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The Journal of Neuroscience, April 15, 1999, 19(8):3171-3182
Hypothalamic Hypocretin (Orexin): Robust Innervation of the
Spinal Cord
Anthony N.
van den Pol
Department of Neurosurgery, Yale University School of Medicine, New
Haven, Connecticut 06520
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ABSTRACT |
Hypocretin (orexin) is synthesized by neurons in the lateral
hypothalamus and has been reported to increase food intake and regulate
the neuroendocrine system. In the present paper, long descending axonal
projections that contain hypocretin were found that innervate all
levels of the spinal cord from cervical to sacral segments, as studied
in mouse, rat, and human spinal cord and not previously described. High
densities of axonal innervation are found in regions of the spinal cord
related to modulation of sensation and pain, notably in the marginal
zone (lamina 1). Innervation of the intermediolateral column and lamina
10 as well as strong innervation of the caudal region of the sacral
cord suggest that hypocretin may participate in the regulation of both the sympathetic and parasympathetic parts of the autonomic nervous system. Double-labeling experiments in mice combining retrograde transport of diamidino yellow after spinal cord injections and immunocytochemistry support the concept that hypocretin-immunoreactive fibers in the cord originate from the neurons in the lateral
hypothalamus. Digital-imaging physiological studies with fura-2
detected a rise in intracellular calcium in response to hypocretin in
cultured rat spinal cord neurons, indicating that spinal cord neurons
express hypocretin-responsive receptors. A greater number of cervical cord neurons responded to hypocretin than another hypothalamo-spinal neuropeptide, oxytocin. These data suggest that in addition to possible
roles in feeding and endocrine regulation, the descending hypocretin
fiber system may play a role in modulation of sensory input,
particularly in regions of the cord related to pain perception and
autonomic tone.
Key words:
hypothalamus; neuroendocrine; spinal cord; autonomic
nervous system; pain; food intake; energy regulation; lamina 1
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INTRODUCTION |
We recently reported a new
hypothalamic peptide, hypocretin, in neurons of the lateral
hypothalamus-perifornical region (de Lecea et al., 1998 ). On the basis
of immunocytochemistry, in situ hybridization, and Northern
blots, synthesis of the peptide was restricted to cell bodies in the
lateral hypothalamic region of the brain and not elsewhere (Gautvik et
al., 1996; de Lecea et al., 1998; Sakurai et al., 1998; van den Pol et
al., 1998). Injections of this peptide, also called orexin, into the
brain caused rats to increase their food intake; food restriction
caused an increase in peptide synthesis, suggesting a role for the
peptide in energy metabolism (Sakurai et al., 1998). Innervation of the
arcuate nucleus and electrophysiological responses of identified
neuroendocrine neurons suggested that the peptide might also be
involved in regulation of the neuroendocrine system (van den Pol et
al., 1998). This is consistent with the finding of a large number of
hypocretin axons in synaptic contact with neuropeptide Y cells of the
arcuate nucleus, suggesting that the activity of the hypothalamic
neuropeptide Y system may be modulated by hypocretin (Horvath et al.,
1999). The region of the brain with the highest density of
hypocretin-containing neurons, the lateral hypothalamus, has been
implicated in control of feeding, arousal, and autonomic control
relating to both sympathetic and parasympathetic systems.
Hypocretin-immunoreactive axons project to many regions of the brain,
including the nucleus of the solitary tract, dorsal motor nucleus of
the vagus, and multiple hypothalamic nuclei (Peyron et al., 1998),
regions that may contribute to the regulation of the autonomic nervous system.
Ultrastructural analysis demonstrates that within axon terminals,
hypocretin is probably localized to dense core vesicles, suggesting a
possible transmitter function (de Lecea et al., 1998). Two receptors
have been cloned that respond to hypocretin (Sakurai et al., 1998),
further substantiating a transmitter role for the peptide. Whole-cell
recordings in culture show that hypocretin increases synaptic activity
(de Lecea et al., 1998). Hypocretin acts both on postsynaptic receptors
to increase cytosolic calcium and on presynaptic receptors to enhance
release of amino acid neurotransmitters (van den Pol et al., 1998). The
hypocretin gene localizes to chromosome 17q21 (de Lecea et al., 1998;
Sakurai et al., 1998), a region that has been implicated in some forms of rare but severe human brain disease (Wilhelmsen et al., 1994; Wijker
et al., 1996). Hypocretin projections to the spinal cord have
not previously been described. In the present study, long descending
axons that contain hypocretin immunoreactivity were found innervating
all levels of the spinal cord. Both immunocytochemistry with normal and
colchicine-treated rats and in situ hybridization studies
have located hypocretin-positive cells only in the lateral hypothalamic
area. Based on this and on double labeling with retrograde tracers and
immunostaining, the hypocretin-immunoreactive axons in the spinal cord
appear to originate only from these neuronal cell bodies in the hypothalamus.
