* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Orexin (Hypocretin)-Like Immunoreactivity in the Cat Hypothalamus
Biochemistry of Alzheimer's disease wikipedia , lookup
Neuroplasticity wikipedia , lookup
Aging brain wikipedia , lookup
Biological neuron model wikipedia , lookup
Electrophysiology wikipedia , lookup
Holonomic brain theory wikipedia , lookup
End-plate potential wikipedia , lookup
Single-unit recording wikipedia , lookup
Artificial general intelligence wikipedia , lookup
Neuromuscular junction wikipedia , lookup
Multielectrode array wikipedia , lookup
Neural oscillation wikipedia , lookup
Neural coding wikipedia , lookup
Axon guidance wikipedia , lookup
Metastability in the brain wikipedia , lookup
Endocannabinoid system wikipedia , lookup
Mirror neuron wikipedia , lookup
Caridoid escape reaction wikipedia , lookup
Neurotransmitter wikipedia , lookup
Nonsynaptic plasticity wikipedia , lookup
Stimulus (physiology) wikipedia , lookup
Neural correlates of consciousness wikipedia , lookup
Development of the nervous system wikipedia , lookup
Activity-dependent plasticity wikipedia , lookup
Anatomy of the cerebellum wikipedia , lookup
Central pattern generator wikipedia , lookup
Apical dendrite wikipedia , lookup
Molecular neuroscience wikipedia , lookup
Sexually dimorphic nucleus wikipedia , lookup
Synaptogenesis wikipedia , lookup
Premovement neuronal activity wikipedia , lookup
Nervous system network models wikipedia , lookup
Optogenetics wikipedia , lookup
Pre-Bötzinger complex wikipedia , lookup
Chemical synapse wikipedia , lookup
Neuroanatomy wikipedia , lookup
Feature detection (nervous system) wikipedia , lookup
Channelrhodopsin wikipedia , lookup
Circumventricular organs wikipedia , lookup
Synaptic gating wikipedia , lookup
OREXIN (HYPOCRETIN)—LIKE IMMUNOREACTIVITY Orexin (Hypocretin)-Like Immunoreactivity in the Cat Hypothalamus: A Light and Electron Microscopic Study Jian-Hua Zhang PhD, Sharon Sampogna, Francisco R. Morales MD, and Michael H. Chase PhD* Department of Physiology and the Brain Research Institute, UCLA School of Medicine, Los Angeles, CA 90095, USA Abstract: Orexin-A-like immunoreactive (OrA-ir) neurons and terminals in the cat hypothalamus were examined using immunohistochemical techniques. OrA-ir neurons were found principally in the lateral hypothalamic area (LHA) at the level of the tuberal cinereum and in the dorsal and posterior hypothalamic areas. In the LHA the majority of the neurons were located dorsal and lateral to the fornix; a small number of OrAir neurons were also present in other regions of the hypothalamus. OrA-ir fibers with varicose terminals were detected in almost all hypothalamic regions. The high density of fibers was located in the suprachiasmatic nucleus, the infundibular nucleus (INF), the tuberomamillary nucleus (TM) and the supra- and pre-mamillary nuclei. Ultrastructural analysis revealed that OrA-ir neurons in the LHA receive abundant input from non-immunoreactive terminals. These terminals, which contained many small, clear, round vesicles with a few large, dense core vesicles, made asymmetrical synaptic contacts with OrA-ir dendrites, indicating that the activity of orexin neurons is under excitatory control. On the other hand, the terminals of OrA-ir neurons also made asymmetrical synaptic contact with dendrites in the LHA, the INF and the TM. The dendrites in the LHA were both non-immunoreactive and OrAir; conversely, the dendrites in the INF and the TM were non-immunoreactive. In these regions, OrA-ir terminals contained many small, clear, round vesicles with few large, dense core vesicles, suggesting that orexinergic neurons also provide excitatory input to other neurons in these regions. Key words: Orexin-A, immunoreactivity; ultrastructure; anatomy; hypothalamus; cat; sleep; wakefulness INTRODUCTION been examined in the central nervous system of a variety of species including the rat,1,2,4-16 mouse,4,7,16 monkey,10,11 and human.7,16 In the rat brain, it has been found that neurons containing either prepro-orexin mRNA or orexin immunoreactivity are exclusively localized in the hypothalamus, mostly within the lateral hypothalamic area (LHA), a region that has long been regarded as a “feeding area” as well as subserving a variety of vegetative functions.17,18 For example, Sakurai et al.2 reported that introcerebroventricular injection of orexin stimulates food intake and that the expression of orexin mRNA is increased by food deprivation. These observations suggest that a major function of the orexins is likely to be involved in the regulation of feeding behavior (reviewed in Ref. 3). Although neurons containing orexins are located exclusively in the LHA, axon terminals of orexin-containing neurons and orexin receptors are distributed widely throughout the mammalian central nervous system.6,14,19 For example, using immunohistochemical techniques with an antibody against prepro-orexin, Peyron et al.14 found labeled fibers in many hypothalamic as well as extrahypothalamic regions of the rat brain. Some of these regions are known to be important for the regulation of blood pressure, neuroendocrine release, body temperature, as well as sleep and wakefulness,6,14,19 indicating that orexins may also be involved in these physiological functions. Recently, using positional cloning techniques, Lin et al.20 identified an autosomal recessive mutation in the well-established canine model of narcolepsy; it was reported that this mutation caused the disruption of the OX2R. At the same time, using gene knockout techniques, Chemelli et al.21 found that mice lack- OREXIN-A (OrA) AND OREXIN-B (OrB) (ALSO KNOWN AS HYPOCRETIN-1 AND-21) ARE TWO CLOSELY RELATED HYPOTHALAMIC NEUROPEPTIDES that have been recently discovered using an intracellular calcium influx assay on multiple cells expressing individual “orphan” G protein-coupled receptors.2 Both orexins are derived from the same 130amino acid residue (rodent) or 131-amino acid residue (human) polypeptide (prepro-orexin) by proteolytic processing. OrA is a 33-amino acid peptide with a sequence that is identical in the human, rat, mouse, and bovine species2 (reviewed in Ref. 3), whereas OrB is a 28-amino