal forebrain acetylcholine release during REM sleep is significantly greater than during waking. Am. 1. Physiol. Regul. Integr. Comp. Physiol., 280, R598-R60\. Williams, J.T., Henderson; G. & North, RA. (1985) Characterization of alpha 2-adrenoceptors which increase potassium conductance in rat locus coeruleus neurones. Neuroscience, 14,95---10\. © 2003 Federation of European Neuroscience Societies, European Journal of Neuroscience, 18, 723-727 Neuroscience 129 (2004) 803-810 GABAERGIC NEURONS WITH a2-ADRENERGIC RECEPTORS IN BASAL FOREBRAIN AND PREOPTIC AREA EXPRESS C-FOS DURING SLEEP M. MOOIRROUSTA, L. MAINVILLE AND B. E. JONES· SWS (Oetari et al., 1984; Szymusiak and McGinty, 1986; Koyama and Hayaishi, 1994; Alam et al., 1996). More recently by examining c-Fos expression as a reflection of neural activity, a restricted collection of neurons was identified within the ventrolateral POA (VLPO) that was active during sleep and thus proposed to be the principal sleep-promoting cell group of the POA and forebrain (Sherin et al., 1996). In immunohistochemical studies, many cells in the VLPO, lateral POA (LPO) and BF, including projection neurons, were shown to contain glutamic acid decarboxylase (GAD), the synthetic enzyme for GABA (Gritti et al., 1993, 1994; Sherin et al., 1998). The possibility was thus raised that GABAergic neurons in both POA and BF could comprise sleep-active and promoting cells. Indeed, in studies employing juxtacellular labeling of recorded neurons in urethane-anesthetized rats, a majority of immunohistochemically identified GABAergic BF neurons were found to tire maximally with cortical slow wave activity, whereas ail cholinergie neurons tired maximally with stimulation-induced cortical activation (Manns etaI., 2OOOa,b). Wake-active versus sleep-active neurons most likely respond differently to the major neurotransmitters of the ascending arousal systems, most notably noradrenaline (NA) contained in locus coeruleus neurons projecting to the BF and POA (Jones and Moore, 1977; Jones and Cuello, 1989; Chou et al., 2002). Indeed, it was tirst found in vivo that NA excited wake-active neurons, whereas it inhibited sleep-active neurons through <l2_adrenergic receptors (<l2-AR) in the POA and adjacent BF (Osaka and Matsumura, 1995). It was also found in vitro that whereas ail cholinergie neurons were excited by NA in the BF, sorne co-distributed non-cholinergie neurons (approximately 15%) were inhibited by NA (Fort et al., 1995, 1998). It was subsequently discovered that the majority of neurons in the VLPO (approximately 60%) were inhibited by NA and also contained mRNA for GAD (Gallopin et al., 2000). Most recently, we found that immunohistochemically identified GABAergic BF neurons which fired maximally with slow wave activity in urèthane-anesthetized rats were immunestained for <l2A-AR (Manns et al., 2003). These results suggested that GABA and <l2-AR may reflect a corn mon phenotype as weil as mechanism for a population of sleeppromoting neurons distributed across the BF and POA. ln the present study, c-Fos expression was examined Montreal Neurological Institute, McGill University, 3801 University Street, Room 897, Montreal, Quebec, Canada H3A 284 Abstract-The basal forebrain (BF) contains cholinergie neu· rons that stimulate cortical activation during waking. In addi· tion, both the BF and adjacent preoptic area (POA) contain neurons that promote sleep. We examined c:-Fos expression in cholinergie and GABAergic neurons in the BF and POA to detennine whether they are differentially active following sleep deprivation versus recovery and whether the GABAerglc neu· rons are active during sleep, Whereas the numbers of c-Fos+ cells and proportions of c:-Fos+ cells that were cholinergie were decreased, the proportions that were GABAergic were in· creased following sleep recovery across BF and POA nuclei. Moreover, the sleep-active GABAergic neurons were immunostained for <lzA-adrenergic receptors. We conclude that GABAergic neurons that c:ommonly bear ~-adrenergic receptors comprise sleep-active cells of the BF and POA. These GABAergic cells would be inhibited by noradrenaline (NA) raleased from locus coeruleus neurons during waking; they wou Id be disinhibited through diminished NA release during drowsiness and thus become active to promote sleep by inhibiting in tum wake-promotlng neurons. © 2004 IBRO. Published by Elsevier Ltd. Ali rights reserved. Key words: cholinergie, waking, slow wave sleep, REM sleep, noradrenaline, rat. Since early studies, the basal forebrain (BF) has been known to play an important role in cortical activation as the ventral extrathalamic relay to the cerebral cortex tram the brainstem ascending activating system (Starzl et al., 1951; Jones, 20(0). Yet, electrical stimulation in the region of the BF and adjacent preoptic area (POA) also elicited cortical slowwave activity with slow wave sleep (SWS; Sterman and Clemente, 1962a,b), and lesions therein produced insomnia (McGinty and Sterman, 1968). From single unit recording studies, it became apparent that whereas many neurons in the BF and POA increase their firing with cortical activation or waking, others increase their firing with cortical slow wave activity or *Corresponding author. Tel: +1-514-398-1913; fax: +1-514-398-5871. E-mail address:[email protected] (B. E. Jones). Abbreviations: AR, adrenergic receptor; BF, basal forebrain; ChAT, choline acetyltransferase; OBB, diagonal band of Broca nuclei; GAD. glutamic acid decarboxylase; LPO, lateral preoptic area; MCPO, magnocellular preoptic area; MnPO, median preoptic nucleus; MPO, medial preoptic area; NA, noradrenaline; NOS, normal donkey serum; non-REMS, non-rapid eye movement sleep; POA, preoptic area; REMS, rapid eye movement sleep; SC, sleep control; 50, sleep deprivation; Sla, substantia innominata, anterior part; Slp, substantia innominata, posterior part; SR, sleep recovery; SWS, slow wave sleep; VLPO, ventrolateral preoptic area. in sleep deprived versus sleep recovery groups of rats to test the hypotheses that sleep-promoting neurons 1) are distributed across nuclei of the BF and POA, 2) are not composed of cholinergie neurons and 3) are composed of GABAergic neurons that are inhibited by NA and th us bear 0306-4522/04$30.00+0.00 © 2004 IBRO. Published by Elsevier Ltd. Ali rights reserved. doi: 10.1 016fJ.neuroscience.2004.07.028 803 804 M. Modirrousta et al. 1 Neuroscience 129 (2004) 803-810 a2-AR. A portion of these results was presented in abstract form (Modirrousta et aL, 2003, 2004). EXPERIMENTAL PROCEDURES Male Wistar rats (Chari es River Canada, St. Constant, Quebec, Canada) weighing between 200 and 250 9 were housed individually in cages with free access to food and water. Three different groups of four rats per group were treated in their home cages for: 1) total sleep deprivation (SO) by gentle touching for 3 h (12:0015:00 h), 2) total SO for 3 h (09:00-12:00 h) followed by sleep recovery (SR) for 3 h (12:00-15:00 h), or 3) undisturbed sleep and waking as sleep control (SC) for 3 h (12:00-15:00 h). Visual observation was used to record the behavioral state every 20 s as wake, non-rapid eye movement sleep (non-REMS) or REMS, whereby non-REMS was scored when the animal was recumbent with eyes closed and showing little or no movement and REMS when the animal was recumbent with eyes closed and showing rapid movements or twitches of the eyes, whiskers, ears or paws (as previously determined to correspond to polygraphically scored sleep states (Maloney et al., 1997». Animais were prevented from sleeping during SO by gentle touching (with a small soft paint brush inserted through the bars ofthe cage top) upon eye closure. The deprivation procedure resulted in the total absence of sleep during 3 h for the SO and SR groups and was followed in the SR group by an increase in total sleep relative to both the SO group and SC group. At the end of the experimental period (15:00 h), the rats were immediately killed under pentobarbital anesthesia (100 mgfkg, Lp.) by intra-aortic perfusion with a fixative solution of 3% paraformaldehyde. Ali procedures were approved by the McGiII University Animal Care Committee and the Canadian Council on Animal Care. Our