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MATERIALS AND METHODS |
Immunocytochemistry. Six- to 8-week-old rats (albino
Sprague Dawley, n = 5) and mice (Swiss Webster
background, n = 12) were anesthetized with sodium
pentobarbital (80 mg/kg) and perfused transcardially with a fixative
containing 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M phosphate buffer. Sections were cut either on a vibratome
at a thickness of 30-50 µm or on a cryostat at 20-30 µm. After
washing in normal buffer containing 0.1% lysine, 1% bovine serum
albumin, 1% normal goat serum, and 0.3% Triton X-100, sections were
incubated overnight in primary rabbit antiserum against the neuroactive
peptide hypocretin-2. After washing in buffer, sections were incubated
in biotinylated goat anti-rabbit Ig, washed, and then treated
with avidin-biotin-peroxidase complex (Vector Laboratories,
Burlingame, CA), as described in greater detail elsewhere (van den Pol,
1985). Sections were then reacted with diaminobenzidine and hydrogen
peroxide to reveal the horseradish peroxidase complex. Some sections
were counterstained with neutral red to reveal spinal cord cells. Most
of the analysis was done with cross-sections, but some series of
sections were also cut in horizontal or sagittal planes.
Human tissue (n = 2) was obtained within 10-18 hr
after death. Death was caused by cardiac arrest. Subjects were 50-70
years old. Slices of cervical cord were immersed in Bouin's fixative for several days and then cut on a cryostat in 30-50 µm sections.
The primary hypocretin antiserum used binds to the neuroactive peptide
hypocretin-2, as described in detail elsewhere (van den Pol et al.,
1998). Control adsorption with the antigen blocked staining, as did
elimination of incubation in primary antiserum. Both immunoperoxidase
and immunofluorescence gave similar patterns of staining. Use of a
second antiserum raised in a different rabbit against hypocretin-2 also
gave similar patterns of staining. Furthermore, a third antiserum (a
generous gift from Drs. T. Kilduff, G. Sutcliffe, and L. de Lecea,
Scripps Research Institute, La Jolla, CA) raised against the
preprohypocretin sequence (de Lecea et al., 1998) showed similar
patterns of immunoreactive axons in the spinal cord, as described in Results.
Tissue culture. To determine whether spinal cord neurons
showed a physiological response to hypocretin, cultures were made from
embryonic day 18 rat spinal cords. The cervical cord was removed and
cultured as previously described for other regions of the CNS (van den
Pol et al., 1998). Cells were plated on glass coverslips precoated with
poly-L-lysine (540 kDa; Collaborative Research,
Bedford, MA) and allowed to grow for 7-12 d before being used for
digital imaging.
Calcium digital imaging. Cultures were loaded with fura-2 AM
(5 µM; Molecular Probes, Eugene, OR) and studied on a
Nikon (Tokyo, Japan) Diaphot microscope with objectives that passed 340 nm light (Olympus Optical, Tokyo, Japan; 40×) using a 150 W xenon bulb for illumination. Light was controlled by a Lambda 10 filter wheel (Sutter Instruments, Novato, CA) interfaced with a lab computer. Software from Universal Imaging (Media, PA; Image 1/Fluor) was used to
collect data, which was then analyzed off-line with Igor Pro software
(WaveMetrics, Lake Oswego, OR). The equation developed by Grynkiewicz
et al. (1985) was used to determine relative cytosolic calcium levels.
During imaging, coverslips were loaded into a laminar flow chamber
(Forscher et al., 1987) that had a volume of ~180 µl and eight
inlet ports that allowed switching between different agonists. Details
of fura-2 calcium digital imaging are described elsewhere (van den Pol
et al., 1996, 1998; Obrietan and van den Pol, 1997). Hypocretin-2 was
made in-house and purified to 98% purity; the same peptide was used
for generating the antisera for immunocytochemistry and for the
physiological studies with calcium imaging. Physiological studies with
hypocretin-1 were done with the peptide sequence described by Sakurai
et al. (1998), and this peptide was obtained from Phoenix Pharm
(Mountain View, CA).
Most photomicrographs here were taken with a Spot-2 digital camera
(Diagnostic Instruments, Sterling Heights, MI) in the monochrome mode.
Some were taken with film, and the film was digitally scanned with a
Polaroid (Waltham, MA) film scanner. Images were printed on an Eastman
Kodak (Rochester, NY) 8650 dye sublimation printer. In some
micrographs, a dark-field condenser was used, and in some the
immunoreactive fibers were illuminated by fiber optics through the side
of the glass slide. Contrast and gray scale were digitally corrected.
Retrograde transport studies. To ensure that hypocretin
axons in the spinal cord arose from hypothalamic origins, the
fluorescent dye diamidino yellow (Sigma, St. Louis, MO) was injected
into six to eight regions of the cervical and thoracic cord of five mice by microinjections with a Hamilton (Reno, NV) microsyringe. Two to
4 d after injections, mice were anesthetized as above and perfused
with 4% paraformaldehyde. Forty micrometer sections were cut in the
lateral hypothalamus, and hypocretin-immunoreactive cell bodies were
immunostained with Alexa546 goat anti-rabbit Ig.
