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Sleep Research Online 2(3): 79-82, 1999 http://www.sro.org/1999/Datta/79/ Printed in the USA. All rights reserved. 1096-214X © 1999 WebSciences Brainstem Afferents of the Cholinoceptive Pontine Wave Generation Sites in the Rat Subimal Datta, Elissa H. Patterson and Donald F. Siwek Sleep Research Laboratory, Department of Psychiatry, Boston University School of Medicine, Boston, Massachusetts 02118, USA, The present study was designed to investigate the distribution of brainstem neurons projecting to the pontine wave (Pwave)-generating sites in the rat. In six rats, biotinylated dextran amine (BDA) was microinjected into the physiologically identified cholinoceptive P-wave generation site. In all cases, microinjections of BDA in the cholinoceptive P-wave generating site resulted in retrograde labeling of cell bodies in many parts of the brainstem. The majority of those retrogradely labeled cells were in the pedunculopontine tegmentum, pontine reticular nucleus oralis, parabrachial nucleus, vestibular nucleus, and gigantocellular reticular nucleus. The results presented in this study provide anatomical evidence that the cholinoceptive P-wave generation site in the rat receives anatomical projections from other parts of the brainstem known to be involved in the REM sleep-generation mechanism. CURRENT CLAIM: Distribution of brainstem neurons projecting to the P-wave generator in the rat. A group of neurons in the pontine tegmentum generates a prominent field potential just prior to the onset of and throughout REM sleep (Datta and Hobson, 1994, 1995; Datta et al., 1998). These field potentials have been recorded from the pons, lateral geniculate body, and occipital cortex. Since, in the cat, these potentials originate in the pons (P) and propagate to the lateral geniculate body (G) and occipital cortex (O), they are called ponto-geniculo-occipital (PGO) waves (Brooks and Bizzi, 1963; Jeannerod et al., 1965). These field potentials have also been recorded from many other parts of the brain which receive excitatory inputs from the P-wave-generation site (Datta, 1997). The P-wave in the rat is equivalent to the pontine component of the PGO waves in the cat (Marks et al., 1980; Sanford et al., 1995; Datta et al., 1998). In the rat, these field potentials are absent in the lateral geniculate body (LGB) due to the lack of afferent inputs from P-wave-generating cells (Stern et al., 1974; Datta et al., 1998). Since these field potentials are absent in the LGB of the rat, in this report we call these field potentials P-waves rather than pontine PGO waves. In addition to REM sleep induction, PGO waves have been implicated in several other important brain functions such as sensorimotor integration, learning, memory, cognition, dreaming, self-organization, development of the visual system, visual hallucination and startle responses (reviewed in Datta, 1997). In our earlier studies we localized cholinoceptive Pwave generation sites both in the cat and rat (Datta et al., 1992, 1998). More recently we have identified brain regions receiving efferent projections from the physiologically identified P-wave generation sites in the rat (Datta et al., 1998). On the basis of this efferent mapping study, we have produced anatomical evidence indicating that the P-wave generating cells are involved in sleep-dependent memory consolidation. In the present study, we investigated the distribution of brainstem neurons projecting to the P-wave generating sites in the rat. Identification of afferent inputs potentially may identify other brain regions that are capable of modulating P-wave generating cells and P-wave production. Although PGO-wave generating sites in the cat have been mapped in previous studies (Quattrochi et al., 1998), this is the first study to our knowledge that maps afferents of functionally identified Pwave generating sites in the rat. METHODS Experiments were performed on 6 male Sprague-Dawley rats (Charles River, Wilmington, MA) weighing between 200 and 300 gm. Animals were deeply anesthetized (chloral hydrate, 400 mg/kg, i.p.) and placed in a stereotaxic apparatus (Kopf, model 1730). A scalp incision was made, the skin retracted, and