experiments meet or exceed ail guidelines of the Association for Assessment and Accreditation of Lab Animal Case International. Following immersion in a 30% sucrose solution, the brains were frozen and stored at -80 oC. They were cut in coronal sections at 20 J1.m, which was determined to be the greatest thickness that would allow full penetration and staining with the antibodies employed. Adjacent series of sections were collected and processed for double immunohistochemical staining using peroxidase-antiperoxidase for 1) c-Fos (rabbit antiserum;1: 10,000; Ab-5; PC38; Oncogene Research Products, La Jolla, CA, USA) with OAB-Ni as chromogen and 2) a) at 400 J1.m intervals, choline acetyltransferase (ChAT; mouse antibody; 1:2000; MAB5270; Chemicon International, Temecula, CA, USA) or b) at 200 J1.m intervals, GAD (mouse antibody, detecting both GA065 and GA067 isoforms; 1:100;. MSA-225; Stressgen Biotechnologies, Victoria, BC, Canada) with pink, a-naphthol pyronin B. Other series taken at 800 J1.m intervals were processed for triple immunostaining for 1) c-Fos in the first position using OAB-Ni, 2) <X2A-AR (goat-purified antiserum; 1:50; sc-1478; Santa Cruz Biotechnology, Santa Cruz, CA, USA) in the second position using red Cy3-conjugated donkey antigoat antiserum (Jackson ImmunoResearch Laboratories, West Grove, PA, USA), and 3) GAO (rabbit antiserum; 1:1000; AB5992; Chemicon) using green Cy2conjugated donkey antirabbit antiserum (Jackson) in the third position. Incubations with primary antibodies were performed at room temperature ovemight for c-Fos, ChAT and GAO antibodies and three nights at 4 oC for <X2A-AR antibody using a Tris-saline solution (0.1 M) containing 1% normal donkey serum (NOS; following initial blocking with 3% NOS). Sections were viewed by light and fluorescence microscopy (Leica OMLB microscope Leica Microsystems (Canada, Inc., Richmond Hill, Ontario, Canada) equipped with an x/y/z movement-sensitive stage and video camera at!ached to a computer). Cells immunopositive (+) for single c-Fos+, double c-Fos+1 ChAT + or c-Fos+/GAO+ and triple c-Fos+/GAO+/<X2A-AR+ were counted by applying stereology using the Optical Fractiona- tor program of Stereo Investigator (2003; MicroBrightField, Williston, VT, USA). Cells were counted within the contours of eight nuclei in the BF and POA, respectively: substantia innominata, anterior part (Sla), substantia innominata, posterior part (Slp), magnocellular POA (MCPO), diagonal band of Broca nuclei (OBB) and LPO, VLPO, medial POA (MPO), median preoptic nucleus (MnPO). The nuclei were delineated in a computer resident atlas (modified from Gritti et al., 1993) that was placed in register ~ith each section. For each nucleus, labeled cells were counted bllaterally in at least three sections (three to 14, depending upon the length of each nucleus) at 400 J1.m intervals for the c-FosfChATimmunostained series through the BF region nuclei or 200 J1.m for the c-Fos/GAO-immunostained series through the BF and POA regions. Through ail nuclei, cells were counted under a 63x oil objective (with 1.4 numerical aperture) within a counting frame of 125x125 J1.m. In most nuclei, cells were counted through 25% of the area at each level by setting the grid size (within which the counting frame is automatically inserted) to 250x250 J1.m. For the smaller nuclei (VLPO and MnPO), cells were counted through the entire area of the nucleus by setting the grid size to 125x125 IJ.m. Counts of triple-Iabeled cells immunostained for c-Fos/GADI <X2A-AR were combined across nuclei for estimates of total cell numbers within the BF and POA regions. By counting nuclei that came into focus beneath the surface of the section, c-Fos+ nuclei were counted within a counting block of 8 IJ.m in depth (in the dehydrated, delipidated, mounted and coverslipped sections that were on average 10 J1.m thick). Sleep and cell counts were analyzed between groups and across nuclei or regions using one and two way ANOVA in Systat (v10.2, Richmond, CA, USA). Ali main effects were confirmed by nonparametric rank order tests (Kruskal-Wallis, P<0.05) to insure that the distribution of variance in groups (containing zeros as a result of the experimental condition) did not distort the parametric statistics. Figures were composed using Adobe Photoshop 6 and lIIustrator 9 (Adobe, San Jose, CA, USA). RESULTS Rats submitted to SD by gentle touehing did not sleep du ring the 3 h prior to kil! at 15:00 h, whereas those perrnitted SR during the same period after 3 h deprivation in the moming (09:00-12:00 h) slept approximately 91 % of the time. The amount of total sleep differed significantly aeross conditions of SD, SR and SC (Table 1). Recovery and control sleep were comprised predominantly of behaviorally quiet, non-REMS (non-REMS: 77.23:±:2.94% in SR and 68.68:±:0.10% in SC with REMS: 13.58:±:1.30% in SR and 7.10:±:0.66% in SC, mean:±:S.E.M. of total time). ln SR and/or SD rats, c-Fos was expressed in ChATimmunopositive (+) and GAD+ cells as weil as in ChATimmunonegative (-) and GAD- cells within the BF and POA (Fig. 1A-O). The numbers of c-Fos+ cells varied significantly aeross conditions, differing between SR or SC and SD but not between SR and SC (Table 1), According to a two-way ANOVA with condition and nuelei as factors, c-Fos+ cells were significantly deereased in SR (like SC) as compared with SD (see Fig. ZA, Band 3B, Table 1). Given a significant interaction between condition and nuclei, post hoc comparisons were perforrned to deterrnine if the deerease was significant in ail nuelei (Table 1). The difference was found to be significant in ail nuelei, exeept the MnPO. The proportions of e-Fos+ eells that were ChAT + were also signifieantly deereased in SR (Iike SC) as eompared with SD (Fig. 2C, D and 3C, Table 1). In this 805 M. Modirrousta et al. / Neuroseience 129 (2004) 803-810 Table 1. Average percent sleep and e-Fos expression aeross nuelei (Nue) of the BF and POA under conditions (Cond) of SC, SO and SR in three groups of ratsa Variable SC SO % Sleep Total number c-Fos+ ceUs Percent c-Fos+/ChAT + Percent c-Fos+/GAO+ 75.75:!:0.61 650.53:!: 86.29 3.57:!:1.61 44.00:!:3.66 O.OO:!:O.OO 2462.70:!:341.40 11.68:!:2.64 17.16:!:2.71 SR (SC,SR) (SC,SR) (SC. SR) (SC,SR) 90.80:!:2.47 575.80:!:95.02 O.OO:!:O.OO 41.16:!:4.04 (SC, SO) (SO) (SO) (SO) F (Cond) F (Nue) F (CondxNue) 1095.40'" 70.72'" 8.95'" 25.86'" 16.67'" 0.33 ns 6.53'" 4.77'" 0.16 ns 1.35 ns • Mean:!:S.E.M. values from four rats per group. % Sleep was calculated as percentage of 3 h observation period prior to killing and was examined across groups by a one-way ANOVA. For number or percent of c-Fos expressing ceUs, two-way ANOVAs were performed with Cond (3) and Nue (8) as factors. For each variable, there was a significant main effect of condition. Post hoc paired comparisons with Bonferroni correction were peIformed to examine differences between conditions (indicated in parentheses, P<.05). For % Sleep, SO and SR both differed from SC and from each other; for c-Fos+ ceU variables, SO differed from SC and SR but SR did not differ from SC. For total number of c-Fos+ ceUs (calculated as c-Fos+ plus c-Fos+/GAD+ ceUs in the c-Fos/GAO dual-immunostained series), there was a significant difference across nuclei and a significant interaction of condition with nuclei. Post hoc comparisons between conditions in each nucleus revealed a main effect of condition in every nucleus except the MnPO. For percent of c-Fos+ ceUs (as c-Fos+/ChAT + or c-Fos+/GAO+ of the total), there was a significant difference for GAD + ceUs between nuclei, but there was no significant interaction between condition and nuclei for GAO+ or ChAT + cells, thus contraindicating post hoc comparisons for condition in each nucleus. ('" indicates F-ratio with P<.001; ns means not significant.) case, there was no significant interaction between condition and nuclei, indicating that the effect of condition was not different across individual nuclei of the BF. In contrast to those being ChAT +, the proportions of c-Fos + cells that were GAD + were significantly increased in SR (like SC) as compared with SD (Fig. 2E, F and 3D; Table 1). In this case, there was also no significant interaction between condition and nuclei, indicating that the main effect of condition did not differ across the nuclei of the BF and POA. The incidence of <XzA-AR labeling was subsequently examined on c-Fos+/GAD+ cells in the SD and SR groups by triple-immunostaining (Fig. 1E and F). The proportions of c-Fos+/GAD+ cells that were <XzA-AR+ were small in the SD group and significantly higher in the SR group across the BF and POA regions (with no significant difference across the regions or interaction of condition with region, Fig. 4). On average across regions, 3.88:±: 1.73% of c-Fos+/GAD+ cells were Ot2A-AR+ in the SD group and 77.70:±:22.07% in the SR group. DISCUSSION Fig. 1. Labeled ceUs in the nuclei of the BF or POA. (A) Single-labeled c-Fos+ cell (stained black with OAB-Ni, black arrowhead), single-labeled ChAT + cell (stained pink with cr.