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The Journal of Neuroscience, October 1, 1998, 18(19):7962-7971
Presynaptic and Postsynaptic Actions and Modulation of
Neuroendocrine Neurons by a New Hypothalamic Peptide,
Hypocretin/Orexin
Anthony N.
van den Pol1, 2,
Xiao-Bing
Gao1,
Karl
Obrietan2,
Thomas S.
Kilduff2, and
Andrei B.
Belousov2
1 Department of Neurosurgery, Yale University Medical
School, New Haven, Connecticut 06520, and 2 Department of
Biological Sciences, Stanford University, Stanford, California 94305
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ABSTRACT |
A new orexigenic peptide called hypocretin (orexin) has recently
been described in neurons of the lateral hypothalamus and perifornical
area. The medial and lateral hypothalamus have been loosely called
satiety and feeding centers of the brain, respectively. Approximately
one-third of all medial and lateral hypothalamic neurons tested, but
not hippocampal neurons, show a striking nanomolar sensitivity to
hypocretin. As studied with calcium digital imaging with fura-2,
hypocretin raises cytoplasmic calcium via a mechanism based on
G-protein enhancement of calcium influx through plasma membrane
channels. The peptide has a potent effect at both presynaptic and
postsynaptic receptors. Most synaptic activity in hypothalamic circuits
is attributable to axonal release of GABA or glutamate. With whole-cell
patch-clamp recording, we show that hypocretin, acting directly at axon
terminals, can increase the release of each of these amino acid
transmitters. Two hypocretin peptides, hypocretin-1 and hypocretin-2,
are coded by a single gene; neurons that respond to one peptide also
respond to the other. In addition to its effect on feeding, we find
that this peptide also regulates the synaptic activity of
physiologically identified neuroendocrine neurons studied in
hypothalamic slices containing the arcuate nucleus, suggesting a second
function of hypocretin in hormone regulation. The widespread
distribution of hypocretin axons, coupled with the strong response to
the peptide at both presynaptic and postsynaptic sites, suggests that
the peptide probably modulates a variety of hypothalamic regulatory
systems and could regulate the axonal input to these regions
presynaptically.
Key words:
hypothalamus; presynaptic; neuroendocrine; neuromodulation; neuropeptide; glutamate; GABA; feeding
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INTRODUCTION |
The hypothalamus has been called the
control center of the brain for homeostasis, and neurons here
regulate a substantial part of the internal milieu. Although the medial
hypothalamus has been a fertile ground for the discovery of many
neuroactive peptides, the lateral hypothalamus (LH) has been
relatively barren. We recently described a new peptide, hypocretin,
which shows robust expression in a restricted subset of lateral
hypothalamic and perifornical neurons, with little apparent expression
in neurons outside the hypothalamus (de Lecea et al., 1998 ). Axons from
these cells show a strong innervation of the hypothalamus and ramify widely throughout the brain, including in areas that also have wide
axonal distributions, such as the septal nuclei, preoptic area, central
gray, and locus ceruleus. These data suggest that the cells that
synthesize the peptide may exert a strong modulatory action on many
different brain functions.
Subsequent to our description of hypocretin, a different group
independently described the same peptide, which they called orexin, and
found that it increased feeding in rats and that the peptide mRNA was
upregulated in hungry animals (Sakurai et al., 1998). The hypocretin
gene maps to a presumptive locus at chromosome 17q21 (de Lecea et al.,
1998; Sakurai et al., 1998), a site that has been implicated in severe
human neurodegenerative disorders (Wilhelmsen et al., 1994; Wijker et
al., 1996), raising the possibility that a hypocretin deficit may
contribute to this problem. Despite the excitement this peptide is
generating on many fronts, there previously has been no
characterization of its physiological actions on neurons and no
assessment of neuroendocrine involvement. These are the focus of the
present paper.
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MATERIALS AND METHODS |
Whole-cell recording in culture. Whole-cell
patch-clamp experiments were performed in synaptically coupled medial
hypothalamic neurons cultured for 10-21 d in vitro
as described previously (Gao et al., 1998). The bath solution contained
(in mM): 150 NaCl, 2.5 KCl, 2 CaCl2, 10 HEPES, and 10 glucose, pH 7.3 with NaOH. Whole-cell voltage
clamp was used to observe spontaneous and miniature postsynaptic
currents at  60 mV with a List (HEKA Elektronik) EPC-7
amplifier. Patch pipettes had a tip resistance of 4-6 M when filled
with pipette solution, which contained (in mM): 145 KCl, 1 MgCl2, 10 HEPES, 1.1 EGTA, 4 Mg-ATP, and 0.5 Na2-GTP, pH 7.3 with KOH. Only cells with an input
resistance >0.8 G were used. The series resistance was <30 M
and was partially compensated by the amplifier. All data were sampled
at 3-10 kHz and filtered at 1-3 kHZ with an Apple Macintosh computer
using AxoData software (Axon Instruments). Data were analyzed with
Axograph 3.5 (Axon Instruments), plotted with Igor Pro (WaveMetrics,
Lake Oswego, OR), and reported as the mean ± SEM. Student's
t test was used to compare two groups of data.