acid peptide with two amino acids that are different in the human sequence and rodent (rat/mouse) sequences.2,3 Both OrA and OrB are endogenous ligands for two closely related (previously) orphan G protein-coupled receptors: orexin1 and orexin-2 receptors (OX1R and OX2R)2. OrA has a high affinity for both OX1R and OX2R, while OrB has a10-fold higher affinity for OX2R than for OX1R2 (reviewed in Ref. 3). Since the discovery of orexin peptides, their distribution has Accepted for publication October 2000 Address correspondence to: Michael H. Chase, PhD, Department of Physiology, UCLA School of Medicine, 53-231 CHS, University of California, Los Angeles, CA 90095; Tel: 310-825-3417; Fax: 310-206-3499; E-mail: [email protected]. SLEEP, Vol. 24, No. 1, 2001 67 Orexin (Hypocretin)-Like Immunoreactivity —Zhang et al ing orexin exhibit sleep abnormalities similar to those observed in narcoleptics. These studies indicate that orexins may play an important role in the control of sleep and wakefulness. The cat has long been used by neurophysiologists, behaviorists, pharmacologists, and others to study the mechanisms that are responsible for sleep and wakefulness and their attendant physiological processes. However, an examination of orexin-like immunoreactive neurons and terminals in the cat brain has not previously been carried out. Accordingly, the present experiment was undertaken to determine the location and ultrastructural characteristics of orexin-immunoreactive neurons and axonal terminals in the hypothalamus of the cat by means of immunohistochemical techniques in conjunction with combined light and electron microscope analyses. identify structures within the hypothalamus. A rabbit polyclonal antibody, raised against the full-length human orexin-A peptide (Phoenix Pharmaceuticals, Mountain View, CA), was used to identify OrA-ir neurons in the hypothalamus. The specificity of the orexin-A antibody has been described in detail in two recently published studies which indicate that the orexin-A antibody exhibits a 100% cross-reactivity with the human orexin-A; no cross-reactivity with human orexin-B or other related peptides was detected.5,9 An Olympus microscope equipped with a CCD camera was used to localize the areas of interest. Using a Macintosh computer (G3) with NIH image software, the cross-sectional areas of OrA-ir neurons were measured by circling the perimeter of labeled neuronal soma at a final magnification of 215X. The average cross-sectional areas were expressed as mean±S.D. MATERIALS AND METHODS Immunohistochemical procedures: electron microscopy Animals and tissue preparation Two adult male cats (two to three-years-old) were used in the electron microscopic examination. The animals were perfused using the same procedures and fixative as described above for the light microscopic study. After perfusion, the hypothalamus was removed and postfixed overnight in the same fixative at 4°C. Tissue blocks containing the hypothalamus were then cut into 50 m sections using a vibratome. These vibratome sections were immunostained with antibody against orexin-A using the procedure that was employed for the light microscopic examination.22 After immunostaining, the sections were further postfixed with 1% osmium tetroxide in 0.1 M PBS for 40 min at room temperature; they were then rinsed in distilled water. The sections were consequently dehydrated in graded ethanols and propylene oxide and flat-embedded in Epon-Araldite epoxy resin. After polymerization at 65ºC for 48 hours, the sections were first observed under the light microscope. Those areas which contained OrA-ir neurons (the perifornical regions of the LHA) and terminals (the infundibular nucleus and the tuberomammillary nucleus) were photographed, removed and mounted on blank blocks. Finally, ultrathin sections (500-600 Å) were cut using a ultramicrotome and counterstained with 8% saturated uranyl acetate (10 minutes) and 0.3% lead citrate (2 minutes). These ultrathin sections were examined using a Hitachi electron microscope. In the present experiment, four two to three-year-old adult male cats were employed. All animals were obtained from and determined to be in good health by the UCLA Division of Laboratory Animal Medicine. Animal treatment and handling in the experiments conformed with the policy of the American Physiological Society. The cats were deeply anesthetized with pentobarbital sodium (45 mg/kg, i.p.) and perfused transcardially with 1 liter of ice-cold saline (containing 1000 units of heparin) followed by 2.5 liters of a fixative containing 4% paraformaldehyde, 15% saturated picric acid and 0.25% glutaraldehyde in 0.1 M phosphate buffer (PBS) (pH 7.4). The hypothalamus was then removed and postfixed overnight in fresh fixative at 4 °C. Next, these tissues were immersed overnight in 20% sucrose (w/v) in 0.1 M PBS at 4 °C. After freezing with dry ice, it was cut into 15 m coronal sections with a Reichert-Jung cryostat. All sections were collected and stored in a solution of 0.1 M PBS containing 0.3% Triton X-100 and 0.1% sodium azide at 4 °C for later use. Immunohistochemical procedures: light microscopy The sections were immunostained with antibody against orexin-A according to previously published procedures.22 Briefly, free-floating sections were rinsed several times in ice-cold PBST (0.1 M PBS with 0.3% Triton X-100); they were then incubated with antibody against orexin A (Phoenix Pharmaceuticals, Mountain View, CA; diluted 1:1500—1:8000) in PBST solution overnight. On the following day, the sections were rinsed four times in PBST for a total duration of 30 minutes; the sections were then incubated for 90 min in PBST containing biotinylated anti-rabbit IgG (Vector Laboratories, Burlingame, CA; diluted at 1:300) followed by incubation in the ABC complex (Vector Laboratories, Burlingame, CA; diluted at 1:200) for 90 minutes. The color reaction was carried out by incubating the sections in 50 mM Tris buffer (pH 7.5) containing 0.02% 3,3’-diaminobenzidine (DAB) and 0.015% H2O2 for 15-30 min. After the DAB reaction, the sections were rinsed in PBST several times, mounted on gelatin-coated glass slides and airdried. The sections were then