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RESULTS |
White matter
In both rat and mouse, hypocretin-immunoreactive axons descended
the entire length of the cord, with the highest concentration of
descending axons in the dorsal region of the lateral white matter (Fig.
1A). Long descending
immunoreactive axons were most clear in horizontal and sagittal
sections of the cord but could also be found in cross-sections. In
single sections, hundreds of immunoreactive axons could be found
traveling in this region of the white matter (Fig. 1). More ventrally
in the lateral white matter, descending axons ran parallel with the
long axis of the cord and often turned medially to enter the gray
matter (Fig. 1B). A low density of isolated axons
were found in all other regions of the white matter also, often
oriented in a radial manner, parallel with large dendrites that
protrude from the gray matter into the white matter in lateral and
ventral white matter. In the dorsal cord, some descending axons entered
the cord via Lissauer's tact (Fig. 1C). Occasional solitary
axons were found traversing the dorsal columns in all levels of the
cord. This was particularly common in the sacral cord where the dorsal
columns are relatively small. Axons were medium-size in the white
matter, with a diameter of 1-2 µm. When turning medially and
entering the gray matter, axonal diameter ranged from 2 down to 0.2 µm.
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Figure 1.
Hypocretin-immunoreactive axons in rat white
matter, horizontal section. A, In the dorsal region of
the lateral white matter, a high density of immunoreactive axons is
found in this dark-field micrograph. B, More ventrally
in the lateral white matter (LW), some axons turn
medially to enter the gray matter (GM,
top). C, In the dorsal part of the cord
in the region of Lissauer's tract (LT), some
axons leave a group of descending axons at the bottom
and enter into part of the marginal zone (MZ) where
boutons are found (arrows). Scale bars:
A, 30 µm; B, 25 µm; C,
15 µm.
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Gray matter
Immunoreactive axons were found in all 10 laminae of the cord,
with a higher level of innervation in the dorsal and midline cord than
in the ventral horn. Identification of the laminae of the spinal cord
was facilitated by descriptions by Molander et al. (1984, 1989) in
rodents, patterned after Rexed's work on the cat (1952, 1954). At any
particular level of the cord, both large and small immunoreactive
boutons were found, with large boutons approaching 3 µm diameter and
small ones of 0.5 µm diameter. In some regions, thin axons with a
fixed diameter appeared to pass through, with little indication of
boutons (Fig. 2A). In
others, frequent boutons en passant (Fig. 2B,C,D,F)
and termineaux (Fig. 2E) were identified.
Whereas in the white matter axons maintained a more homogeneous size,
in the gray matter a wide variety of thick and thin shapes and
diameters were adopted (Fig. 2), suggesting that the local milieu may
play a role in determining the local shape of these axons. In the same
section, axons included those that showed a paucity of boutons and a
diameter of <1 µm (Fig. 2A), axons that were
unbranched and fairly straight and run over long distances through
several laminae with large numbers of boutons at regular intervals
(Fig. 2B), thick axons with a diameter approaching 2.5 µm with large numbers of boutons (Fig. 2D), axons that
showed a substantial number of branches (Fig. 2E),
and thin axons that tended to wander with intermittent and infrequent
boutons.
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Figure 2.
Hypocretin axons with a variety of shapes in rat
cord. A, Thin parallel axons entering the gray matter
from the dorsolateral white matter. B, Long axons
descend from lamina 3/4 down to lamina 7 with boutons at regular
intervals in each lamina the axon traverses. C, Axons
may traverse the intermediolateral column with frequent boutons.
D, Some axons are thick with large numbers of tightly
packed boutons investing a small region of lamina 5. E,
Lateral to the central canal, axons have frequent branches.
F, In the ventral horn, thin axons maintain only a few
boutons. Scale bar, 20 µm.
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Dorsal horn
Throughout the entire length of the spinal cord in rats and mice,
the dorsal horn received a proliferation of immunoreactive fibers
(Figs. 3,
4). Within the dorsal horn, lamina 1 received the strongest innervation (Fig. 3), with immunoreactive axons surrounding lamina 1 cells that have a slightly elliptical cell body
that is oriented parallel with the surface of the cord, or in the
medial and lateral part of lamina 1, with the external border of the
gray matter. Fibers had numerous boutons en passant and termineaux,
characterized by asymmetrical swelling on one side of the axon. Axons
were found that descended along the most superficial part of the cord
over lamina 1, sometimes turning from the external localization to
penetrate into lamina 1 (Fig. 1C). The true extent of the
innervation of lamina 1 is best appreciated in sagittal or horizontal
sections (Fig. 4A), which reveal a great abundance of
hypocretin axons and a large number of boutons in single sections.
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Figure 3.
Lamina 1 of the dorsal horn in rat. A single
50-µm-thick section was used to trace hypocretin-immunoreactive axons
in a cross-section of the midcervical dorsal horn. Boutons were
particularly prevalent in lamina 1. DC, Dorsal columns.
