ISRAEL JOURNAL OF
VETERINARY MEDICINE
Vol. 56 (4) 2001
DISTRIBUTION OF OREXIN-A- AND OREXIN-BCONTAINING NEURONES AND FIBRES IN THE RAT
BRAIN AND SPINAL CORD
Summary
Orexin-A and orexin–B are recently identified neuropeptides that are localized exclusively to the lateral
hypothalamic area, the function of which is stimulate feeding. In this study, we employed
immunohistochemistry to map the distribution of neurones and fibres containing orexin-A and orexinB in fifteen adult rat brains and spinal cords. Orexin-A and orexin-B cell bodies were concentrated in
the lateral hypothalamic and perifornical nuclei and throughout the spinal cord. Orexin-A- and orexinB-immunoreactive fibres were observed extensively in the hypothalamus and spinal cord.
The results indicate that orexin-A and -B-immunoreactive neurones contribute to the control of
energy homeostasis. The widespread projections of orexin-A and -B fibres within the hypothalamus
and other feeding-related centres outside of the hypothalamus, suggests that orexin-A and orexin-B
may have an important role in the regulation of feeding.
Key Words: Orexin-A, orexin-B, peptide, hypothalamus, feeding.
Introduction
The hypothalamus represents an important tissue involved in the regulation of nutritional
status via the coordination of many neurotransmitter systems implicated in food intake and
energy expenditure (1-4). Orexin-A and Orexin-B are, respectively, 33- and 28-amino acid
residue peptides, identified from rat brain extracts that activated a G-protein-coupled ‘orphan’
receptor, i.e. that had no known ligand (1). These peptides were independently identified and
termed hypocretin-1 and hypocretin-2, although the sequence deduced for the orexins (1).
Immunocytochemical studies using antisera against the synthetic fragment orexin-A (14-33)
showed that orexin-positive neurones are located in lateral hypothalamic area (LHA) (1). In
another study, the antibodies, which were against the synthetic C-terminal sequence of the
prepro-hypocretin peptide, labelled a population of neurones with a similar pattern of
distribution (5). Immunocytochemical observations on the location of orexin fibres have
described the presence of orexin-A and orexin-B immunoreactive processes in the LHA,
arcuate, and perifornical nuclei (PeF) and various sites beyond the hypothalamus, such as
the thalamic paraventriculer and reuniens nuclei, locus coeruleus, central grey, nucleus of the
solitary tract, raphe nuclei and septal nuclei (5-7). The widespread distribution of orexin fibres
suggests that the orexins may also be implicated in functions other than feeding and further
raises the possibility that these peptides have extensive influences on many target sites in the
central nervous system (CNS).
The present study was undertaken to characterise the pattern of the distribution of the orexin-A and
orexin-B immunoreactivities in the rat brain using immunohistochemistry. Furthermore, we also
examined the distribution of orexin fibres in the spinal cord.
Materials and Methods
Wistar rats of both sexes (200-250 g) were group housed in standard cages and maintained
on a 12 h light/dark cycle, 55% relative humidity, with food and water ad libitum. Polyclonal
anti-orexin-A and anti-orexin-B antisera were used for the immunohistochemical experiments
were raised in New Zealand white rabbits against synthetic peptide C-terminal sequences. All
antisera were diluted in a solution of 0.1 M phosphate buffered saline (PBS; pH 7.2), 2.5%
bovine serum albumin, 0,25% sodium azide and 2% triton X-100. Antisera specificity was
determined in control experiments in which the primary antiserum was (1) omitted or (2) preabsorbed with an excess of anti-orexin-A+control peptide orexin-A (1:100) and anti-orexin-B+
control peptide orexin-B peptide (1:500).
Fifteen rats were deeply anaesthetised with sodium pentobarbitone (150 mg/kg, i.p; Rh™ne M?rieux
Harlow, UK) and perfused transcardially via the left ventricle with Krebs solution followed by 4%
paraformaldehyde in 0.1M PBS. All subsequent steps were performed at room temperature unless
indicated otherwise. Brain and spinal cord were removed, post-fixed with 4% paraformaldehyde in
PBS (4-6 h), cryoprotected with 30% sucrose in PBS (16 h). Brain block was cut serially into 40µm
thick coronal sections on a freeze knife microtome. Spinal cord sections were cut into 10µm in a
sample order on a cryostat.
Immunofluorescence protocol was used to visualize orexin immunoreactivity. Free-floating sections
were washed five times with PBS (15 min per wash), incubated with 10% donkey serum in PBS (1 h)
and washed once with PBS prior to incubation with anti-orexin-A (1:100) or anti-orexin-B (1:500)
antiserum overnight at 4 0C. Sections were washed five times with PBS before incubation (1 h)
with either biotinylated rabbit anti-species IgG as appropriate (1:500) then fluorescein (DTAF)conjugated streptavidin (1:400) (both from Jackson, U.S.A.) or anti-rabbit CY3 (1:150)
(Jackson) and finally mounted on chrome alum gelatin-coated microscope slides and
coverslipped with Vectashield (Vector, U.K.). Sections were examined by conventional
fluorescence microscopy (596 nm excitation, 615 nm emission).