dehydrated and coverslipped in Permount. At least one set of sections (15 mm) from each animal was counterstained by neutral red before dehydration in order to SLEEP, Vol. 24, No. 1, 2001 RESULTS In a variety of species the hypothalamus is divided into three distinct longitudinal zones (periventricular, medial, and lateral).23,24 Each zone contains four rostro-caudal regions (preoptic, anterior [or supra optic], tuberal, and mammillary).23,24 In the present study, we adopted this reference system for our description of the location of orexin-A-like immunoreactive (OrA-ir) neurons and terminals (Table 1). The demarcations and nomenclature of cell groups in the cat hypothalamus in the present study is based on Bleier’s classic work on the cat hypothalamus.25 Light microscope Under the light microscope, labeled neurons exhibited a localized pattern of bilateral symmetric distribution in coronal sections of hypothalamus (Figure 1). OrA-ir fibers, on the other hand, were seen throughout the hypothalamus with different 68 Orexin (Hypocretin)-Like Immunoreactivity —Zhang et al Table 1—Relative densities of orexin-A-like immunoreactive somata and fibers in the hypothalamus of the cat Cell group Soma 1. Periventricular zone Preoptic level Anterior periventricular n. Paraventricular n.(anterior component) Anterior level Anterior periventricular n. Suprachiasmatic n. Paraventricular n. (dorsal component) Tuberal level Periventricular n. (tuberal component) Infundibular n. Mamillary level Periventricular n. (tuberal component) 2. Medial zone Preoptic level Anterior hypothalamic a. Anterior level Anterior hypothalamic a. Anterior hypothalamic n. Dorsal hypothalamic a. Parvocellular n. Tuberal level Dorsal hypothalamic a. Area of the tuber cinereum Ventromedial n. Dorsomedial n. Mamillary level Dorsal hypothalamic n. Posterior hypothalamic a. Mamillary complex Medial mamillary n. Lateral mamillary n. Supramamillary n. Premamillary n. Tuberomamillary n. 3. Lateral zone Preoptic level Lateral hypothalamic a. (anterior division) Anterior level Lateral hypothalamic a. (anterior division) Supraoptic n. (anterior component) Tuberal level Lateral hypothalamic a. (tuberal division) Nucleus of the fields of Forel Supraoptic n.(posterior component) Mamillary level Lateral hypothalamic a. (mamillary division) Nucleus of the fields of Forel Relative density Fiber/terminals + + + + + +/++ + + ++ + + + + ++ + + + + + + - +/++ + + + ++ + + + +/++ + + +/++ + ++ +/++ +/++ + + + - + + ++ ++ +++ + +/++ +/++ - +/++ + +++ + + +/++ + + + - + + Scale rating: - none, + very few, ++ few, +++ many SLEEP, Vol. 24, No. 1, 2001 69 Orexin (Hypocretin)-Like Immunoreactivity —Zhang et al Abbreviations used in Figure 1 AHA: anterior hypothalamic area AHN: anterior hypothalamic nucleus BST: bed nucleus of stria terminalis CEM: centromedial nucleus of the thalamus cp: cerebral peduncle DHA: dorsal hypothalamic area DHN: dorsal hypothalamic nucleus DMH: dorsomedial nucleus EP: entopeduncular nucleus fd: descending column of the fornix FF: nucleus of the fields of Forel fr: fasciculus retroflexus ic: internal capsule INF: infundibular nucleus ITP: bed nucleus of the inferior thalamus peduncle LHA: lateral hypothalamic area MA: anterior mamillary nucleus MM: medial mamillary nucleus M.rec. mamillary recess of the third ventricle mt: mamillothalamic tract oc: optic chiasm ot: optic tract PAA: paraventricular nucleus, anterior component PAD: paraventricular nucleus, dorsal component PAR: paraventricular nucleus of the thalamus PEM: perimamillary nucleus PET: periventricular nucleus, tuberal component PHA: posterior hypothalamic nucleus PM: premamillary nucleus pmt: principal mamillary tract PV: parvocellular nucleus PVa: anterior periventricular nucleus PT: parataenial nucleus of the thalamus Re: reticular complex of the thalamus SCh: suprachiasmatic nucleus SOA: supraoptic nucleus, anterior component SOT: supraoptic nucleus, tuberal component ST: subthalamic nucleus of Luys SUM: supramamillary nucleus TCA: area of the tuber cinereum TM: tuberomamillary nucleus VA: ventroanterior nucleus of the thalamus VB: ventrobasal nucleus of the thalamus V.III.: third ventricle VMH: ventromedial nucleus ZI: zona incerta Figure 1—Schematic drawings of rostro-caudal coronal sections showing the localization and relative density of orexin-A-like immunoreactive neurons (large dots in the left half of drawings) and terminals (small dots in the right half of drawings) at different levels of the cat hypothalamus: A: preoptic level; B: anterior level; C: tuberal level; D: junction of tuberal and mamillary levels; E: mamillary level. All cells at specific levels in 15- m-thick sections have been plotted. Bar = 1 mm. intensities in discrete regions (Table 1, Fig 1). (Figure 2B). In contrast, there were only a few fibers or terminals in other nuclei in the periventricular zone (Table 1, Fig.1). 1. Periventricular zone 2. Medial zone In the periventricular zone, OrA-ir neurons were lightly scattered throughout several nuclei which included the suprachiasmatic nucleus, the paraventricular nucleus, the periventricular nucleus and the infundibular nucleus (Figure 1). These neurons were fusiform or bipolar cells (260.19±114.86 m2) with two primary dendrites (Figure 2A). OrA-ir products were observed as dark brown granules in the cytoplasm of neuronal somata and dendritic processes (Figure 2A). The nuclei of these cells were not stained. OrA-ir fibers and terminals were abundant in the suprachiasmatic nucleus and the infundibular nucleus (INF, which is the same as the arcuate nucleus in other species) (Figure 2B); some OrA-ir fibers in these nuclei had varicose terminals SLEEP, Vol. 24, No. 1, 2001 As described above for the periventricular zone, most nuclei in the medial zone contained either very few or no OrA-ir neurons (Table 1, Figure 1). However, many labeled neurons were found in the dorsal hypothalamic area and in the posterior hypothalamic area (Table 1, Figure 1 and Figure 2C). These OrA-ir neurons were fusiform or bipolar cells (194.82±46.45 m2) (Figure 2C). A large number of OrA-ir fibers and terminals were seen in the tuberomammillary nucleus (TM) (Figure 2D); the remainder