Scale bar, 70 µm.
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Figure 4.
Dorsal horn, horizontal sections in rat.
A, A substantial number of hypocretin-immunoreactive
axons are found in 40-µm-thick horizontal sections of the midcervical
cord, shown here from camera lucida drawings. B, In
contrast to the marginal zone above, in laminae 2 and 3, relatively few
axons are found, and those that do appear show little branching and few
boutons. Both regions were drawn with the same size dimensions. Scale
bar, 45 µm.
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Fibers also entered laminae 2 and 3 after passing through lamina 1, but
these were not as dense as those in lamina 1 (Figs. 3,
4B). In the mouse, relatively few fibers were found
in layers 2 and 3 of the substantia gelatinosa; in the rat there were
more, but still fewer than found in lamina 1. Some dorsal roots were also examined to determine whether any immunoreactive axons would be
present there, but immunoreactivity was not found in the root. There
were, however, numerous immunoreactive axons found at the dorsal root
entry zone, which descended from cell bodies in the hypothalamus and
turned ventrally to enter into the marginal zone of the dorsal horn.
Middle cord
Whereas there were relatively few axons in the dorsal white
matter, directly under the ventral aspect of the dorsal columns and
blending between the edge of the gray and white matter a number of
immunoreactive axons were found (Fig.
5B). One of the strongest regions of innervation was around the central canal in lamina 10, shown
in a dark-field micrograph in Figure 5A. Axons here were
sometimes closely associated with the ventricular ependymal cells. In
some regions of the thoracic and lumbar cord, the plexus of axons in
lamina 10 stretched out into the intermediomedial and intermediolateral
sympathetic cell groups.
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Figure 5.
Hypocretin-immunoreactive axons in rat gray
matter, horizontal section. A, In the midcord, the
highest density of hypocretin-immunoreactive axons is found around the
central canal (long arrows). The short
arrows indicate axons in lamina 10 immediately above the
central canal. B, At a slightly higher plane of
horizontal section, the ventral aspect of the dorsal columns is seen
and continues to the right in the direction of the
small arrow. On the right, just under the
dorsal column white matter (large arrow), the axon
density increases. C, In lamina 7, a
hypocretin-immunoreactive axon and some of its collaterals
(arrows) are found among cells stained with nuclear red.
D, In the ventral horn, occasional axons are found,
sometimes with swellings, as indicated by the arrow.
Scale bars: A, 20 µm; B, 22 µm;
C, D, 18 µm.
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Ventral horn
Immunoreactive axons wandered through the ventral horn into
laminae 7-9 (Fig. 5C). The density of axons was not high
here. Dispersed boutons en passant and termineaux were found among
different groups of cells in laminae 8 and 9 (Fig. 5D).
Longitudinal innervation
Hypocretin-immunoreactive axons with numerous boutons were found
in all levels of the spinal cord from C1 to the caudal part of the
sacral region. Differences between different segments were noted,
particularly related to cell groups such as the intermediolateral column of the thoracic and lumbar cord that received hypocretin innervation. In addition, axons and boutons were found just lateral to
this area in the white matter, a region where the long dendrites of
these cells may extend. The highest density of fibers per segment was
found in the caudal sacral spinal cord. Here the long descending axons
in the dorsal region of the lateral white matter turn into the gray
matter. As soon as the axons turned, boutons began to proliferate.
Axons crossed the midline with high frequency and were found in all
laminae, although the dorsal half of the cord was more heavily
innervated than the ventral half. Camera lucida drawings from the
sacral cord demonstrate the different morphological patterns that axons
assume even within one segment (S3) of the cord (Fig. 2). A similar
heterogeneous quality of axonal shape was found in rostral regions of
the gray matter.
Hypocretin-synthesizing neurons were found in the same lateral
hypothalamic area in mouse (Fig. 6) as previously described in the rat
(Peyron et al., 1998). Although the rat spinal cord, being considerably
larger, has more hypocretin-immunoreactive axons than the mouse, the
general pattern of axonal innervation was similar in rat and mouse.
The mouse spinal cord was used to construct a descending series of camera lucida drawings of the cord
from C2 to S4. These are shown in Figures
7 and 8. In
all segments shown, immunoreactive axons could be found in the
superficial region of the dorsal horn and in lamina 10 around the
central canal. Some of the axons that innervated lamina 10 traveled in a medial direction from the lateral white matter, and others descended through lamina 1 and continued ventrally along the interface of the
lateral aspect of the dorsal columns and the gray matter before entering into and branching in lamina 10.
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Figure 6.
Hypocretin-immunoreactive perikarya in mouse. In
the lateral hypothalamus and perifornical area,
hypocretin-immunoreactive cell bodies and their processes are labeled.
The midline is on the left, and lateral is on the
right. Scale bar, 60 µm.
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Figure 7.
Hypocretin axons in mouse spinal cord, C2-T4.