A low magnification of coronal sections through
the hypothalamus labeled with orexin-B antisera.
Figure 1.
Results
Antisera specificity:
To check the specificity
of the orexin antisera,
anti-orexin-A antiserum
was preabsorbed with
control peptide orexin-A
and
anti-orexin-B
antiserum with control
peptide orexin-B and
omission of primary
antibody.
Immunoreactivity
for
orexin-A or orexin-B was
not observed in the
antiserum
omission
control
experiment.
Orexin-A and orexin-B
immunoreactivities were
abolished entirely by preabsorbtion with orexin-A
or -B control peptide.
Orexin-Bimmunoreactivity:
Orexin-B
immunoreactivity
detected
in
A higher magnification showing the distribution of
orexin-B-immunoreactive neurones and their processing in
was the lateral hypothalamic area and perifornical nucleus.
Figure 2.
the
cytoplasm of neurones
that were restricted to
the
hypothalamus.
Orexin-B
immunoreactive
neurones
are
concentrated bilaterally
and symmetrically in
the LHA and PeF, and
more
sparsely
distributed medially in
the dorsal aspects of
the
anterior
hypothalamic area and
the
dorsomedial
hypothalamic nucleus
(DMH) (Fig. 1, 2).
Morphologically,
the
majority of orexin-B
positive
neurones,
which were similar in
size
and
shape,
appeared
typically
spherical
and
had
several
primary
dendrites
with
few
secondary branching
(Fig. 2).
Axons immunoreactive
for
orexin-B,
were
observed in the brain and
spinal cord, were both
varicose and non-varicose
in appearance. In the
hypothalamus,
an
extensive network of
orexin-B immunoreactive
fibres was observed in the
LHA and PeF, in close
apposition to the orexin-B
containing neurones (Fig.
1,
2).
Fibres
immunoreactive
for
orexin-B
were
also
A low magnification of coronal sections through
the hypothalamus labeled with orexin-A antisera.
Figure 3.
A higher magnification showing orexin-Aimmunoreactive neurones and their processing are
distributed in the lateral hypothalamic area and perifornical
nucleus.
Figure 4.
abundant
throughout
much of the rest of the
hypothalamus
with
moderately
dense
projections
to
the
paraventricular nucleus,
supraoptic
nucleus,
DMH, parastrial nucleus
and preoptic areas. With
the noticeable exception
of
arcuate
nucleus,
median eminence and
other subregions of the
hypothalamus where a
fairly dense network of
immunoreactive fibres is
noted.
Orexin-Bimmunoreactive
fibres
were abundant in the
septal nuclei. In the
thalamus, the midline
nuclei
exhibited
a
distinctive distribution of
orexin-B-immunoreactive
fibres. Orexin-B fibres
were spread throughout
the spinal cord at all
levels
investigated
(cervical,
thoracic,
lumbar and sacral) with
obvious localisation of
fibres in the superficial
dorsal horn.
Orexin-Aimmunoreactivity: The
distribution of orexin-A
immunoreactive
neurones in the LHA
and PeF was similar to
that of orexin-B (Fig. 3,
4).
Fibres
immunoreactive
for
orexin-A were also
similar to that of orexinB in the brain (Fig 4)
and spinal cord (Fig.5).
Discussion
With the use of
antibodies specific
against orexin-A or
orexin-B peptides, the
present
immunocytochemical
Photomicrograph showing the pattern of
fluorescein-labelled orexin-A immuno-reactive fibres in
lamina I-II of the spinal cord.
Figure
5.
investigation
determined the
distribution of orexincontaining neurones
and processes in the
rat brain and spinal
cord. Populations of
orexin-A- and orexin-Bimmunoreactive
neurones were found in
the hypothalamus
concentrated in
LHA/PeF region; orexin
neurones were not
found elsewhere in the
rat brain and spinal
cord. These findings
are in agreement with
recent studies on the
localisation in the rat
brain of neurones
containing the orexin
peptides (1, 3-7), or
prepro-orexin-mRNA
(1,5). With the
exception of the testis,
prepro-orexin mRNA
has not been described
elsewhere in the
central or peripheral
nervous systems (1).
Both varicose and nonvaricose orexin-B fibres
were distributed
extensively throughout
the hypothalamus but
also projected to
numerous other sites
(e.g. the septal nuclei,
the midline thalamic
nuclei). A similar
pattern of fibre
distribution has been
described recently in
studies utilised antisera
raised against the
orexin-A, orexin-B,
hypocretin-2 or preprohypocretin peptide
sequences (5-7).