of the hypothalamic nuclei contained only a few terminals (Table 1, Figure 1). In the TM, a few OrA-ir terminals were found having close contacts with non-immunoreactive 70 Orexin (Hypocretin)-Like Immunoreactivity —Zhang et al Figure 2—Photomicrographs showing orexin-A-like immunoreactive (OrA-ir) neurons and terminals in the cat hypothalamus. A: A bipolar OrA-ir neuron in the periventricular nucleus at the tuberal level. The immunoreactive products is within the cytoplasm of soma (S) and dendrites (D), but not in the nucleus (N). B: OrA-ir terminals in the suprachiasmatic nucleus. A labeled fiber with numerous varicosities (double arrows) are also present. C: OrA-ir neurons in the dorsal hypothalamic area at the tuberal level. The majority of neurons in this region have fusiform somata with short dendrites. A few bipolar neurons with relatively long dendrites are also seen (arrows). D: OrA-ir terminals in the tuberomammillary nucleus. Some terminals exhibit close contacts with a non-immunoreactive soma (counterstained by neutral red) and its dendrites (double arrows). E: Camera lucida drawing of a neuron indicated by double arrows in D at high magnification showing close contacts of OrA-ir terminals with a non-immunoreactive neuronal soma and its dendrites. Bar in A. B. D. and E are 25 µm; Bar in C is 50 µm. Figure 3—Photomicrographs illustrating the localization of orexin-A-like immunoreactive (OrA-ir) neurons and terminals in the lateral hypothalamic area (LHA). A: OrA-ir neurons and terminals in the area dorsal to the fornix. Many neurons in this area have round or fusiform somata with short dendrites. However, multipolar (arrow) and bipolar (double arrows) neurons are also seen. B: OrA-ir neurons in the region lateral to the fornix and dorsal to the tuberomammillary nucleus. Many neurons in this area are bipolar cells with long dendrites (arrows). C: Two OrA-ir neurons with long primary (arrowheads) and secondary (arrows) dendrites in the area lateral to the fornix. D shows close contacts between OrA-ir terminals and either a OrA-ir dendrite (1) or a nonimmunoreactive soma (2) (arrows). The contacts on cells 1 and 2 are clearly seen under higher magnification as shown in panels E and F, respectively. Bar in A and B are 50 µm; bar in C-F are 25 µm. soma and dendrites (Figure 2D and E). the perifornical area of the LHA for somata and the INF and the TM for terminals) (see Materials and Methods). In the perifornical area, OrA-ir neurons had an elongated soma with an invaginated nucleus that occupied most of the cell body (Figure 4A). Within the cytoplasm of the somata, there were abundant organelles including rough endoplasmic reticulum (RER), mitochondria, Golgi apparatus, and lysosomes (Fig. 4A). Many large, dense core vesicles (DCV) were also seen in the cytoplasm. OrA-ir reaction products were scattered within the cytoplasm of OrA-ir neurons (Fig. 4A). Most of the surface of the somata of OrA-ir neurons were covered by glia cell bodies and their processes (Fig 4A and B); thus, only a small portion of the soma surface of these neurons was covered by terminals (Fig. 4C). These terminals, which contained small, clear, round vesicles, usually made asymmetric synaptic contact with the somata of OrA-ir neurons (Fig, 4C). Under the electron microscope, most of immunostained dendrites in the LHA were either small or medium-sized and contained a few mitochondria and some microtubules (Fig. 5A and B). Unstained synaptic terminals usually made contact with these dendrites (Fig. 5A). Occasionally, several large synaptic terminals were observed converging onto a single OrA-ir dendrite (Fig. 5B). These terminals contained many small, clear, round vesicles and a few large, dense core vesicles (Fig 5A and B). The majority of these terminals made asymmetric synapses with OrA-ir dendrites (Fig. 5A and B). In addition to mitochondria and microtubles, large OrA-ir dendrites in the LHA also con- 3. Lateral zone The largest number of OrA-ir neurons (232.77 ± 59.20 m2) in the hypothalamus were found in the LHA at the tuberal level and at the junction of the tuberal and mamillary areas. Neurons in these regions were located dorsal and lateral to the fornix (Figure 1C and D; Figure 3A) and dorsal to the TM (Figure 1D and E; Figure 3B). The immunoreactive neurons in the region dorsal to the fornix were fusiform cells with short dendrites (Figure 3A), a few bipolar and mutipolar neurons were also seen in this region (Figure 3A). On the other hand, most of the OrAir neurons in the area dorsal to the TM were bipolar cells with long primary dendrites and secondary dendrites (Figure 3B and C). In contrast to the many stained somata that were observed, OrA-ir fibers and terminals were only sparsely distributed in the lateral hypothalamic area. Few of these OrA-ir terminals were found having close contacts with either non-immunoreactive or OrA-ir somata or dendrites in the LHA (Figure 3D, E, and F ). The rest of the nuclei in the lateral zone contained very few or no OrA-ir neurons or terminals (Table 1, Figure 1) Electron microscope Regions that contained a majority of the OrA-ir neurons or terminals were selected for electron microscopic examination (e.g., SLEEP, Vol. 24, No. 1, 2001 71 Orexin (Hypocretin)-Like Immunoreactivity —Zhang et al Figure 4—A: Photomicrographs showing an orexin-A-like immunoreactive (OrAir) neuronal soma with an invaginated nucleus (N) in the lateral hypothalamic area. This neuron contains many different types of organelles including mitochondria (M), Golgi apparatus (GA), lysosome (Ly) and rough endoplasmic reticulum (RER). Large dense core vesicles (DCV) are seen in the area containing Golgi apparatus. Immunoreactive products are randomly distributed throughout the cytoplasm (curved arrows). The surface of this neuron is covered by a glia cell and its processes. GN: glia nucleus; GP: glia process. B: Enlargement of framed region in A showing in detail the glia processes (arrowheads). S: soma. C: one non-immunoreactive axonal terminals that make contact with the OrA-ir neuron shown in A. The terminals contain many small, clear, round vesicles. Asymmetrical synapses are formed between the terminal (t) and soma (s). Bars in A and B are 1 µm; bar in C is 0.5 µm. Figure 5—Photomicrographs showing orexin-A-like immunoreactive (OrA-ir) dendrites in the lateral hypothalamic area. A: a small OrA-ir dendrite (d) receives an asymmetric synaptic input from a non-immunoreactive axonal terminal (t). The arrowhead indicates the synaptic site. Note that both the dendrite and the terminal contain mitochondria (M). The axonal terminal also contains many small, clear, round vesicles and two large dense core vesicles (DCV). Curved arrow indicates immunoreactive products. B: Seven non-immunoreactive terminals (t1-t7) converge onto a medium-sized OrA-ir dendrite (d). All these terminals contain small, clear, round vesicles. Five terminals (t1-t2 and t5-t7) make synaptic contacts with the dendrite (arrowheads) and all of these synapses are asymmetric. There are no clear synaptic sites between the t3-t4 and the dendrite. Curved arrows indicate immunoreactive products. C: A large OrA-ir dendrite (d) contains many types of organelles within the cytoplasm including mitochondria (M), Golgi apparatus (GA), lysosome (Ly) and rough endoplasmic reticulam (RER). The surface of this large dendrite is covered by eight axonal terminals (t1-t8) and glia processes (GP). Curved arrows indicate immunoreactive products. D is the enlargement of the framed region in panel C showing the detail of glia processes (GP) and one asymmetric synaptic contact (arrowhead) between the dendrite (d) and t1 terminal. Note that the t1 terminal contains many small, round, clear vesicles and one large dense core vesicle (DCV). M: mitochondria. Bars in A and D are 0.5 µm; bars in B and C are 1 µm. tained ERE and Golgi apparatus (Fig. 5C). In contrast to the somata, a large portion of the surface of these large dendrites was covered by terminals and only a small portion by glial processes (Fig. 5C and D). Most terminals contained many small, clear, round vesicles and made asymmetric synaptic contact with the large dendritic surface (Fig. 5C and D). In addition to the somata and dendrites observed in the perifornal area of the LHA, OrA-ir axon, and terminals were also found in the TM, the INF, as well as the LHA. Most of the OrAir axons were myelinated fibers with thin myelin sheaths. A few unmyelinated OrA-ir axons were also observed. In all three regions, OrA-ir terminals were found to contain many small, clear, spherical synaptic vesicles with a few large, dense core vesicles (DCV) (Fig. 6A—6C). OrA-ir reaction product was observed within the large dense-core vesicles and the cytosole of the terminals (Fig. 6A—6C). OrA-ir axon terminals mainly SLEEP, Vol. 24, No. 1, 2001 made asymmetric synaptic contact with medium-sized or small dendrites and occasionally with somata (Fig. 6B and C). In the INF and TM, none of the postsynaptic structures contained OrAir products (Fig. 5B). However, in the LHA, although the majority of postsynaptic dendrites and somata were not immunolabeled, a few post synaptic dendrites did contain OrA-ir products (Fig. 6C). 72 Orexin (Hypocretin)-Like Immunoreactivity —Zhang et al fibers in the LHA, INF, and TM. In the LHA, OrA-ir neurons contained a large number of RER and other organelles in their soma and primary dendrites, while the medium-sized and small dendrites contained many microtubules and only a few mitochondria. Non-immunoreactive terminals containing many small, clear, round vesicles often made synaptic contact with OrA-ir dendrites or somata. The majority of these synapses were asymmetric. Asymmetric synapses with small, round, clear vesicles are considered to be indicative of excitatory neural transmission.26,27 In addition, axo-somatic synapses were less frequently found than axo-dendritic synapses because a large portion of the somata of OrA-ir neurons was covered by glial processes. Therefore, OrA-ir neurons in the LHA appeared to receive excitatory synaptic input mainly on their dendritic surfaces. Some of the synaptic input may be mediated by neuropeptides because some non-immunoreactive terminals contained large dense-core vesicles.28 In addition, it has been reported that orexin neurons in the LHA are innervated by neuropeptide Y (NPY)-, agouti-related peptide (AgRP)-, and a-melanin-stimulating hormone (a-MSH)-immunoreactive fibers.4,7,10 These fibers likely originate from the INF, a nucleus involved in the regulation of body weight.4,7,10 We also examined OrA-ir axon terminals in the LHA, INF and TM under the electron microscope. In all of these regions, OrAir terminals contained many small, clear, round vesicles with a few large, dense core vesicles. OrA-ir products were found within the large dense core vesicles and the cytosole of the terminals. These results indicate that orexin-A may be stored in the large, dense core vesicles and the cytosole of terminals, while the small, clear, round vesicles are likely to contain other excitatory neurotransmitters. Further investigations are necessary to determine whether orexin coexists with other neurotransmitters in the same neuron and synaptic terminals. In the LHA, INF, and TM, almost all of the OrA-ir terminals made asymmetric synaptic contact with small to medium-sized non-immunoreactive dendritic shafts and a few somata. Similar OrA-ir axon terminals and synapses have been reported in the periaqueductal gray,1 the locus coeruleus, and the arcuate nucleus of the rat and monkey.10,11 In addition, there have been recent electrophysiological findings indicating that orexinergic terminals exert an excitatory influence on their post synaptic neurons in both the hypothalamus1 and the locus coeruleus.11 Therefore, orexinergic terminals in both the INF and TM may be excitatory with respect to neurons in these areas. In the cat LHA, in addition to making synaptic contact with non-immunoreactive postsynaptic structures, some of the OrA-ir terminals also made synaptic contact with OrA-ir dendrites. Similar connections have been detected in the LHA of the hypothalamus of the rat and monkey.10 These finding suggests that recurrent collaterals from orexin-containing axons make excitatory synapses with orexincontaining neurons in the LHA. These connections may act to synchronize