Camera lucida drawings were made in regular intervals from 30 µm
cross-sections. The outline of the cord and the outline of the gray
matter are indicated by the thick lines. To allow
visibility of axons when reduced in size, the diameter of the axons is
greater on the drawing than it actually appeared in the microscope.
Axons that were descending the cord, particularly in dorsolateral white
matter, and oriented parallel with the long axis of the cord, which
consequently would appear only as a single point in these
cross-sections, were not included.
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Human spinal cord
In the human cervical cord, the general pattern of immunoreactive
fibers was similar to that of rodents. In the dorsal cord, immunoreactive axons were found at the surface of the cord (Figs. 9A,B,
10) penetrating down into Lissauer's
tract, as described by Truex and Carpenter (1969). In the dorsal horn,
immunoreactive axons formed a half-ring in the region of the marginal
zone, shown in the micrograph in Figure 9C and in the camera
lucida trace in Figure 10. Both thick and thin axons traversed lamina
10 in the region of the central canal (Figs. 9D,F-H, 10).
One aspect of hypocretin innervation of the human cord that differed
slightly from rat and mouse was the finding of immunoreactive axons
that grouped together at the surface of the ventrolateral white matter in an orientation perpendicular to the outer surface of the white matter (Figs. 9E, 10).
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Figure 9.
Hypocretin-immunoreactive axons in human spinal
cord, photomicrographs. A, In the dorsal region of the
human cervical cord, immunoreactive axons (large and
small arrows) are found at the surface of the cord and
down into Lissauer's tract. B, Higher magnification of
an axon in the same region as A. C, In
the dorsal horn the immunoreactive axons (arrows) form a
semicircle in the area of lamina 1. D, In lamina 10 a number of thin and thicker axons are found (arrows) in
this dark-field micrograph. E, Immunoreactive axons
(arrows) travel to the edge of the cord in the
ventrolateral white matter. The beaded appearance of immunoreactive
axons in the human cord is probably attributable to inadequate
immersion fixation, which may result in some axonal degeneration before
axonal stabilization. F, In lamina 10 axons and boutons
are found. Two sets of small terminal boutons are shown at higher
magnification in G and H, as indicated by
the arrows. Scale bars: A, E, 12 µm;
B, 7 µm; C, 20 µm; D,
18 µm; H, 15 µm.
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Figure 10.
Human cervical spinal cord. Camera lucida drawing
was made of hypocretin-immunoreactive axons in two adjacent 40 µm
sections of a C4-C5 cross-section of a human cord. DH,
Dorsal horn; VH, ventral horn; DC, dorsal
columns; cc, central canal; LW, lateral
white matter; VW, ventral white matter;
LT, Lissauer's tract. Scale bar, 1 mm.
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Retrograde transport from the spinal cord to the hypothalamus
After injections of diamidino yellow in the cervical and lumbar
spinal cord of mouse, hypothalamic neurons that maintained long
descending projections to the cord were labeled, mostly in their cell
nuclei (Fig. 11B).
The nuclear label was yellow. Hypocretin-immunoreactive neurons were
stained red with Alexa546. With fluorescence microscopy, neurons were
found that had a red cytoplasm and yellow nuclei, indicating that they
were immunoreactive for hypocretin and retrogradely transported the
diamidino yellow back to the lateral hypothalamus from the spinal cord.
These double-labeled cells were identified then as
hypocretin-synthesizing neurons (Fig. 11A), which
sent long axons to the spinal cord. In addition to cells that were double-labeled, other cells were found that showed yellow nuclei but
were not immunoreactive for hypocretin or that showed immunoreactivity for hypocretin but did not have yellow nuclei (Fig. 11). Within the
field of hypocretin-immunoreactive cell bodies (Fig. 6), no preferential distribution of those that projected to the spinal cord
was noted.
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Figure 11.
Retrograde transport of tracer to
hypocretin-immunoreactive cells in lateral hypothalamus. In the same
section, neurons immunoreactive for hypocretin
(A) and also labeled with diamidino yellow
(D.Yellow) (B) transported from the spinal
cord back to the hypothalamus. The two vertical arrows
in A and B show hypocretin-immunoreactive
neurons in which the nucleus is labeled with diamidino yellow. The
short arrowhead shows a hypocretin-immunoreactive neuron
in the same field that shows no nuclear labeling for diamidino yellow.
Scale bar, 5 µm.
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Calcium responses to hypocretin in cord neurons
We previously showed that hypocretin evokes a robust calcium rise
in some hypothalamic neurons but not in hippocampal neurons, and that
this calcium rise is dependent on activation of plasma membrane calcium
channels (van den Pol et al., 1998). To determine whether spinal cord
neurons would show any physiological response to hypocretin, the
calcium response of cultured rat cervical cord neurons to hypocretin-2
was tested after at least 10 d in vitro. Using fura-2
ratiometric imaging with a criterion of at least a 20 nM
rise at the time of peptide application, a rise in calcium in response
to hypocretin was found in 14% of 235 cells recorded in five sets of
experiments. Addition of hypocretin (1 µM) evoked a
calcium rise that recovered to lower baseline levels after hypocretin washout (Fig. 12A).