-naphthol pyronin B, white arrowhead) and double-labeled c-Fos+/ChAT+ cell (double arrowhead) in the MCPO. (8-0) Single-Iabeled c-Fos+ cells (stained black, black arrowheads), single-Iabeled GAO+ cells (stained pink, white arrowheads) and double-labeled c-Fos+/GAO+ cells (double arrowheads) in the MCPO (B), MPO (C) and VLPO (0). (E, F) A triple-labeled c-Fos+/GAD+/~ AR+ cell shown in images of c-Fos staining (stained black with OAB-Ni, black arrowhead in E) and ~-AR (stained by red fluorescence with Cy3) together with GAD staining (stained by green fluorescence with Cy2, white arrowhead in F). The ~-AR labeling is avident over the GAD + cell body (in orange or yellow). Scale bars=2Q ....m (for A-O, shown in D, and E-F, shown in F). The present results indicate that across the BF and POA fewer neurons are active during sleep than during waking, and that of those active during sleep, none are cholinergic and a substantial proportion are GABAergic. Moreover, the majority of the sleep-active GABAergic neurons appear to bear Ot 2A-AR. ln ail nuclei of the BF and ail but the MnPO of the POA, more neurons accumulated c-Fos protein following 3 h of waking produced by gentle deprivation of sleep than following 3 h of sleep either during recovery from deprivation or control condition. These results suggest that the majority of neurons in the BF and POA are more active during waking than during sleep. Similar conclusions had been reached by other studies employing c-Fos immunohistochemistry or in situ hybridization in the past (Pompeiano et al., 1992; Cirelli et al., 1993; Ledoux et aL, 1996; Sastre et aL, 2000). Only recently was it reported first for the VLPO and then for the MnPO in the POA, that more neurons expressed c-Fos during sleep than during waking (Sherin et aL, 1996; Gong et al., 2000). It is possible that the particular experimental conditions in those studies, such as the housing, surgery or method of SD, which can be associated with certain degrees of stress and associated c-Fos expression at 806 M. Modirrousta et al./ Neuroscience 129 (2004) 803-810 ..... A8.6 E .. ..... A9.0 F (·Fos+/GAD+ SO SR SO SR Fig. 2. Distribution of c-Fos+ cells in one SD and one SR representative brain through the BF and POA (at approximately A8.6 in A, C and E and approximately A9.0 in B, 0 and F; Gritti etat, 1993). (A, B) C-Fos+ cells (blaekdots)within BF and POA nuelei. (C, 0) Double-Iabeled c-Fos+/ChAT+ cells (red circ/es) distinguished from single-Iabeled c-Fos+ cells (black dots) within the BF. (E, F) Double-Iabeled c-Fos+/GAD+ cells (blue triangles) distinguished from single-Iabeled c-Fos+ cells (black dots) within the BF and POA nue/ei. Atlas images show cells plotted in one 20 ""m thick section per level (approximately A 8.6 on left and approximately A 9.0 on right) from adjacent series (separated by 200 ""m) dual-immunostained for c-Fos/ChAT (C, 0) or c-Fos/GAD (A, Band E, F). Sip not shown. (Note that ail nuelei examined are contained within the sections iIIustrated in Fig. 2, except Slp, which is located in the same relative position as Sla at more caudal levels.) particular times (Oragunow and Faull, 1989; Cullinan et al., 1995; Maloney et aL, 1999), resulted in relatively higher c-Fos expression in those nuclei during sleep than in the present study. Here, we employed conditions that would minimize stress prior to and during the experimental period by studying the animais in their home cages, using behavioral observation to record sleep so as to avoid prior surgery and applying gentle touching with a soft brush for preventing sleep during a relatively short (3 h) period. Under these conditions, an increase in the total number of neurons expressing c-Fos was not observed in any nucleus of BF or POA during recovery or control sleep as compared with enforced waking. Indeed, the number of neurons active in SR was on average <25% of that active in SO. The proportions did differ however across nuclei with the highest proportions being in the MnPO (approximately 75%) and VLPO (approximately 50%) and lower proportions in the BF and LPO nuclei (approximately 20%), suggesting different concentrations of sleep- versus wake-active neurons with the highest concentrations in MnPO and VLPO. These results are supported by both in vivo and in vitro electrophysiological studies showing relatively high concentrations of putative sleep-promoting neurons in these POA nuclei (Szymusiak et aL, 1998; Gallopin et aL, 2000; Suntsova et aL, 2002). Single unit recording studies have also found relatively smaH proportions of neurons that are maximaHy active specifically during non-REMS or SWS in BF and POA nuclei (Oetari et aL, 1984; Szymusiak and McGinty, 1986; Koyama and Hayaishi, 1994; Szymusiak et aL, 2000; Suntsova et aL, 2002; Lee et al., 2004). On the M. Modirrousta et al. / Neuroscience 129 (2004) 803-810 807 A Bt 1 ~ 1 ! i ~ ~ 1 ! C i ! + ~ '# 0 Fig. 3. Bar charts showing the percentage of sleep (in A) and the total numbers of e-Fos+ ceUs (in B) or proportions of o-Fos+ ceUs that were cholinergie or GABAergic (in C or 0) within each nucleus of the BF and/or POA in SO and SR groups. (A) The percentage of sleep (representing percentage of total sleep of 3 h period prior to kiU) was significantly inereased in SR as compared with SO. (B) The total numbers of e-Fos+ ceUs (obtained trom c-Fos/GAO dual-immunostained series as the total of o-Fos+ and e-Fos+/GAO+ ceUs) were significantly deereased across nuelei of the BF and POA in SR as compared with SO (with a significant differenee across nuclei and a significant interaction between condition and nuelei, due to a significant decrease in ail nuelei except MnPO, Table 1). (C) The percentages of e-Fos+ cells that were ChAT + (obtained trom the o-Fos/ChAT dual-immunostained series as the percentage of e-Fos+ plus o-Fos+/ChAT + cells that were e-Fos+/ChAT +) were significantly decreased aeross nuclei of the BF in SR as compared with SO (with no significant difference across nuelei or interaction betwèen condition and nUclei, Table 1). (0) The percentages of o-Fos+ cells that were GAD + (caleulated from total numbers shown in B) were significantly inereased in SR as compared with SO (with a significant difference across nuelei but no significant interaction between condition and nuelei, contraindicating post hoc comparisons in individual nuelei, Table 1). For each group (n=4), mean±S.E.M. are presented. M. Modirrousta et al. 1 Neuroscience 129 (2004) 803-810 808 BF A ~~ cr:+ oc:( 1 oc:( ~+ 0 oc:( ~ ~ ~ 6 ~ 0 SO SR Condition POA B !Il. ~ + cr: oc:( 1 oc:( ~+ 0 oc:( ~ ~ ~ ô ~ 0 SO SR Condition Fig.4. Bar charts showing the proportions of c-Fos+/GAO+ ceUs that bear «2A-AR in the BF (A) and POA (B) in SO and SR groups. There was a significant increase in c-Fos+/GAO+/«2A-AR+ ceUs in SR as compared with SO (ANOVA for main effect of condition. F=7.168. P<O.05 with no significant difference across the two ragions and no significant interaction between condition and region, P>O.05). other hand in the sa me studies, a large and varying proportion of neurons has been characterized through these regions as active during REMS, some as nonREMS/REMS-active, some selectively REMS-active and others as Wake/REMS-active. In a study examining cFos in pharmacologically produced predominant REMS versus predominant non-REMS conditions, it was found that REMS was