the activity of orexin neurons in the LHA. The restricted localization of orexin-containing neurons in the LHA indicates that orexin may be involved in the central regulation of feeding behavior and energy homeostasis, which are classical functions of the LHA.1,7,18,29,30 Recent studies have reported that food consumption is increased following acute injections of orexins into the lateral ventricle of rat2,31 (reviewed in Ref. 3). Similar effects have also been found after the injection of OrA into the LHA, the perifornical area, the paraventricular nucleus, Figure 6—A: A non-immunoreactive terminal (t1) and an orexin-A-like immunoreactive (OrA-ir) axonal terminal (t2) in the lateral hypothalamic area. Both t1 and t2 terminals contain many small, round, clear vesicles, while t2 terminals also contain large dense, core vesicles (DCV). Orexin-A-like immunoreactive products are found within the DCV and the cytosole of t2 terminals. Arrowhead indicates the asymmetric synaptic contact between the t1 terminal and dendrites (d). B: OrA-ir (t1) and non-immunoreactive (t2) axonal terminals in the tuberomammillary nucleus. Both terminals contain small, clear, round vesicles and make asymmetric synapses (arrowheads) with the same dendrite (d). C: OrA-ir (t1) and non-immunoreactive (t2) axonal terminals making asymmetric synapses (arrowheads) with one OrA-ir dendrite (d) in the lateral hypothalamic area. Both terminals contain small, clear, round vesicles, while t1 also contains a large dense core vesicle (DCV). Bars in all figures are 0.5 µm DISCUSSION In the present study, the distribution of orexin-A-containing neurons in the hypothalamus of the cat was examined using immunohistochemical techniques with an antibody against orexin-A. OrA-ir neurons were located principally at the tuberal level of the lateral hypothalamus. Most of these neurons were restricted to regions dorsal and lateral to the fornix at the tuberal level of the hypothalamus. Some OrA-ir cells were also detected in the dorsal and posterior hypothalamic areas. The rest of the hypothalamic regions contained very few OrA-ir neurons. In general, a similar localization of OrA-ir neurons has been reported in the hypothalamus of other species including the rat,1,2,4-16 mouse,4,7,16 monkey10,11 and human.7,16 In addition to OrA-ir cell bodies, labeled fibers and terminals were observed in almost all hypothalamic regions. However, the density of labeled fibers was not uniform. Many OrA-ir fibers were found in the suprachiasmatic nucleus, the INF, the tuberomammillary nucleus, and the supra- and pre-mamillary nuclei. The rest of the hypothalamic areas contained only a small number of OrA-ir fibers. This distribution pattern of OrA-ir terminals resembled that reported in the rat12,14 and monkey.10 The ultrastructure of OrA-ir neurons and terminals have not previously been examined in the cat.1,10 Consequently, in the present experiment, using the electron microscope to view immunohistochemically stained material, we examined the ultrastructure of OrA-ir neurons in the lateral hypothalamic area and OrA-ir SLEEP, Vol. 24, No. 1, 2001 73 Orexin (Hypocretin)-Like Immunoreactivity —Zhang et al and the dorsomedial nucleus.32,33 In addition, it has been reported that the prepro-orexin mRNA level and both OrA and OrB concentrations are significantly up-regulated in the hypothalamus of fasted rats when compared to that of fed rats.2,13 Finally, orexin neurons in the rat LHA are activated during insulin-induced hypoglycemia, as indicated by the significant increase of preproorexin mRNA and the expression of fos-like immunoreactivity in orexin-containing neurons under these conditions.34,35 In summary, these results suggest that the orexinergic system in the LHA and its adjacent regions is important in the regulation of feeding behavior. Given the similarity in the distribution of orexins between rat and cat, it is reasonable to suspect that the orexinergic system in the cat LHA may also be involved in the regulation of food consumption. Although investigations into the physiological role of orexins have been focused primarily on the regulation of feeding and energy homeostasis, recent studies suggest that orexins may also be involved in the regulation of other behaviors.6,14,19 Based on the widespread localization of orexin terminals and receptors in the CNS and a number of physiological experiments, it has been suggested that orexins participate in the regulation of the neuroendocrine system,14,36 the cardiovascular system,37 drinking behavior,38 and body temperature, as well as sleep and wakefulness.6,8,11,14 Among the preceding functions, perhaps the most interesting newly proposed role for orexin is its possible involvement in the regulation of the sleep and wakefulness. Recently, Chemelli et al.21 (reviewed in Ref. 39 and 40) developed an orexin gene knockout mouse preparation. These mice which had no labeled orexinergic neurons in the hypothalamus, exhibited a behavior similar to that observed in humans and dogs with narcolepsy. This disease is characterized by prolonged daytime sleepiness, cataplexy, and rapid transition from wakefulness into REM sleep. In addition, by means of double immunostaining of orexin and cFos, an anti-narcoleptic drug, modafinil, was found to activate orexin-containing neurons in the LHA.21 Concurrently, using positional cloning to search for the molecular mechanisms of canine narcolepsy, Lin et al.20 found that the disruption of the orexin receptor 2 (OX2R) gene can produce canine narcolepsy. These data suggest that orexin may be a major neurotransmitter in the regulation of sleep and wakefulness. Neurons in the lateral hypothalamus, specifically the posterior lateral hypothalamus (PLH), have long been known to be involved in the maintenance of the waking state.41,42 For example, electrical or chemical lesions of the PLH result in somnolence and hypersomnia in the rat,43,44,45 cat,46 and monkey;47 electrophysiological recording studies have found that histaminergic neurons that are located in the TM of both the cat and rat are involved in the maintenance of wakefulness.48,49,50 These neurons discharge at highest rates during