Some cells showed a stable plateau of calcium during hypocretin
activation, and others showed a substantial increase in calcium
transients during peptide treatment. Two different peptides may be
derived from preprohypocretin, hypocretin 1 and 2. The response of
single neurons to each of the peptides was examined. As a control, a
C-terminal 17-amino acid peptide derived from the preprohypocretin
sequence with no expected physiological action was also used. Neurons
responded to both hypocretin 1 and hypocretin-2 with a calcium rise but
not to the C-terminal control (Fig. 12B) or to
stopcock changes in normal buffer. After peptide washout, calcium
levels returned to baseline values.
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Figure 12.
Spinal cord neurons show calcium rise in response
to hypocretin. A, Hypocretin-2 (H, 1 µM) was added by bath perfusion. In the presence of
hypocretin, cytoplasmic calcium increased as measured with fura-2
imaging. Horizontal lines mark the presence of the
peptide. B, A neuron responds both to hypocretin-1
(H-1) and hypocretin-2 (H-2) but not to
the 17-amino acid inactive C-terminal fragment
(17) of preprohypocretin. C, D, In
two neurons recorded simultaneously, the response to hypocretin-2
(H, 1 µM) and oxytocin
(Oxy, 1 µM) was compared. In
C, the neuron responds to hypocretin but shows little
response to oxytocin. The neuron in D responds to both
oxytocin and hypocretin.
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Oxytocin is another hypothalamic peptide that has been described in the
spinal cord. Some of the regions of oxytocin innervation in the cord
are similar to that for hypocretin. Because the number of oxytocin
neurons projecting to the cord from the hypothalamic paraventricular
nucleus is greater than the number of vasopressin neurons, which also
innervate the cord (Sawchenko and Swanson, 1982), and because oxytocin
has been reported to have an excitatory role in the hypothalamus
(Theodosis, 1985), the responses of hypocretin were compared with those
of oxytocin. The calcium response to hypocretin was compared with
equimolar concentrations of oxytocin (1 µM) on the same
neurons. Almost twice as many cells responded to hypocretin compared
with oxytocin (Fig. 12C,D). Of 182 total cells in four sets
of experiments, 14.8% responded to hypocretin, but only 7.6%
responded to oxytocin.
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DISCUSSION |
The present study describes for the first time a strong projection
from hypocretin-immunoreactive cells in the lateral hypothalamic area
to all levels and all laminae of the spinal cord. Although some fibers
can be found in all regions of the cord, some regions receive a more
substantial and selective innervation. Hypocretin innervation shows
similar patterns of innervation in the mouse, rat, and human,
suggesting some functional importance facilitating evolutionary
conservation of this tract.
Melanin-concentrating hormone (MCH) is another peptide synthesized by
neurons in the same region of the lateral hypothalamus where
hypocretin-containing cells are found (Skofitsch et al., 1985; Zamir et
al., 1986; Fellmann et al., 1987, 1989). Similar to hypocretin, MCH has
been implicated in the control of feeding behavior (Qu et al., 1996).
Although no figures were presented, an MCH projection to the spinal
cord was noted (Bittencourt et al., 1992). This raises the question of
whether the cells that express hypocretin are the same as those that
synthesize MCH. Double-labeling studies failed to find any neurons that
expressed both peptides, suggesting that in fact the two peptides are
found in different populations of neurons (Elias et al., 1998; Peyron et al., 1998). Furthermore, Bittencourt et al. (1992) reported that the
MCH projection to the cord had approximately similar levels of
innervation in the marginal zone, substantia gelatinosa, ventral horn,
and central gray (Bittencourt and Elias, 1998) and smaller projections
to the intermediolateral column. This is in striking contrast to
hypocretin, which showed a strong projection to the intermediolateral
column, a higher innervation of the central gray than the ventral horn,
and a much higher innervation of the marginal zone than of the deeper
substantia gelatinosa.
That some neurons were immunoreactive for hypocretin but were not
retrogradely labeled with dye transported from the cord could suggest
that not all hypocretin-containing neurons project to the spinal cord.
An alternate explanation is that not all neurons that send axons to any
specific region would necessarily be labeled with retrogradely
transported dye, possibly because of damage during dye injection or to
small-caliber fibers with limited transport. Some cells showed
diamidino yellow labeling but no hypocretin immunoreactivity. These
could represent other classes of neurons in the lateral hypothalamus
that project to the spinal cord, for instance, the neurons that contain MCH.
Hypocretin function in spinal cord
What is the physiological role of hypocretin in the spinal cord?
This may in part be addressed by examining the role of the lateral
hypothalamus in general. It has been implicated in the regulation of
feeding (Powley and Keesey, 1970; van den Pol, 1982) and
detection of circulating glucose levels (Oomura, 1983). This is
consistent with suggestions that hypocretin injections may enhance food
intake (Sakurai et al., 1998). Hypocretin may also participate in
endocrine regulation (van den Pol et al., 1998).