a hole was drilled in the skull overlying the cholinoceptive P-wave generation site (stereotaxic, AP: -0.50; L: 1.25; H: 2.0) as identified earlier (Datta et al., 1998). A single carbachol microinjection (50 ng in 50 nl saline solution) was made within the cholinoceptive pontine P-wave generation zone as described earlier (Datta et al., 1998). These microinjections were made to positively identify the cholinoceptive pontine P-wave generation site. Following positive identification, using the same chemitrode, these cholinoceptive pontine P-wave generation sites were microinjected with 50 nl of 4% Biotinylated Dextran Amine in saline (BDA, Molecular Probes, Inc., Eugene, OR). The scalp skin incisions were then sutured and the animals were allowed to recover from anesthesia. Correspondence: Subimal Datta, Ph.D., Sleep Research Laboratory, Department of Psychiatry, Boston University School of Medicine, M 934, 715 Albany St., Boston, MA 02118, USA, Tel: 617-638-5863, Fax: 617-638-5862, E-mail: [email protected]. 80 DATTA ET AL. from a Vectastain kit (Vectastain, ABC Elite Kit, Vector Labs., Inc., Burlingame, CA). On the following day, the sections were rinsed well in 5 changes of PBS and then reacted in a DAB reagent kit (DAB-Plus reagent set; Zymed Laboratories, San Fancisco, CA) for approximately 25 minutes. The progress of this reaction was monitored under the microscope to prevent excess non-specific staining. Sections were rinsed well in several changes of PBS, mounted onto chrom-alum subbed glass slides and allowed to air dry. Slides were then sequenced through distilled water for 4 minutes, 70% ethyl alcohol for 3 minutes, two washes of 95% ethyl alcohol for 3 minutes each, two two-minute washes of 100% ethyl alcohol, a four-minute Histoclear wash, and a final Histoclear wash. From the final Histoclear wash, the slides were coverslipped using Permount. All consecutive sections were processed for quantitative analysis, except in two cases, in which one in every four sections was counterstained with cresyl violet to demonstrate nuclear boundaries. All sections were examined microscopically under light field illumination. Using camera lucida tracings of these sections and with the aid of a rat brain atlas (Paxinos and Watson, 1986), brainstem nuclear divisions were identified. Cells retrogradely labeled with BDA are identified by the dark brown color in the surroundings of pale unstained cells (see Fig. 1). These boundaries and the position of labeled cells and injection sites were plotted on the drawings. Following microscopic examination, retrogradely labelled cell counts were obtained from the plots for well-known groups of neurons in the brainstem. Counts from the two sides of the brain were combined. Combined mean and standard deviations were calculated from the six individual cases. RESULTS Figure 1. Digitized brightfield photomicrographs (Optimas, Bioscane) showing examples of neurons that are retrogradely labeled after injection of BDA into the cholinoceptive P-wave generation site. (A) Labeled neurons in the laterodorsal tegmentum. (B) Labeled neurons in the vestibular nucleus. (C) Labeled neurons in the gigantocellular reticular nucleus. Scale bar = 100 micrometer. After ten days, rats were deeply re-anesthetized with chloral hydrate, and their brains were fixed by transcardial perfusion of approximately 60 ml 0.9% saline containing 1% sodium nitrite followed by 200 ml 4% paraformaldehyde and 0.25% gluteraldehyde in 0.1M phosphate buffer (pH of 7.4). The brains were then removed and cut in serial sections of 50 micrometer thickness with a vibratome. These sections were treated to reveal the presence of BDA as follows. Sections were first rinsed in three changes of phosphate buffered saline (PBS), followed by immersion in 0.3% hydrogen peroxide for 15 minutes to remove endogenous peroxidases and to lyse red blood cells. Sections were then carried through four rinses in PBS or until small bubbles were no longer present. Sections were then incubated overnight at 4°C in a solution of 10 ml Triton-X, 20 ml PBS, and 8 drops each of solution A and B All injections were placed within the stereotaxic coordinates of antero-posterior -0.30 to -0.80, lateral 1.1 