associated with increased c-Fos expression in many forebrain regions (Sastre et al., 2000). We assume that in the present study the major percent of time spent in non-REMS (approxirhately 77%) as compared with the small percent of time spent in REMS (approximately 14%) in the 3 h preceding kHI served as the major determinant in c-Fos expression and accu mu- lation during recovery as weil as control conditions. We thus conclude that the c-Fos+ neurons seen following SR or control largely represent non-REMS-active neu~ rons (which would include non-REMS/RE MS-active but not selective REMS-active or Wake/REMS-aetive neurons) that are distributed aeross BF and POA as a sm ail population though with relatively greater representation in certain nuelei of the POA. ln parallel with the total number of neurons expressing e-Fos, the proportions of those neurons that were eholinergic aeross BF nuelei were diminished during sleep. In fact, no c-Fos+/ChAT + neurons were detected in the SR condition. These results confirm electrophysiological studies indicating that presumed cholinergie neurons are silent during SWS (Jones, 2004; Lee et aL, 2004). They also confirm that cholinergie neurons are active during waking. Aecording to release of aeetyleholine and firing of presumed cholinergie BF neurons, however, they should also be active during REMS (Marrosu et aL, 1995; Jones, 2004). As for ail e-Fos+ ce"s (above), we assume that the laek of e-Fos immunostaining in cholinergie neurons in the recovery condition, as weil as control, is due to the predominance of non-REMS in the 3 h prior to kill and the small am ou nt of time spent in REMS (14%) as weil as waking (approximately 9%) that would be inadequate for accumulation of the Fos protein during that period (Dragunowand Faull, 1989). ln contrast to the proportion of cholinergie neurons, the proportion of GABAergic neurons expressing c-Fos increased significantly with SR as compared with SD across BF and POA nuclei. These results indicate that many GABAergic neurons distributed across these regions remain or become active in association with sleep following waking. They support claims that neurons in the BF and POA including the VLPO that project to the posterior hypothalamus and contain GAD may be sleep-active and promoting neurons and may act by inhibiting wake-promoting neurons of that region (Gritti et aL, 1994; Sherin et aL, 1998). They also support the hypothesis that identified GABAergie BF neurons that discharge in association with cortical slow wave activity in urethane-anesthetized rats and project either to posterior hypothalamus or cortex, if not locally, as determined by antidromic activation, could be sleep-active and promoting as weIl (Manns et aL, 2OOOa). Finally, they confirm similar results most recently reported by others for e-Fos expression in GAD+ neurons of the MnPO and VLPO (Gong et al., 2004). GABAergic neurons of different BF and POA nuclei could thus exert an inhibitory influence upon wake-active and promoting systems in the posterior hypothalamus, cortex and/or BF. However, in single unit recording studies, it was also found that another physiologically distinct group of GABAergic neurons discharged maximally in association with cortical activation (Manns et aL, 2000a). Therefore, different groups of GABAergic neurons in the BF and POA are likely active in association with cortical activation of waking and others in association with SWS. The major proportion (approximately 78%) of GAD+ neurons that expressed c-Fos with SR in the present study were immunostained for the Cl2A-AR, whereas only a min- M. Modirrousta et al. 1 Neuroscience 129 (2004) 803-810 imal percentage (approximately 4%) of those that did so with SO were so labeled. These results confirm the hypothesis. that GABAergic neurons in the BF and POA that are active during sleep bear <x2 -AR and would accordingly be inhibited by NA (Osaka and Matsumura, 1995; Bai and Renaud, 1998; Manns et al., 2003; Saint-Mieux et al., 2004). This particular group of GABAergic neurons would be he Id under inhibition during waking by release of NA from afferent locus coeruleus neurons (Jones and Cuello, 1989) that fire maximally during active waking, decrease firing during quiet waking, and further diminish firing during SWS to cease firing during REMS (McCarley and Hobson, 1975; Aston-Jones and Sloom, 1981). With decremental release of NA during drowsiness, these GABAergic neurons would be disinhibited to become active and promote sleep by inhibiting in tum the noradrenergic neurons together with other neurons of the brainstem and forebrain activating systems (Gritti et al., 1994; Luppi et al., 1995; Sherin et al., 1998; Steininger et al., 2001; Chou et al., 2002). Acknowledgments-The research was supported by a grant From the Canadian Institutes of Health Research (13458). 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(Acœpted 14 July 2004) (Avai/able online 18 Oatober 2004) European Journal of Neuroscience, Vol. 21, pp. 2807-2816, 2005 © Federation of European Neuroscience Societies Orexin and MCH neurons express c-Fos differently after sleep deprivation vs. recovery and bear different adrenergic receptors Mandana Modirrousta, Lynda Mainville and Barbara E. Jones Montreal Neurological Institute, McGili University, Montreal, Quebec, Canada H3A 284 Keywords: hypocretin, hypothalamus, noradrenaline, rat, waking Abstract Though overlapping in distribution within the posterior hypothalamus, neurons containing orexin (Orx) and melanin concentrating hormone (MCH) may play different roles in the regulation of behavioural state. In the present study in rats, we tested whether they express c-Fos differently after total sleep deprivation (SO) vs. sleep recovery (SR). Whereas c-Fos expression was increased in Orx neurons after SO, it was increased in MCH neurons after SR. We reasoned that Orx and MCH neurons could be differently modulated by noradrenaline (NA) and accordingly bear different adrenergic receptors (ARs). Of ail Orx neurons (estimated at R:l6700), substantial numbers were immunostained for the <X1KAR, including ceUs expressing c-Fos after SO. Yet, substantial numbers were also immunostained for the <X2A-AR, also including cells expressing c-Fos after SO. Of ail MCH neurons (estimated at R:l12 300), rare neurons were immunostained for the <X1A-AR, whereas significant numbers were immunostained for the <X2A-AR, including cells expressing c-Fos after SR. We conclude that Orx neurons may act to sustain waking during sleep deprivation, whereas MCH neurons may act to promote sleep following sustained waking. Sorne Orx neurons would participate in the maintenance of waking during deprivation when excited by NA through ()(1-ARs, whereas MCH neurons would participate in sleep recovery after deprivation when released from inhibition by NA through <X2-ARs. On the other hand, under certain conditions, Orx neurons may also be submitted to an inhibitory influence by NA through <X2-ARs. Introduction The posterior hypothalamus has long been known to play a critical role in the maintenance of wakefulness (Jones, 2000). Recently two peptides, orexin (Orx, also called hypocretin) and melanin concentrating hormone (MCH) have been found to be contained in distinct populations of magnocellular neurons that partially overlap in their distribution through the mid to posterior hypothalamus and commonly give rise to widespread projections through the central nervous system (Bittencourt et al., 1992; Broberger et al., 1998; de Lecea et al., 1998; Peyron et al., 1998). They were both initially found to have a stimulatory influence upon eating and thus to be considered as contingents of the hypothalamic 'orexigenic' systems (Williams et al., 2004). Yet, as became evident in knock out experiments eliminating the peptides or their receptors, their normal roles appeared to extend beyond influences upon eating and to differ with regard to energy homeostasis. Most notably, mice lacking the gene for orexin or dogs lacking the gene for its receptor manifested a syndrome of narcolepsy (Chemelli et al., 1999; Lin et al., 1999) and humans with narcolepsy were found to 1ack the gene, peptide or neurons containing Orx (Peyron