Spinal cord and other extrahypothalamic projections from the
hypothalamus have been described previously, particularly a projection from parvocellular neurons of the paraventricular nucleus that contains
oxytocin, vasopressin, and their associated neurophysins (Hancock,
1976; Swanson, 1977; Sofroniew and Weindl, 1978; Hosoya and Matsushita,
1979; Armstrong et al., 1980; Nilaver et al., 1980; Swanson and
Sawchenko, 1983; van den Pol and Collins, 1994). These axons innervate
the intermediolateral column of the thoracic spinal cord (Swanson,
1977) and also innervate laminae 1 and 10, a partial overlap with the
projection from hypocretin axons described in the present study.
Although the projection from the paraventricular nucleus has received
the most attention within the hypothalamus, the spinal projection from
the lateral hypothalamus is greater, based on a larger number of
lateral hypothalamus (LH) cells labeled after retrograde dye transport
from the cord (Hosoya, 1980). Projections from the paraventricular
nucleus (Ono et al., 1978) and LH in general (Saper et al.,
1976) have been suggested to be ipsilateral; although this may be a
general rule, in the present study hypocretin axons were found crossing
the midline particularly in the dorsal region of the central gray
(lamina 10) at all levels of the cord. This is particularly striking in
the sacral cord where axons cross the midline frequently, suggesting
that some LH cells innervate the cord bilaterally. Another point of
comparison relates to the response of cervical cord neurons to
hypocretin and oxytocin. Twice as many cells responded to hypocretin as
to oxytocin, suggesting that more cervical spinal cord cells may
express receptors responsive to hypocretin than to oxytocin. Because
thoracolumbar neurons receive a strong oxytocin innervation, the
relative response to oxytocin and hypocretin may be different in
neurons there. Alternately, hypocretin receptors may more closely
couple to calcium regulation than do oxytocin receptors.
Based on the innervation patterns of hypocretin in the spinal cord, it
appears that the peptide may modulate both the sympathetic and
parasympathetic parts of the autonomic nervous system. Innervation of
the intermediolateral cell group in thoracic and lumbar cord suggests
an involvement in sympathetic regulation. Hypocretin innervation of the
nucleus of the solitary tract and dorsal motor nucleus of the vagus
(Peyron et al., 1998), coupled with the innervation of the caudal
sacral cord described here suggest that a hypocretin may be involved in
regulation of the parasympathetic system.
Inferences about hypocretin function can be made on the basis of the
laminae in the spinal cord that hypocretin preferentially innervates.
The innervation of the marginal zone, lamina 1, throughout all segments
of the cord suggests that hypocretin may be involved in modulation of
pain and thermal sensation. Although most of the axons in Lissauer's
tract, dorsolateral to the dorsal horn, are primary sensory axons
(Chung and Coggeshall, 1982), hypocretin axons descend here before
entering lamina 1. Previous work has shown fine-caliber primary
afferents terminating in lamina 1 (Kumazawa and Perl, 1978). Three
types of cells in lamina 1 were found in physiological studies: those
that respond to mechanical or thermal nociception and a type that
responds to both noxious stimuli and non-pain-related temperature
changes (Christensen and Perl, 1970). In some sections of the present
study, some cells of the marginal zone were surrounded by
hypocretin-immunoreactive boutons, with neighboring cells in the same
lamina showing no innervation. This suggests that hypocretin fibers
selectively innervate subpopulations of neurons in the marginal zone,
perhaps either related to a functional sensory modality, such as a
c-fiber bearing pain information, or based on specific ascending
projections, which have been found innervating the locus coeruleus
(Cedarbaum and Aghajanian, 1978) or contributing to the
spinothalamic tract (Narotzky and Kerr, 1978). Hypocretin fibers may
therefore modulate pain sensation. This is consistent with observations
that stimulation of the lateral hypothalamus in the region of
hypocretin neurons produces pain analgesia (Dafny et al., 1996; Franco
and Prado, 1996), raising the interesting question of whether
hypocretin might possess antinociceptive properties in the cord.
Some of the same superficial dorsal horn regions innervated by
hypocretin axons receive an innervation from oxytocin- or
vasopressin-containing fibers from the paraventricular nucleus (Swanson
and McKellar, 1979), in contrast to long descending axons from
the cortex, which preferentially innervate the deeper layers of the
dorsal horn (Brown, 1981). Whereas the paraventricular nucleus is
reported to send only a sparse innervation to the sacral cord (Swanson and McKellar, 1979), a large hypocretin projection is found here. This
indicates that although there is some overlap between the descending
projections from different fiber systems of the hypothalamus to the
cord, there also appear to be some substantial differences.
Hypocretin axons with numerous boutons can be found in lamina X around
the central canal. Similar to lamina 1, cells here also respond to
nociceptive sensory information (Honda and Lee, 1985; Honda and Perl,
1985; LaMotte, 1987) and are also involved in autonomic function.