wakefulness, low rates during non-REM sleep, and tend to cease firing during REM sleep.48,51 A recent study also found a similar pattern of discharge of neurons in an area dorsal to the TM, at the caudal tuberal level of the hypothalamus.49 In the same region, we also found OrAir neurons in the cat. These data support the hypothesis that orexinergic neurons are involved in regulation of sleep and wakefulness.49 In the LC of both the rat and monkey, orexin-containing terminals were found to make asymmetrical (excitatory) synaptic SLEEP, Vol. 24, No. 1, 2001 contacts with tyrosine hydroxylase-immunopostive cells.11 In addition, in vitro studies show that OrA increases cell firing of noradrenergic neurons in the LC.11 Intracerebroventricular injections of OrA in conscious animals at the onset of the normal sleep period increases the proportion of time spent awake and reduces the time spent in REM sleep8 These data suggest that orexin may be involved in maintaining the waking state through an interaction with the noradrenergic system in the LC.8,11 In the current experiments, we found that neurons in the TM of the cat are densely innervated by OrA-ir terminals. Furthermore, our ultrastructural study showed that these OrA-ir terminals contain small, clear, round vesicles and that they make asymmetric synaptic contact with non-immunoreactive soma and dendrites, suggesting that orexins exert an excitatory drive on postsynaptic neurons, which are likely to include histaminergic neurons in the TM. The excited TM neurons may, in turn, activate cholinergic neurons in the LDT or noradronergic neurons in the LC, thereby promoting cortical arousal.49,50,51,54 At present, it is not clear how orexins participate in the regulation of sleep and waking state. One possibility is that orexins may act through modulating other sleep and wakefulness-related neurotransmitter systems (e.g., histaminergic neurons in the TM, serotonergic neurons in the dorsal raphe [DR], cholinergic neurons in the laterodorsal tegmemtal nucleus [LDT], or noradrenergic neurons in the locus coeruleus [LC] [reviewed in Ref. 52, 53]), all of these areas are densely innervated by orexin terminals.6,14 ACKNOWLEDGMENTS We wish to thank Dr. J. K. Engelhardt for critically edit this manuscript. This study was funded by grants NS 23426, NS 09999 and MH 43362. Financial disclosure: This study was funded by grants NS 23426, NS 09999 and MH 43362. REFERENCES 1. de Lecea L, Kilduff TS, Peyron C, Gao X-B, Foye PE, Danielson PE, Fukuhara C, Battenberg ELF, Gautvik VT, Bartlett FS, Frankel WN, van den Pol AN, Bloom FE, Gautvik KM, Sutcliffe JG. The hypocretins: hypothalamus-specific peptides with neuroexcitatory activity. PNAS USA 1998;322-27. 2. Sakurai T, Amemiya A, Ishii M, Matsuzaki I, Chemelli RM, Tanaka H, Williams SC, Richardson JA, Kozlowski GP, Wilson S, Arch JRS, Buckingham RE, Haynes AC, Carr SA, Annan RS, McNulty DE, Liu WS, Terrett JA, Elshourbagy NA, Bergsma DJ, Yanagisawa M. Orexins and orexin receptors: a family of hypothalamic neuropeptides and G protein-coupled receptor that regulate feeding behavior. Cell 1998;92:57385. 3. Sakurai T. Orexins and orexin receptors: implication in feeding behavior. Regulatory Peptides. 1999;85:25-30. 4. Broberger C, de Lecea L, Sutcliffe JG, Hökfelt T. Hypocretin/orexinand melanin-concentrating hormone-expressing cells form distinct populations in the rodent lateral hypothalamus: relationship to the neuropeptide Y and agouti gene-related protein systems. J Comp Neurol 1998;402:460-474. 5. Chen CT, Dun S. L, Kwok E.H, Dun N.J, Chang JK. Orexin A-like immunoreactivity in the rat brain. Neurosci Letters 1999;260:161-64. 6. Date Y, Ueta Y, Yamashita H, Yamaguchi H, Matsukura S, Kangawa K, Sakurai T, Yanagisawa M, Nakazato M. Orexins, orexigenic hypothalamic peptides, interact with autonomic, neuroendocrine and neuroregulatory systems. PNAS USA 1999;96:748-53. 74 Orexin (Hypocretin)-Like Immunoreactivity —Zhang et al 7. Elias CF, Saper CB, Maratos-Flier E, Tritos NA, Lee C, Kelly J, Tatro JB, Hoffman GE, Ollmann MM, Barsh GS, Sakurai T, Yanagisawa M, Elmquist JK. Chemically defined projections linking the mediobasal hypothalamus and the lateral hypothalamic area. J Comp Neurol 1998:402:442-59. 8. Hagan JJ, Leslie RA, Patel S, Evans ML, Wattam TA, Holmes S, Benham CD, Taylor SG, Routledge C, Hemmati P, Munton RP, Ashmeade TE, Shah AS, Hatcher JP, Hatcher PD, Jones DNC, Smith MI, Piper DC, Hunter AJ, Porter RA, Upton N. Orexin A activates locus coeruleus cell firing and increases arousal in the rat. PNAS USA 1999; 96:10911-916. 9. Harrison TA, Chen CT, Dun N.J, Chang JK. Hypothalamic orexin Aimmunoreactive neurons project to the rat dorsal medulla. Neurosci Letters 1999;273:17-20. 10. Horvath TL, Diano S, van del Pol AN. Synaptic interaction between hypocretin (orexin) and neuropeptide Y cells in the rodent and primate hypothalamus: a novel circuit implicated in metabolic and endocrine regulations. J Neurosci. 1999a;19:1072-87. 11. Horvath TL, Peyron C, Diano S, Ivanov A, Aston-Jones G, Kilduff TS, van den Pol AN. Hypocretin (orexin) activation and synaptic innervation of the locus coeruleus noradrenergic system. J Comp Neurol 1999b;415:145-59. 12. Nambu T, Sakurai T, Mizukami K, Hosoya Y, Yanagisawa M, Goto K. Distribution of orexin neurons in the adult rat brain. Brain Res 1999;827:243-60. 13. Mondal MS, Nakazato M, Date Y, Murakami N, Yanagisawa M, Matsukura S. Widespread distribution of orexin in rat brain and its regulation upon fasting. Biochem Biophys Res Commun 1999;256:495-9. 14. Peyron C, Tighe DK, van den Pol AN, de Lecea L, Heller HC, Sutcliffe JG, Kilduff TS. Neurons containing hypocretin (orexin) project to multiple neuronal systems. J Neurosci 1998;18:9996-10015. 15. Taheri S, Mahmoodi M, Opacka-Juffry J, Ghatei MA, Bloom SR. Distribution and quantification of immunoreactive orexin A in rat tissues. FEBS Lett 1999;457:157-61. 16. van den Pol AN. Hypothalamic hypocretin (orexin): robust innervation of the spinal cord. J Neurosci 1999;19:3171-82. 17. Bernardis LL, Bellinger LL. The lateral hypothalamic area revisited: neuroanatomy, body weight regulation, neuroendocrinology and metabolism. Neurosci Biobehav Rev, 1993;17:141-93. 