Download Orexin (Hypocretin)-Like Immunoreactivity in the Cat Hypothalamus

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
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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
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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
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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.,
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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).
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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
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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.
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