et al., 2000; Thannickal et al., 2000). In addition, Orx knock out mice became obese despite being hypophagic, attributed to a decrease in activity and basal metabolic rate. As shown by c-Fos expression and Orx release, Orx cells also appeared to be most active during the night, when animaIs were awake and engaged in motor Correspondence: Dr Barbam E. Jones, as above. E-mail: [email protected] Received 22 December 2004. revised 18 February 2005. accepted 14 March 2005 doi:IO.llll/j.l460-9568.2005.04104.x activity (Estabrooke et al., 2001; Yoshida et al., 2001). Orx thus appeared to stimulate arousal with motor activity and increased energy expenditure. In contrast, although MeH knock out mice were also hypophagic, they manifested a decrease in body weight attributed to an increase in activity and basal metabolic rate (Shimada et al., 1998; Marsh et al., 2002). MCH thus appeared to decrease arousal and energy expenditure. According to these results, Orx and MCH neurons may perform opposite roles in wake vs. sleep promotion and/or maintenance. Here, we examined whether Orx and MCH ceUs respond differently to total sleep deprivation (SD) vs. sleep recovery (SR) by using c-Fos expression as an indicator of neuronal activity. Different activity profiles could he due to different responses of the Orx and MCH neurons to the major neurotransmitters of the ascending arousal systems including most importantly noradrenaline (NA). In the basal forebrain and preoptic areas, cholinergic wake-active neurons are excited by NA through <Xl adrenergic receptors (<XI-ARs), whereas GABAergic putative sleep-active neurons are inhibited by NA through <xrARs (Fort et al., 1995; Fort et al., 1998; Gallopin et al., 2000; Manns et al., 2003; Modirrousta et al., 2004). We thus examined whether the Orx and MCH neurons bear different adrenergic receptors while possibly expressing c-Fos under different conditions of sustained waking vs. recovery sleep. Materials and methods AH procedures were approved by the McGill University Animal Care Committee and the Canadian Council on Animal Care, whose 2808 M. Modirrousta et al. standards meet those of the Association for Assessment and Accreditation of Animal Care International. Male Wistar rats (200-250 g) were used in minimal numbers to make up three test groups. The rats were housed individual!y with free access to food and water at al! times and maintained on a 12-h light : 12-h dark schedule (with lights on from 07:00 to 19:00 h). As previously described (Modirrousta et al., 2004), the experiment was designed to deprive rats of sleep under non-stressful conditions and was thus performed in the home cages. The three different groups (n = 4 per group) were submitted to: (i) total sleep deprivation (SD) for 3 h (12:00-15:00 h); (ii) total sleep deprivation for 3 h (09:00-12:00 h) followed by sleep recovery (SR) for 3 h (12:00-15:00 h) or (iii) undisturbed sleep and waking as sleep control (SC) for 3 h (12:0015:00 h). The experimenter (MM) brought each rat in its cage (in groups of three) to an experimental room in the animal facility and habituated each rat to her presence and to a paint brush inserted through the cage top for 3 days prior to the experimenta1 day. During the experiment for the SD and SR groups, she deprived each rat of sleep by gently touching it with the brush when it closed its eyes. She deprived and/or observed one rat at a time (on one day) and scored by visua1 observation its behavioura1 state every 20 s as comprised in the majority by wake, non-rapid eye movement sleep (non-REMS) or REMS. The behavioura1 scoring was based upon previous studies in the current lab (Maloney et al., 1997) together with demonstrations in other labs (Bergmann et al., 1987; Espana et al., 2003), showing that behaviour can be used in normal rats to reliab1y score the major states of W, non-REMS and REMS. Thus, as previously determined to correspond to polygraphically scored sleep states in our 1aboratory (Maloney et al., 1997), non-REMS was scored when the animal was recumbent with eyes closed and showing little or no movement and REMS when the animal was recumbent with eyes closed and showing rapid movements or twitches of the eyes, whiskers, muzzle, ears or paws. The deprivation procedure resulted in the total absence of sleep during 3 h for the SD and SR groups and was followed in the SR group by an increase in total sleep relative to both the SD and SC groups. At the end of the experimental period (15:00 h), the rats were immediately killed under pentobarbital anaesthesia (100 mglkg, Lp.) by intra-aortic perfusion with a fixative solution of 3% paraformaldehyde. Following immersion in a 30% sucrose solution, brains were frozen and stored at -80 oC. They were cut in coronal sections at 20 Ilm, which was determined to be the greatest thickness that would allow full penetration and staining with the antibodies employed. Adjacent series of sections were collected at 400-llm intervals and processed for double immunohistochemical staining using peroxidase-anti-peroxidase (PAP) for: (i) c-Fos (rabbit antiserum, 1 : 10 000, Ab-5, PC38, Oncogene Research Products, La JolIa, CA) with DAB-Ni as chromogen and (ii) Orexin (Orexin-A; C-19, goat antibody, 1 : 500, sc-8070, R230, Santa Cruz Biotechno10gy, Santa Cruz, CA) or MCR [MCR (E-16) goat antibody, 1 : 500, sc-14507, L281, Santa Cruz Biotechno10gy] with pink, a1pha-naphthol pyronin B. Other series were processed for triple immunostaining for: (i) c-Fos in the first position using DAB-Ni; (ii) (l(IKAR (goat purified antiserum, 1: 50, sc-1475, R310, Santa Cruz Biotechnology) or (l(2A-AR (goat purified antiserum, 1 : 50, sc-1478, K0702, Santa Cruz Biotechnology) in the second position using Cy3-conjugated donkey anti-goat antiserum (Jackson ImmunoResearch Laboratories, West Grove, PA) and (iii) Orx (Orexin A, rabbit antiserum, 1 : 2000, R261-3, Phoenix Pharmaceuticals, Belmont, CA) or MCR (rabbit antiserum, 1: 5000, Rl77-6, Phoenix Pharmaceuticals) in the third position using Cy2-conjugated donkey anti-rabbit antiserum (Jackson). Incubations with primary antibodies were performed at room temperature overnight for c-Fos, Orx and MCR antibodies and two nights at 4 oC for (l(IKAR and (l(2KAR antibodies using a Tris-saline solution (0.1 M) containing 1% normal donkey serum (NOS, following initial blocking with 3% NDS). Sections were viewed by light and fluorescence microscopy with a Leica DMLB microscope equipped with an x/y/z movementsensitive stage and video camera attached to a computer. Single-, double- and triple-immunostaining were evaluated for c-Fos, Orx or MCR and (l(IKAR or 1X2A-AR. Single-, double- and triple-laheUed cel!s were counted by applying stereology using the Optical Fractionator program of Stereo Investigator (2003, MicroBrightField, Williston, VT). Cells were sampled and counted within delineated contours at appropriate levels of a computer resident atlas (expanded from Gritti et al., 1993). On each side, a large contour was employed to include the nuclei in which the Orx neurons were located [lateral hypothalamus (LR), perifornical area (PF), and dorsomedial hypothalamus (DMR») or those in which the MCR neurons were located [LR, PF, DMH and zona incerta (ZI»). Partially overlapping through the hypothalamus, the Orx and MCR cells were distributed within these contours and nuclei over approximately 1600-1800 Ilm from anterior to posterior. Accordingly, celIs, single-, double- or triple-iabelled for c-Fos and/or peptide (Orx or MCR) and/or receptor «(l(IA-AR or a2A-AR) were sampled and counted bilaterally within the contours 'in three section-levels at 400-llm intervals from anterior to posterior. Within the stereology program, the sizes of the counting frame, within which cells are counted, and the grid, within which the counting frames are automatically placed, were optimized for each single-, double- or triple-labeUed series so that a suitable number of cells were counted per level in the deprived or recovery condition. Accordingly, the counting frame (125 x 125 Jlffi) and grid size (300 x 300 Jlffi or 350 x 350 Ilm) were set to sample ~13 or 17% of the area for single-labeIled c-Fos, Orx and MCR cell counts; the counting