Injections of retrograde dyes into the cord label neurons in the
lateral hypothalamus (Veening et al., 1987). Anterograde transport of
tritiated substances from injections in the hypothalamus labeled axons
terminating in the intermediolateral column of the thoracic cord (Saper
et al., 1976). Coupled with the finding of hypocretin innervation of
the intermediolateral column of cells, this suggests that hypocretin
innervates a number of spinal cord regions that may be involved in
autonomic function. This contrasts with the view for other lateral
hypothalamic neurons containing a different peptide, MCH, for which
only a sparse innervation of autonomic preganglionic regions is
reported, leading to the suggestions that the "MCH system is not apt
to play a major direct role in modulating autonomic functions"
(Bittencourt et al., 1992).
The fact that some spinal neurons respond to hypocretin application
suggests that receptors may be expressed by some subpopulations of
neurons. In our earlier physiological studies with whole-cell voltage-clamp recording and digital imaging, we found that hypocretin receptors were located both on the cell body and on axon terminals (van
den Pol et al., 1998). The frequency of both glutamate- and GABA-mediated miniature postsynaptic currents was enhanced in the
presence of hypocretin, with no change in amplitude, suggesting the
presence of presynaptic hypocretin receptors on axon terminals. This
raises the interesting and testable hypothesis that hypocretin may
modulate sensory information from the primary afferents arising from
the dorsal roots and innervating lamina 1. If so, and if hypocretin
actions are similar in the cord, then hypocretin may hypothetically
increase the release of the neurotransmitter released by the primary afferents.
General role of hypocretin
Although projections from hypocretin-containing neurons to the
spinal cord have not been previously reported, in more rostral regions
of the brain hypocretin-containing axons have been found to selectively
innervate a number of brain regions that may play a role in setting a
general tone for brain activation or inactivation; this includes a
strong innervation of the locus coeruleus and innervation of the dorsal
motor nucleus and nucleus of the solitary tract, the raphe, and most
regions of the hypothalamus, including regions involved in sleep and
temperature regulation in the posterior hypothalamus and preoptic
area (de Lecea et al., 1998; Peyron et al., 1998; van den Pol et
al., 1998). Together with the results of the present study
demonstrating an innervation of both sympathetic and parasympathetic
regions of the autonomic regions of the cord, these results suggest
that hypocretin-containing neurons may play a widespread role in
setting a general level of activation of a large number of interrelated
systems related to the autonomic nervous system. This would include
systems regulating energy balances, as suggested by the feeding studies
of Sakurai et al. (1998). With this perspective, the enhancement of
feeding previously reported could be attributable to a general level of
autonomic activation, known to induce a variety of behaviors, including
but not restricted to feeding (Smith et al., 1997).
| |
FOOTNOTES |
Received Dec. 10, 1998; revised Jan. 29, 1999; accepted Jan. 29, 1999.
I thank Dr. C. LaMotte for helpful suggestions and Dr. Y. Yang and J. Belousova for excellent technical assistance.
Correspondence should be addressed to Anthony N. van den Pol,
Department of Neurosurgery, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520.
| |
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B. E. Levin
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T. Mochizuki, E. B. Klerman, T. Sakurai, and T. E. Scammell
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Hypocretin/Orexin Peptide Signaling in the Ascending Arousal System: Elevation of Intracellular Calcium in the Mouse Dorsal Raphe and Laterodorsal Tegmentum
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J. H. Peever, Y.-Y. Lai, and J. M. Siegel
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Gonadal Steroids Differentially Regulate the Messenger Ribonucleic Acid Expression of Pituitary Orexin Type 1 Receptors and Adrenal Orexin Type 2 Receptors
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M. J Follwell and A. V Ferguson
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Orexin-A Augments Voltage-Gated Ca2+ Currents and Synergistically Increases Growth Hormone (GH) Secretion with GH-Releasing Hormone in Primary Cultured Ovine Somatotropes
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Hypocretin release in normal and narcoleptic dogs after food and sleep deprivation, eating, and movement
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Selective Action of Orexin (Hypocretin) on Nonspecific Thalamocortical Projection Neurons
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Induction of Active (REM) Sleep and Motor Inhibition by Hypocretin in the Nucleus Pontis Oralis of the Cat
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Direct and Indirect Excitation of Laterodorsal Tegmental Neurons by Hypocretin/Orexin Peptides: Implications for Wakefulness and Narcolepsy
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Orexins/hypocretins excite rat sympathetic preganglionic neurons in vivo and in vitro
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Prepro-Orexin and Orexin Receptor mRNAs Are Differentially Expressed in Peripheral Tissues of Male and Female Rats
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Orexin-A Regulates Body Temperature in Coordination with Arousal Status
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Randomized trial of modafinil as a treatment for the excessive daytime somnolence of narcolepsy
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TOP
ABSTRACT
INTRODUCTION