Furthermore, we also
report here the novel
observations that
orexin-A and orexin-B
fibres are located at all
levels of the spinal
cord. Physiological
studies with whole-cell
voltage-clamp
recording and digital
imaging, have
indicated that
hypocretin receptors
were located both on
the cell body and on
axon terminals (8).
Finding that some
spinal neurones
respond to hypocretin
application suggests
that receptors may be
expressed by some
subpopulations of
neurones.
The
hypothalamic
area
has
been
implicated
in
the
regulation of feeding
behaviour and energy
homeostasis ever since
the classic experiments
showing that animals
with
lateral
hypothalamic lesions
had decreased food
intake and lower set
point for body weight
(9-13). Since it has
been
demonstrated
that orexin stimulates
feeding (1,14) and
orexin
mRNA
is
increased with fasting
(1), we shall focus on
the
possible
participation of orexin
in feeding.
The hypothalamic area has been implicated in the regulation of feeding behaviour and
energy homeostasis ever since the classic experiments showing that animals with lateral
hypothalamic lesions had decreased food intake and lower set point for body weight (9-13).
Since it has been demonstrated that orexin stimulates feeding (1,14) and orexin mRNA is
increased with fasting (1), we shall focus on the possible participation of orexin in feeding.
Our observations extend knowledge of these novel peptides in a number of ways. Firstly,
orexin-A and orexin-B processes are located at all levels of the spinal cord with a pattern of
expression similar to that found for hypocretin-2 (14). Secondly, when compared to the
distribution of orexin-B immunoreactive fibres, those processes detected using orexin-A
antiserum were similar pattern in many regions of the brain and the spinal cord.
Despite the increasing understanding of the roles of the mediobasal hypothalamus in
regulating feeding behaviour and energy homeostasis, the role of the LHA is still debated.
The most important feature of the orexin system is that its neuronal cell bodies are localised
only around the LHA and DMH and has a expansive projection. Our findings suggest that
orexin neurones might receive information from the medial hypothalamus and other peripheral
tissues, and send integrated information to outside of the hypothalamus, including the
cerebral cortex, brainstem and limbic system. This distribution pattern is similar to that of
melanin-concentrating hormone (MCH) neurones (15,16), but double staining studies has
showed that these neurones are distinct from each other (17). This finding suggests that the
orexin and MCH systems have a important roles in feeding behavior.
The present study has demonstrated that the distribution of orexin cell bodies and fibres in the LHA
and PeF region of the hypothalamus would allow them to participate in the control of feeding and
energy balance. Our finding that orexin neurones are confined to the LHA region could support this
contention since ~25% of LHA cells are glucose sensitive (stimulated by hypoglycaemia) and affected
by high insulin levels (18,19). Thus, orexins may influence food intake by transmitting information that
influences ingestive behavior from the hypothalamus to these regions. Further experimental
investigation is needed to determine whether these neurones are immunoreactive for orexin.
The morphological distribution of orexin-immunoreactive neurones and processes indicates that the
orexins may participate in a host of other functions. For example, the LHA and perifornical area are
reported to relate to cardiovascular responses to emotional situations or defensive behavior, and
furthermore, orexin-A fibres were observed in nuclei that are involved in cardiovascular control such as
the area postrema, the parabrachial nuclei, the locus coerulus, the nucleus of the solitary tract, the
habenula and the periaqueductal gray (5,11,20). Finally, the LHA/ PeF area is also involved in the
control of other processes such as fluid and temperature homeostasis, salivation, olfaction, pancreatic
function and the regulation of autonomic function (11,12), and furthermore, these potentially diverse
actions of orexin are supported by the extensive distribution of orexin fibres in the brain and spinal
cord.
In conclusion, the hypothalamus has a major role in regulating various behaviors that
contribute to homeostasis such as arousal, feeding, and thermoregulation by integrating
external and internal stimuli (21). Orexins, which have been discovered and characterised a
family of neuropeptides, stimulate food consumption by acting on the lateral hypothalamus
(1). Nahon (16) has discovered one other neuropeptide in the feeding centres of
hypothalamus, MCH, that mediates feeding behavior. The localization of orexin cells bodies
and processes to the LHA/PeF area suggests that the orexins may participate in the control of
feeding and energy homeostasis. Findings of orexin fibres throughout much of the
hypothalamus and at other sites in the brain, which have been implicated in feeding, are
consistent with the appetite-stimulating properties of these peptides. As a result, the spread of
the orexin fibres in the brain and spinal cord raises the possibility that the orexins may also be
implicated in other vegetative and neuroendocrine regulations or related to general arousal
states.
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