frame (140 x 140 Ilm) and grid size (200 x 200 Jlffi) were set to sample 49% of the area for counting double-labeIled c-Fos/Orx or c-FosIMCR ceUs; and the counting frame and grid size (both at 140 x 140 Ilm) were set to sample 100% of the area for double-labelled Orx or MCR and aIA-AR or a2A-AR cell counts as weIl as triple-labeUed profiles with c-Fos. The cells were counted under a 63x oil objective (with 1.4 numerical aperture). Nuclei or cells that came into focus beneath the surface of the section were counted within a counting block of 8 Jlffi in depth (in the dehydrated, delipidated, mounted and coverslipped sections that were on average 10 Jlffi thick). Sleep and cell counts were analysed between groups and across nuclei or regions using one- and two-way ANOVAS in Systat (vl0.2, Richmond, CA). AlI main effects were confirmed by non-parametric rank order tests (Kruskal-Wallis, P < 0.05) to insure that the distribution of variance in groups (containing zeros as a result of the experimental condition) did not distort the parametric statistics. Figures were composed using Adobe Photoshop Creative Suite (CS) and Adobe Illustrator CS (Adobe, San Jose, CA). Results Sleep deprivation and recovery Rats in the SD group did not sleep during the 3 h in the afternoon prior to kiIIing (at 15:00 h), whereas rats in the SR group that were allowed to recover sleep in the afternoon after 3 h of sleep deprivation in the © 2005 Federation of European Neuroscience Societies, European Journal of Neuroscience, 21, 2807-2816 Orx and MCR neurons 2809 moming (09:00-12:00 h) slept ~91% of the time prior to killing (at 15:00 h). The amount of sleep differed significantly across conditions of SD, SR and SC (Table 1). The major proportion of time for the SR and SC groups was spent in non-REMS (77.23 ± 2.94% in SR and 68.68 ± 0.10% in SC) and a minor proportion in REMS (13.58 ± 1.30% in SR and 7.10 ± 0.66% in SC, mean ± SEM of total time). c-Fos expression in Orx and MCH neurons after sleep deprivation and recovery c-Fos was expressed in neurons of the hypothalamus including Orx, MCR, non-Orx and non-MCR cells (Fig. lA and B). According to ANOVA with condition (and peptide series) as factors, total numbers of c-Fos-immunopositive (+) cells varied significantly across TABLE 1. Average percentage sleep and numbers of Orx and MCH cells expressing c-Fos under conditions of sleep control (SC), sleep deprivation (SD) and sleep recovery (SR) Variable SC Sleep (%) 75.75 c-Fos+ cells (n) Orx+ cells (n) c-Fos+/Orx+ cells (n) MCH+ cells (n) c-Fos+/MCH+ cells (n) 2535.67 of; 424.00 4888.75 of; 536.98 25.50 of; 25.50 10906.67 of; 1120.81 25.50 of; 14.72 SD of; 0.61 0.00 of; 0.00 11924.38 of; 1382.56 8042.00 of; 1421.88 675.75 of; 123.84 12711.75 of; 1430.25 38.25 of; 12.75 SD comparisons SR (SC,SR) 90.80 (SC,SR) 5187.88 of; 1112.65 7227.25 of; 397.04 142.50 of; 39.54 13212.00 of; 592.98 382.50 of; 60.70 (SC,SR) (SR) of; 2.47 SR comparisons F (Cond) (SC, SD) 1095.40"· (SD) 19.57*** 3.26 (ns) 20.54·" 1.21 (ns) 30.28*** (SD) (SC, SD) Values represent mean of; SEM for three groups of four rats (n = 12). Cell numbers were obtained from stereologica1 estimates for total numbers on left and right sides. Values across conditions (Cond) were compared by one- or two-way ANOVAS (with condition and peptide series as factors), followed by one-way ANOVAS for condition (with P < 0.001 indicated by ... in the table; ns, non significant) and posthoc pair-wise comparisons between conditions (using Fisher's LSD with p < 0.05 indicated for SD and SR with respect to SC, SR or SD). The estimated total numbers of c-Fos+ cells differed across conditions in both the Orx and MCH series and did not differ between (or in interaction with) the peptide series (df = 2, 1,2, 18) and are thus presented as the mean values for the two peptide series (n = 24). The numbers of Orx and MCH cells did not differ across (or in interaction with) conditions but did differ between each other, Orx cells (grand mean of; SEM, 6719 of; 622) being significantly fewerthan MCH cells (12 277 of; 649; F = 18.26, df = 1,2,9; P = 0.002). In a two-way ANOVA, the numbers of c-Fos+/Orx+ and c-Fos+/MCH+ neurons differed significantly as a function of condition (F = 18.3; df = 2, 9; P < 0.001), differed between the two peptide series (F = 6.56; df = l, 9; P = 0.03) and differed as a function of an interaction between condition x peptide (F = 25.62; df = 2, 9; P < 0.001). Given the significant interaction, one-way ANOVAS were performed for each series and presented in the table along with posthoc pair-wise comparisons. MCH Orx " ,-, , ~. f . '. f .- FIG. 1. c-Fos expression in Orx and MCH neurons. (A) Single-Iabelled c-Fos+ cell (stained black with DAB-Ni, black arrowhead), single-Iabelled Orx+ cell (stained pink with alpha-naphthol pyronin B, white arrowhead) and double-Iabelled c-Fos+/Orx+ cells (double arrowheads). (8) Single-Iabelled c-Fos+ cell (stained black, black arrowhead), single-Iabelled MCH+ cells (stained pink, white arrowheads) and double-Iabelled c-Fos+/MCH+ cell (double arrowhead). (C) Orx cell distribution in the LH, PF and DMH (stained brown with DAB). (D) MCH cell distribution in the LH, PF, DMH and ZI (stained brown). f, fornix. Magnification bars, 20 !lm (A and B); 1 mm (C and D). © 2005 Federation of European Neuroscience Societies, European Journal of Neuroscience, 21,2807-2816 2810 M. Modirrousta et al. conditions (independent of peptide series) being highest in the SD condition (Table 1). Total numbers of Orx- and MCH-immunopositive neurons did not vary significantly as a function of condition. The Orx+ neurons, which are distributed across the LH, PF and DMH (Figs IC and 2A) numbered on average ::::::6700 (Table 1). MCH neurons, which are distributed across LH, PF, DMH and ZI (Figs ID and 2D) numbered on average R::12 300 and were significantly more numerous than the Orx cells (Table 1). The numbers of c-Fos+/Orx+ and c-Fos+/MCH+ cells varied significantly across conditions but also varied significantly as a function of an interaction between condition and peptide (Table 1). Numbers of c-Fos+/Orx+ cells were significantly decreased' in SR as compared to SD (Figs 2B and C, and 3C; Table 1), whereas numbers of c-Fos+/MCH+ cells were significantly increased in SR as compared to SD (Figs 2E and F, and 3F; Table 1). Numbers of c-Fos+/MCH+ cells were also significantly higher in SR as compared to SC (Table 1). Across the SD and SR groups, the numbers of c-Fos+/Orx+ cells were negatively correlated with the numbers of c-Fos+/MCH+ cells (correlation coefficient for pairwise values, r = -0.82, n = 8, P = 0.01). Relative to all c-Fos+ cells in SD, the c-Fos+/Orx+ cells represented a small proportion (R::6%, Figs 2B, and 3B and C). They also represented a small proportion of the Orx cell population (R::9%, Figs 2A and B, and 3A and C). Similarly, relative to all c-Fos+ cells in SR, the c-Fos+IMCH+ cells represented a small proportion (R::6%, Figs 2F, and 3E and F). They also represented a small proportion of the MCH cell population (3%, Figs 2D and F, and 3D and F). SO Adrenergic receptors on Orx and MCH, including c-Fos expressing, neurons after sleep deprivation and recovery Immunostaining for adrenergic receptors (cxIA-AR or CX2KAR), peptide (Orx or MCH) and c-Fos was performed to examine the incidence of cxIA-AR or CX2A-AR on Orx and MCH cells and also on Orx and MCH cells expressing c-Fos in the ditTerent conditions. Orx cells were immunostained for both types of adrenergic receptors, R:: 1100 for CXIA-AR (Fig. 4A) and R:: 1400 for <X2A-AR (Fig. 4B; Table 2). Orx cells bearing the ditTerent ARs were distributed across the LH, PF and DMH with no ostensible segregation (Fig. 5A and B). Very few MCH neurons were immunostained for <xIKAR (R::160 having minimal positive staining, Fig. 4C), whereas a large number were immunostained for <X2A-AR (R::1800, Table 2; Fig. 4D). MCH cells immunopositive for <X2A-AR were distributed across the LH, PF, DMH and ZI (Fig. 5D). The numbers of Orx and MCH ARimmunopositive cells ditTered significantly between the two peptide cell types (Table 2). There was a significantly higher prevalence of Orx+/<XIA-AR+ cells than MCH+/<xIKAR+ cells. Moreover, whereas