18. Bernardis LL, Bellinger LL. The lateral hypothalamic area revisited: ingestive behavior. Neurosci. Biobehav. Rev., 1996;20:189-287. 19. Trivedi P, Yu H, MacNeil DJ, van der Ploeg LHT, Guan X-M. Distribution of orexin receptor mRNA in the rat brain. FEBS Lett 1998:438:71-5. 20. Lin L, Faraco J, Li R, Kadotani H, Rogers W, Lin X, Qiu X, de Jong PJ, Nishino S, Mignot E. The sleep disorder canine narcolepsy is caused by a mutation in the hypocretin (orexin) receptor 2 gene. Cell 1999; 98:365-76. 21. Chemelli RM, Willie JT, Sinton CM, Elmquist JK, Scammell T, Lee C, Richardson JA, Williams SC, Xiong Y, Kisanuki Y, Fitch TE, Nakazato M, Hammer RE, Saper CB, Yanagisawa M. Narcolepsy in orexin knockout mice: molecular genetics of sleep regulation. Cell 1999:98:437-51, 1999. 22. Zhang J-H, Sampogna S, Morales FR, Chase MH. Age-related intraaxonal accumulation of neurofilaments in the dorsal column nuclei of the cat brainstem: a light and electron microscopic immunohistochemical study. Brain Res 1988;797:333-8. 23. Nauta WJH, Haymaker W. Hypothalamic nuclei and fiber connections. In: Haymaker W, Anderson E, Nauta WJH, eds. The hypothalamus. Springfield, IL: Charles C. Thomas Publisher, 1969:136-209. 24. Swanson LW, The hypothalamus. In: Björklund A, Hökfelt T, Swanson LW, eds. Handbook of chemical neuroanatomy. Vol. 5: integrated systems of the CNS. Part 1. Amsterdam: Elsevier Science Publisher B.V., 1987:1-124. 25. Bleier R, ed. The hypothalamus of the cat. Baltimore, MD: The Johns Hopkins Press, 1961. SLEEP, Vol. 24, No. 1, 2001 26. Bodian D. Synaptic diversity and characterization by electron microscopy. In: Pappas GD, Purpura DP, eds. Structure and function of Synapse. New York: Raven Press, 1972:45-63. 27. Gray EG, Electron microscopy of excitatory and inhibitory synapse: a brief review. Prog Brain Res 1969;31:141-55. 28. Pickel V. General morphological features of peptidergic neurons. In: Björklund A, Hökfelt T, eds. Handbook of chemical neuroanatomy. Vol 4: GABA and neuropeptides in the CNS, part 1. Amsterdam: Elsevier Science Publisher B.V., 1985:72-92 29. Powley TL, Kessey RE. Relationship of body weight to the lateral hypothalamic syndrome. J Comp Physiol Psychol 1970;70:25-36. 30. van den Pol AN. Lateral hypothalamic damage and body weight regulation: role of gender, diet, and lesion placement. Am J Physiol 1982;243:R265-74. 31. Haynes AC, Jackson B, Overend P, Buckingham RE, Wilson S, Tadayyon M, Arch JRS. Effects of single and chronic intracerebroventricular administration of the orexins on feeding in the rat. Peptides 1999;20:1099-105. 32. Dube MG, Kalra SP, Kalra PS. Food intake elicited by central administration of orexins/hypocretins: identification of hypothalamic sites of action. Brain Res 1999;842:473-7. 33. Sweet DC, Levine AS, Billington CJ, Kotz CM. Feeding response to central orexins. Brain Res 1999;821:535-8. 34. Griffond B, Risold P-Y, Jacquemard C, Colard C, Fellmann D. Insulin-induced hypoglycemia increases preprohypocretin (orexin) mRNA in the rat lateral hypothalamic area. Neurosci Lett 1999;262:7780. 35. Moriguchi T, Sakurai T, Nambu T, Yanagisawa M, Goto K. Neurons containing orexin in the lateral hypothalamic area of the adult rat brain are activated by insulin-induced acute hypoglycemia. Neurosci Lett 1999;264:101-4. 36. van den Pol AN, Gao X-B, Obrietan K, Kilduff TS, Belousov AB. Presynaptic and postsynaptic actions and modulation of neuroendocrine neurons by a new hypothalamic peptide, hypocretin/orexin. J Neurosci 1998;18:7962-71. 37. Samson WK, Gosnell B, Chang J-K, Resch ZT, Murphy TC. Cardiovascular regulatory actions of the hypocretins in brain. Brain Res 1999;83:248-53. 38. Kunii K, Yamanaka A, Nambu T, Matsuzaki I, Goto K, Sakurai T. Orexins/hypocretins regulate drinking behavior. Brain Res 1999;842:256-61. 39. Aldrich MS, Reynolds PR. Narcolepsy and the hypocretin receptor 2 gene. Neuron 1999;23:625-31. 40. Siegel JM. Narcolepsy: a key role for hypocretins (orexins). Cell 1999;98:409-412. 41. Goodless-Sanchez N, Moore RY, Morin LP, Lateral hypothalamic regulation of circadian rhythm phase. Physiol. Behav. 1991;49:533-37. 42. Koizumi K, Nishino H. Circadian and other rhythmic activity of neurons in the ventromedial nuclei and lateral hypothalamic area. J Physiol 1976;263:331-56. 43. McGinty DJ. Somnolence, recovery and hyposomnia following ventro-medial diencephalic lesions in the rat. Electroencephalogr Clin Neurophysiol 1969;26:70-9. 44. Nauta WJH. Hypothalamic regulation of sleep in rats: an experimental study. J Neurophysiol 1946;9:285-316. 45. Shoham S, Teitelbaum P, Subcortical waking and sleep during lateral hypothalamic “somnolence” in rats, Physiol Behav 1982;28:323-333. 46. Swett CP, Hobson JA. The effects of posterior hypothalamic lesions on behavioral and electrographic manifestations of sleep and waking in cats. Arch Ital Biol 1968;106:283-93. 47. Ranson SW. Somnolence caused by hypothalamic lesions in the monkey. Arch Neurol Psychiatry 1939;41:1-23 48. Lin J-S, Luppi P-H, Salvert D, Sakai K, Jouvet M. Histamine-containing neurons in the cat hypothalamus. C R Acad Sci Paris 1986;303:371-76. 49. Steininger TL, Alam MN, Gong H, Szymusiak R, McGinty D. Sleep75 Orexin (Hypocretin)-Like Immunoreactivity —Zhang et al waking discharge of neurons in the posterior lateral hypothalamus of the albino rat. Brain Res 1999;840:138-47. 50. Vanni-Mercier G, Sakai K,. Jouvet M. “Waking-state specific” neurons in the caudal hypothalamus of the cat. CR Acad Sc Paris 1984;298:195-200. 51. Lin J-S, Sakai K, Jouvet M. Evidence for histaminergic arousal mechanisms in the hypothalamus of cat. Neuropharmacol 1988;27:11122. 52. Jones BE. Paradoxical sleep and its chemical/structural substrates in the brain. Neurosci 1991;40:637-656. 53. Kryer MH, Roth T., Dement WC, eds. Principle and practice of sleep Medicine. 3rd Edition. Philadephia, PA:W.B. Saunders Company, 2000;1-92. 54. Lin J-S, Hou Y, Sakai K, Jouvet M. Histaminergic descending inputs to the mesopontine tegmentum and their role in the control of cortical activation and wakefulness in the cat. J Neurosci 1996;15:1523-37. SLEEP, Vol. 24, No. 1, 2001 76 Orexin (Hypocretin)-Like Immunoreactivity —Zhang et al