to 1.4, and dorsoventral 1.9 to 2.3 (Paxinos and Watson, 1986). Histological identification showed that microinjections were made in the dorsal part of the nucleus subcoeruleus. The surrounding anatomical landmarks of this cholinoceptive pontine generator are: locus coeruleus, dorsally, the caudal pontine reticular nucleus, ventrally, and the dorsomedial tegmental area and ventral nucleus subcoeruleus, medially, and the mesencephalic trigeminal and motor trigeminal nucleus, laterally. In all cases, small volume injections (50 nl) of BDA in the cholinoceptive PGO generating sites resulted in retrograde labeling of cells in many regions in the brainstem (see Fig. 2). A slightly greater number of labeled cells was located ipsilateral to the injection sites compared to the contralateral side, except for midline structures. The location and extent of BDA injection site diffusion was determined histologically by identifying the tip of the track left by the injection cannula and assessing the spread of the dark brown BDA reaction product. The area of BDA diffusion at the injection sites ranged from 50 to 100 micrometer in diameter. The total number of BDA-labeled brainstem cells projecting to the functionally identified P-wave generation sites ranged between 889 and 941, with a mean of 917±24.32 (S.D.). A high number of retrogradely labeled cells were in the 81 PONTINE WAVE AND AFFERENT CONNECTIONS A. B. Figure 2. Distribution of labeled neurons in the brainstem. Schematic representation of selected coronal brainstem sections illustrating the distribution of labeled neurons (each dot represents one BDA labeled neuron) after a small injection of BDA into the cholinoceptive P-wave generation site. The number in the upper left corner of each section indicates the rostrocaudal distance from interaural line. (2A) Rostral brainstem and (2B) caudal brainstem. Abbreviations: 3, oculomotor nucleus; 7, facial nucleus; 7n, facial nerve or its root; 8vn, vestibular root vestibulocochlear nucleus; Aq, aqueduct; BIC, nucleus brachium inferior colliculus; CnF, cuneiform nucleus; CP, cerebral peduncle, basal; ctg, central tegmental tract; DC, dorsal cochlear nucleus; DR, dorsal raphe; Gi, gigantocellular reticular nucleus; IC, inferior colliculus; icp, inferior cerebellar peduncle; IO, inferior olive nucleus; IP, interpeduncular nucleus; LC, locus coeruleus; LDT, laterodorsal tegmental nucleus; LL, lateral lemniscus; LPGi, lateral paragigantocellular nucleus; LVe, lateral vestibular nucleus; M5, motor trigeminal nucleus; mcp, middle cerebellar peduncle; ml, medial lemniscus; mlf, medial longitudinal fasciculus; MR, median raphe nuclei; MVe, medial vestibular nucleus; PB, parabrachial area; pd, predorsal bundle; PDT, posterodorsal tegmental nucleus; Pn, pontine nuclei; PnC, pontine reticular nucleus, caudal; PnO, pontine reticular nucleus, oral; PPT, pedunculopontine tegmental nucleus; Pr5, priciple sensory trigeminal nucleus; PrH, prepositus hypoglossal nucleus; Py, pyramidal tract; Rm, raphe magnus nucleus; ROb, raphe obscurus nucleus; RTP, reticulotegmental nucleus pons; RVL, rostroventrolateral reticular nucleus; S5, sensory root trigeminal nerve; SC, superior colliculus; scp, superior cerebellar peduncle; SO, superior olive nucleus; Sol, nucleus solitary tract; Sp5, spinal trigeminal tract; Sp5n, spinal trigeminal nucleus; SpVe, spinal vestibular nucleus; SubC, subcoeruleus nucleus; SuVe, superior vestibular nucleus; VC, ventral cochlear nucleus; VTg, ventral tegmental nucleus; Xscp, decussation superior cerebellar peduncle. pedunculopontine tegmentum (PPT, 117.83±7.47), pontine reticular nucleus oralis (PnO, 115±11.14), parabrachial area (PB, 98.83±9.74), vestibular nucleus (Ve, 120.83±8.38), and gigantocellular reticular nucleus (Gi, 108.83±14.48). Within the vestibular nucleus, the majority of those retrogradely labeled cells were in the superior part (SuVe, 53.78%) and the medial part (MVe, 34.45%). Only a small percentage of retrogradely labeled cells were in the lateral (LVe, 7.57%) and spinal part (SpVe, 4.20%) of the vestibular nucleus. A moderate number of retrogradely labeled cells were in the central gray (CG, 39.67±6.86), raphe group nucleus (RN, 40±6.75), laterodorsal