there was no significant ditTerence between the numbers of <XIAAR+ vs. CX2KAR+ Orx cells, there was a significant ditTerence between the numbers of <XIA-AR+ vs. CX2A-AR+ MCH cells (Table 2). Indeed, R::16% of Orx cells bore <XIA-AR and R::21 % bore <X2A-AR, whereas only R::l.5% ofMCH cells bore <XIA-AR and R::15% bore CX2AAR. The numbers of AR-immunopositive Orx and MCH cells did not vary significantly as a firnction of condition (Table 2). Analysis of the triple-immunostaining revealed sorne c-Fos expressing Orx cells in SD SR Orx+cells • c-Fos+ cells • c-Fos+/Orx+ cells -Orx MCH+celis • c-Fos+ cells • c-Fos+/MCH+ cells MCH FIG. 2. Distribution of Orx and MCH neurons expressing c-Fos after SD or SR. (A) Orx cell distribution in the LH, PF and DMH (open triangles). (B and C) Double-Iabelled c-Fos+/Orx+ cells (filled triangles) distinguished from single-labelled c-Fos+ cells (black dots) in one SD (B) and one SR (C) representa-' tive brain. (0) MCH cell distribution in the LH, PF, DMH and ZI (open squares). (E and F) Double-Iabelled c-Fos+/MCH+ cells (filled squares) distinguished from single-Iabelled c-Fos+ cells (black dots) in one SD (E) and one SR (F) representative brain. f, fomix; DMH, dorsomedial hypothalamus; LH, lateral hypothalamus; PF, perifornical area; ZI, zona incerta. Maguification bar, 1 mm. © 2005 Federation of European Neuroscience Societies, European Journal of Neuroscience, 21, 2807-2816 Orx and MCH neurons A 2811 B t t lJ c;; :;, ! + 8 'il ~ 0 o + ~ 6 sc SR SO SR E MCH Condition SO SR Condition FIG. 3. Numbers of Orx and MCH neurons expressing c-Fos after SD or SR. (A) Total numbers ofOrx+ ceUs counted bilaterally and estimated by stereological analysis in the hypothalamus including LH, PF and DMH. (8) Total numbers of c-Fos+ ceUs (obtained from c-Fos/Orx dual-immunostained series as the total of single c-Fos+ and double c-Fos+/Orx+ cells) were significantly decreased in SR as compared to SD (Table 1). (C) Total numbers of c-Fos+/Orx+ ceUs were significantly decreased in SR as compared to SD (Table 1). (0) Total numbers of MCH+ ceUs counted and estimated by stereological analysis in the hypothalamus including LH, PF, DMH and ZI. (E) Total numbers ofc-Fos+ ceUs (obtained from c-Fos/MCH dual-immunostained series as the total of single c-Fos+ and double c-Fos+/MCH+ cells) were significantly decreased in SR as compared to SD (Table 1). (F) Total numbers ofc-Fos+/MCH+ cells were significantly increased in SR as compared to SD (Table 1). Note the sarne scale in A, B, D and E (0-20000) forsingle-labeUed ceUs and the smallerscale in C and F (0-1000) for double-labeUed cells. that bore (XIKAR and some that bore (X2A-AR (Fig. 4E and F). No c-Fos expressing MCH neurons in SR were found to be immunopositive for (XIA-AR, whereas sorne were found to be immunopositive for (X2A-AR (Fig. 4G). Discussion Parallel with a large population of neurons in the posterior hypothalamus, more Orx cells express c-Fos after sleep deprivation than after sleep recovery, whereas more MCH ceUs express c-Fos after sleep recovery. This reciprocal pattern of activity could be explained in part by different adrenergic receptors and thus response to NA in the Orx and MCH neurons. c-Fos differentially expressed in Orx and MCH neurons as a function of sleep A large population ofneurons in the posterior hypothalamus expressed c-Fos following 3 h of total sleep deprivation. As a refiection of neural activity, the c-Fos expression thus confirms unit recordings showing the maximal discharge rate by the vast majority of neurons in this region to be during waking as compared to non-REMS (Steininger et al., 1999; Alam et al., 2002; Koyama et al., 2003). In parallel with the major population of hypothalamic neurons, more Orx neurons also expressed c-Fos followirtg sustained waking than following recovery or control sleep. These results confirm microdialysis studies showing that Orx release is high in association with waking and 10W in association with sleep (Fujiki et al., 2001; Yoshida et al., 2001; Zeitzer et al., 2003). However, the percentage of c-Fos+ cells that wete Orx+ in the present study was very small (~5%) indicating that the Orx cells were not the major contributors in this region to the maintenance of the waking state. Moreover, the percentage of Orx+ ceUs that were c-Fos+ was also very small (~10%), indicating that the Orx cells were not maximally activated for the maintenance of the waking state under the present conditions. In fact, the experimental conditions were intended to deprive rats of sleep without stress and accordingly employed behavioural obseIVations for sleep-wake state scoring to avoid the surgery and tethering needed for polygraphic recording, maintained the animais in their home cages with food and water ad libitum to avoid any alimentary deprivation and used gentle touching with a soft brush when necessary to avoid continuous or stressful stimulation and evoked activity during the day. The results suggest that although other neurons in the posterior hypothalamus are active in association with quiet waking and thus potentially involved in maintaining cortical activation of the waking state, the Orx neurons may only be active with more aroused or stressful waking. In another study, it was found that Orx neurons expressed c-Fos in large numbers during the day only in association with high-arousal and stressful waking induced by continuous auditory stimulation (Espana et al., 2003). Selective deprivation of REM sleep was also found to be associated with increased c-Fos expression in Orx neurons (Verret et al., 2003), perhaps due to stress that may be associated with that procedure. Orx release has also been © 2005 Federation of European Neuroscience Societies, European Journal ofNeuroscience, 21, 2807-2816 2812 M. Modirrousta et al. FIG. 4. Adrenergic receptors (AR) on Orx and MCH neurons including those expressing c-Fos. (A) Cell immunostained for Orx (AI) and IXIA-AR (A2 and in superimposed image in A3). (B) Cell immunostained for Orx (BI) and 1X2A-AR (B2 and B3). (C) Cell immunostained for MCH (CI) that was minima1ly stained for IXIA"AR (C2 and C3). (D) Cells immunostained for MCH (DI) ofwhich one was also stained for 1X2A-AR (D2 and D3). (E) Labelled Orx+ cell (El) that was IXIA-AR+ (E2) and c-Fos+ (E3). (F) Labelled Orx+ cell (FI) that was 1X2A-AR+ (F2) and c-Fos+ (F3). (G) Labelled MCH+ cell (G 1) that was 1X2A-AR+ (G2) and c-Fos+ (G3). Peptides were stained by green fluorescence with Cy2; receptors were stained by red fluorescence with Cy3 and c-Fos by black DAB-Ni. Magnification bar, 20 Iffil. © 2005 Federation of European Neuroscience Societies, European Journal of Neuroscience, 21, 2807-2816 Orx and MCH neurons TABLE 2. Average number of Orx or MCH cens that were immunostained for ~IKAR or ~2A-AR under conditions of Sc., SD and SR Variable SC SD SR Mean Orx+/~I-AR+ 1102.75 ± 122.74 1058.33 ± 456.97 1325.00 ± 450.69 1084.56 ± 179.41 1531.75 ± 297.58 1133.33 ± 475.29 1735.00 ± 761.07 1406.33 ± 290.04 213.25 ± 48.48 166.67 ± 87.00 141.67 ± 117.56 161.44 ± 44.38 1479.25 ± 170.93 2300.00 ± 478.93 1933.33 ± 147.43 1818.56 ± 196.29 Orx+/~- AR+ MCH+htl- AR+ MCH+/~- AR+ 2813 F-value (Condition) F-value (AR) F-value (Condition 0.29 (ns) 1.95 (ns) 0.27 (ns) 2.40 (ns) 81.64** 1.86 (ns) X AR) Values represent mean ± SEM for three groups of three or four rats (n = 10). Cell numbers were obtained from stereological estimates for total numbers on left and right sides. Values of each peptide series (Orx and MCH) were analyzed by two-way ANOVA for condition and adrenergic receptor subtype (AR). Total numbers of Orx and MCH neurons with IXIA-AR or OI2A-AR did not vary significantly as a function of condition (ns, not significant). Whereas the number of neurons immunostained for OIIA-AR vs. OI2A-AR did not differ for the Orx neurons, it did differ for the MCH neurons (**P < 0.01). alAR a2AR '* Orx+/alAR+ cells Orx+/a2AR+ cells Orx '* MCH+/alAR+ cells MCH+/a2AR+ cells MCH FIG. 5. Distribution of OII-AR vs. 0I2-AR immunopositive Orx and MCH neurons. (A) Distribution of OIIA -AR+/Orx+ cens (stars) and (B) OI2A-AR+/Orx+ cens (circles) across LH, PF and DMH. (C) Distribution of OIIA-AR+/MCH+ cens (stars) and (D) OI2A-AR+/MCH+ cells (circles) across LH, PF, DMH and ZI. For abbreviations see Fig. 2. Magnification bar, 1 mm. found to be maximal in association with motor activity (Kiyashchenko et al., 2002; Zeitzer et al., 2003). Effects of intracerebroventricular (lCV) administration of Orx and loss of function in Orx knock out mice moreover indicate that Orx can stimulate energy metabolism along with motor activity by positive influences upon the sympathetic nervous system (stimulating increased heart rate, blood pressure, temperature and thermogenesis), the hypothalamo-pituitary thyroid axis (HPT, stimulating increased basal metabolic rate) and the hypothalamo-pituitary adrenal axis (HPA, stimulating increased corticosteroid levels) (Lubkin & Stricker-Krongrad, 1998; Shirasaka et al., 1999; Ida et al., 2000; Hara et al., 2001; Espana et al., 2002; Monda et al., 2003; Yamanaka et al., 2003). An integral role in stimulating arousal, activity and metabolism can be mediated by excitatory influences of Orx upon multiple central arousal systems, including noradrenergic locus coeruleus neurons and cholinergic brainstem and basal forebrain neurons (Horvath et al., 1999; Bayer et al., 2001; Eggerrnann et al., 2001; Burlet et al., 2002), upon central motor and sympathetic systems (peyron et al., 1998; van den Pol, 1999; Krout et al., 2003; Yamuy et al., 2004) and upon hypothalamic neurons (van den Pol et al., 1998; Ferguson & Samson, 2003). In contrast to the Orx ceUs, MCH ceUs showed more c-Fos activation after sleep recovery than after sleep deprivation. c-Fos expression was specifically associated with recovery sleep, when animais slept ~90% of the time and not with normal sleep in the control condition, when animais slept ~75% of the time. These results suggest that the c-Fos activation is associated with the recovery process and not simply with natural sleep. Nonetheless, the percentage of MCH neurons that expressed c-Fos during recovery foUowing 3 h of gentle sleep deprivation was very small « 5%). In another study examining c-Fos expression following 72 h ofparadoxical sleep (PS) deprivation © 2005 Federation of European Neuroscience Societies, European Journal of Neuroscience, 21, 2807-2816 2814 M. Modirrousta et al. upon inverted flower pots, it was found that a large proportion ofMCR neurons (> 50%) expressed c-Fos during recovery, when animaIs slept 77% of the time, comprised by 33% SWS and 44% paradoxical sleep (Verret et al., 2003). In our paradigm, the recovery sleep was comprised in greatest proportion by non-REMS sleep (estimated at ~77% as compared to 14% REMS), and the percentages ofboth non-REMS and REMS were significantly increased above control (estimated at ~69% and 7%, respectively). Accordingly, the increased c-Fos expression in MCR cells could be explained by the predominance of non-REMS in SR or by a recovery process afforded by total sleep that includes unimpeded non-REMS and REMS along with the loss of postural muscle tone. MCR cells may thus become activated only when there is an increased need as well as possibility for the behavioural inactivity and rest that is pennitted by non-REMS and REMS sleep. Based upon evidence ofMCR administration or MCR knock out, it has been shown that MCR decreases energy metabolism along with activity through a negative influence upon the sympathetic nervous system, the hypothalamo-pituitary thyroid (HPT) axis and the hypothalamo-pituitary adrenal (RPA) axis (Shimada et al., 1998; Kennedy et al., 2001; Marsh et al;, 2002; Ito et al., 2003; Shearman et al., 2003; Zhou et al., 2005). MCR neurons could thus play a role in opposition to Orx neurons by decreasing activity and energy metabolism in association with sleep through inhibition of transmission in the hypothalamus and other arousal systems (Bittencourt et al., 1992; van den Pol et al., 1998; Gao & van den Pol, 2001). Adrenergic receptors differentia/ly distributed on Orx and MCH including c-Fos expressing neurons c-Fos expression was inversely related in Orx and MCR neurons across the deprived and recovery conditions in the present study suggesting a reciprocal relationship in their activity and roles. Orx and MCR cells are partially overlapping in their distribution within the hypothalamus and have reciprocal synaptic relationships, which could mediate reciprocal profiles of activity (Bayer et al., 2002; Guan et al., 2002). It is also possible that they would be under reciprocal influences by other activating systems involved in behavioural state and energy regulation, such as the noradrenergic systems, including importantly the locus coeruleus neurons (Jones & Yang, 1985). It could thus be expected that Orx neurons would bear <Xl-AR, associated with depolarizing responses to NA as present upon cholinergic basal forebrain neurons, whereas MCR neurons would bear <xrAR, associated with hyperpolarizing responses to NA as present upon GABAergic sleep-active basal forebrain neurons (Fort et al., 1995; Fort et al., 1998; Modirrousta et al., 2004). Many Orx neurons were found to be immunostained for the <XIAAR, although unexpectedly many were also inImunostained for the <X2A-AR. In contrast, MCR ceUs were almost exclusively immunostained for the <X2A-AR. The difference in the incidence of <XIA-AR on Orx vs. MCR cells explains one mechanism by which Orx neurons may be stimulated, whereas MCR neurons would be inhibited during waking when noradrenergic locus coeruleus neurons are active (Aston-Jones & Bloom, 1981). The MCR neurons may become active when released from inhibition by noradrenergic input with sleep onset. These conclusions were recently substantiated in electrophysiological studies using slices from rat brain, in which NA was found to have an excitatory effect upon Neurobiotin-labeUed Orx ceUs and to have an inhibitory effect upon Neurobiotin-labeUed MCR ceUs (Bayer et al., 2005). On the other hand, the present finding that sorne Orx neurons bear <X2A"ARs suggests that sorne Orx ceUs may be inhibited by NA. In fact, in transgenic mice expressing GFP in Orx neurons, NA was found to have a hyperpolarizing effect upon the GFP-IabeUed Orx ceUs in vitro (Li et al., 2002) and most recently it was discovered that the excitatory effect of NA on Orx ceUs in the rat slice was transfonned into a hyperpolarizing or biphasic effect after two hours of sleep deprivation (Grivel et al., 2004). It is thus .possible that under certain circumstances in mice and rats, Orx ceUs may mobilize or express <Xz-ARs. In the present experiment, the numbers of ceUs bearing <XlA"ARs or <X2A"ARs did not vary as a function ofsleep-wake condition, thus indicating that mild sleep deprivation or recovery in adult rats does not appear to alter the expression of these receptors. On the other hand, sleep deprivation in younger rats (~15-20 days), as employed in the in vitro studies, would be associated with stress, as indicated by elevated corticosterone (Rairston et al., 2004), and stress or corticosteroids have been shown to be associated with changes in the expression of <xrARs (Jhanwar-Uniyal et al., 1986; Flugge et al., 2003). Thus, the influence ofnoradrenergic input from locus coeruleus or other brainstem cell groups may under nonnal conditions be predominantly excitatory upon Orx neurons through <XI-ARs, however, change foUowing stress to become predominàntly inhibitory upon them through increased expression or mobilization of <XrARs. This dual potential may aUow adaptive changes under conditions of stress, when sleep along with energy conservation, instead of sustained waking along with energy expenditure, could enhance survival. ln summary, the present results show that Orx and MCH neurons are active in a reciprocal manner during sleep deprivation and sleep recovery such as to suggest that they play opposing roles in regulating sleep-wake states along with activity and energy metabolism. These opposing roles may in part be mediated by reciprocal relationships or by differential responses to afferent inputs including importantly noradrenergic inputs. Acknowledgements The research was supported by a grant from the Canadian Institutes of Health Research (13458). 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