tegmentum (LDT, 37.83±12.22), locus coeruleus (LC, 73.33±5.72), subcoeruleus nucleus (SubC, 78.83±9.26), and parvocellular reticular nucleus alpha (PRtA, 39.83±7.08). Of those retrogradely labeled cells in the raphe group, almost half of them were in the dorsal raphe nucleus (DR, 51.16%), and the other half were distributed in the median raphe nucleus (MR, 23.26%), and raphe magnus nucleus (RM, 25.58%). There were no labeled cells in the raphe obscurus nucleus. Some labeled cells were in the central (3.67±2.50) and dorsal (11.50±4.09) tegmental area (ctg and dtg) and in the cuneiform nucleus (CnF, 13±3.74). Even fewer labeled cells were found in the pontine reticular nucleus caudalis (PnC, 5.33±3.33) and facial nucleus (7, 6.67±3.98). DISCUSSION The results presented in this study provide anatomical evidence that the cholinoceptive P-wave generation site in the rat receives anatomical projections from other parts of the brainstem known to be involved in the REM sleep-generation mechanism. We chose to use local microinjection of carbachol to identify P-wave generation sites, because in the past this approach was successfully used to map P-wave generation sites in the cat and rat (Datta et al., 1992, 1998). In this study, BDA was used as the retrograde tracer because it has been shown to be one of the most effective retrograde tracers to study anatomical pathways (Rajkumar et al., 1993). 82 DATTA ET AL. The present study showed that the P-wave generation site receives projections from the PPT, LDT, and CnF. Immunohistochemical studies have shown that the majority of cells in the PPT, LDT, and CnF of rats are cholinergic, suggesting that the P-wave generation site may receive cholinergic inputs (Mesulam et al., 1983; Rye et al., 1987). Besides cholinergic inputs, the P-wave generation site also receives anatomical projections from LC, a nucleus which contains noradrenergic cells and RN, a nucleus which contains serotonergic cells (Swanson, 1976; Grzanna and Molliver, 1980; Steinbusch and Nieuwehuys, 1983). Inputs from both cholinergic and aminergic cells to the P-wave generation site are significant, because they provide anatomical evidence for a recent model of REM sleep generation which proposes that the activation of brainstem cholinergic cells and inactivation of aminergic cells activates individual REM sleep sign generators (Datta, 1995). In the present study, we have shown that many cells in the vestibular nucleus project to the P-wave generation site. Earlier lesion studies have demonstrated that the vestibular nucleus is involved in the generation of clustered PGO waves (Morrison and Pompeiano, 1966). Vestibular nucleus cells projecting to the P-wave generation site provide anatomical evidence of the involvement of the vestibular system in the generation of clustered PGO waves (Morrison and Pompeiano, 1966). This study has demonstrated that cells in the PnO and PB project to the P-wave generation site. It is known that the REM sleep events like hippocampal theta waves and autonomic fluctuations are generated by PnO and PB cells (Vertes et al., 1993; Datta, 1995, 1997). In this study, we have also demonstrated that the P-wave generation site receives projections from Gi cells. In the past, other studies have shown that the activation of Gi induces REM sleep sign EMG atonia (Sakai et al., 1981; Chase et al., 1986; Lai and Siegel, 1988, 1992). These data together indicate that the individual REM sleep sign generators are interconnected. Understanding these interconnections may help to understand the coordinated actions that lead to the state of REM sleep. ACKNOWLEDGMENTS This research was supported in part by National Institutes of Health Grant #NS34004. REFERENCES 1. Brooks DC, Bizzi E. Brain stem electrical activity during deep sleep. Archives Italiennes de Biologie 1963; 101: 648-65. 2. Chase MH, Morales FR, Boxer PA, Fung SJ, Soja PJ. Effect of stimulation of the nucleus reticularis gigantocellularis on the membrane potential of cat lumber motoneurons during sleep and wakefulness. Brain Research 1986; 386: 237-44. 3. Datta S. Neuronal activity in the peribrachial area: relationship to behavioral state control. 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