Two active peptides are cleaved from preprohypocretin, hypocretin-1 and
hypocretin-2 (also called orexin A and orexin B, respectively). All whole-cell recordings and most of the digital imaging studies were
done with the 27 amino acid peptide hypocretin-2,
PGPPGLQGRLQRLLQANGNHAAGILTM-NH2, made in-house.
Hypocretin-1, using the structure described for orexin A (Phoenix
Pharmaceuticals) (Sakurai et al., 1998), was used in some
digital imaging experiments.
Slice recording. Arcuate nucleus (ARC) frontal slices,
350-400 µm thick, were obtained from 3-week-old Sprague Dawley rats. Slices were incubated in an interface chamber with constant flow (4 ml/min) of the oxygenated artificial CSF (ACSF) containing (in
mM): 124 NaCl, 3.0 KCl, 2.0 CaCl2, 2.0 MgCl2, 1.23 NaH2PO4, 26 NaHCO3, 10 glucose, 0.1 D,L-2-amino-5-phosphonovalerate (AP-5), and 0.01 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), pH 7.4, at room
temperature. Whole-cell patch-clamp recordings were made with an
Axoclamp-2B amplifier (Axon Instruments). Glass pipettes were filled
with: 145 mM potassium methylsulfate, 2 mM
MgCl2, 0.1 mM CaCl2,
1.1 mM EGTA, 10 mM HEPES, 2 mM
Na-ATP, 0.3 mM Na-GTP, pH 7.2, 290 mOsm, and 5-7 M
resistance. Hypocretin (30 µM) was dissolved in ACSF and
applied to ARC slices using the microdrop technique (Christian
and Dudek, 1988; Belousov and van den Pol, 1997). A small
drop was pressure ejected from the hypocretin-containing pipette (7-10
µm tip diameter) onto the area of the recorded cell. To stimulate
axons that innervated the recorded ARC neuron, a bipolar electrode was
placed in the dorsal or ventrolateral part of the ARC. Electrical
impulses (0.2 msec, 0.5 Hz, 40-250 µA) were delivered through an
isolation unit from a Grass 44 stimulator. In some slices, a
bipolar electrode was also placed on the median eminence to generate
antidromic responses in ARC neurons, identifying them as neuroendocrine
cells (stimulating current, 50-70 µA). In all slice experiments,
data were monitored and stored on a Macintosh Quadra 800 computer using
AxoData 1.2 with the subsequent off-line analysis by AxoGraph 3.0 (Axon
Instruments) or Igor Pro (WaveMetrics). Recordings were made at 2000 Hz
frequency acquisition.
Immunocytochemistry. Hypocretin-2 was conjugated with
glutaraldehyde to keyhole limpet hemocyanin, dialyzed in phosphate
buffer, mixed with complete Freund's adjuvant, and injected
subcutaneously and intradermally in three rabbits. Subsequent boosts
were made from the hypocretin conjugate mixed with incomplete Freund's
adjuvant. Use of rabbits for this purpose was approved by the Yale
University Committee on Animal Use. Each of the rabbits made antiserum
against the peptide and stained the same cells in the LH. Rats were
deeply anesthetized and perfused transcardially with 4%
paraformaldehyde. Brains were removed, cut into 20-50-µm-thick
sections, and stained with immunoperoxidase as described previously
(van den Pol, 1985). Adsorption of the antigen with the antibody or
elimination of the primary antiserum blocks immunostaining.
Fura-2 imaging. Neurons were loaded with fura-2 AM (5 µM; Molecular Probes, Eugene, OR) and studied on a
Nikon inverted microscope fitted with a 40× Olympus UV objective with
high 340 nm light transmittance (van den Pol et al., 1996a,b).
Fluor software (Universal Imaging Corporation, West Chester, PA) was
used to control a Lambda 10 Sutter filter wheel with 340 and 380 nm
filters and a 150 W xenon light source. Calcium standards from
Molecular Probes were used to calibrate the system according to the
equation of Grynkiewicz et al. (1985). Cells were imaged while in a
linear-flow chamber, allowing a complete change of solution in 5 sec
(Forscher et al., 1987).
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RESULTS |
Hypocretin modulates glutamatergic and GABAergic signaling in
cultured hypothalamic neurons
In our previous studies of hypothalamic neuromodulators,
which included neuropeptide Y, adenosine, metabotropic glutamate receptor, and GABAB receptor agonists, we found primarily
inhibitory actions (Obrietan et al., 1995; Chen and van den Pol,
1996, 1998; van den Pol et al., 1996b; Obrietan and van den Pol,
1997). In striking contrast, hypocretin increases synaptic
activity in hypothalamic cultures. Almost all fast synaptic activity in
the hypothalamus is generated by release of either the excitatory
transmitter glutamate (van den Pol et al., 1990) or the inhibitory
transmitter GABA (Randle and Renaud, 1987; Decavel and van den Pol,
1990; Renaud and Bourque, 1991). In some parts of the brain, peptide
modulation is restricted to one or another of the amino acid
transmitters, as in the hippocampus, in which neuropeptide Y decreases
glutamate release but has little effect on GABA (Bleakman et al.,
1992). For this reason, we studied GABA or glutamate activity
selectively in neurons cultured from the medial hypothalamus, a brain
region that has been suggested as a satiety center. To determine
whether hypocretin responses would be found in GABAergic neurons, we
blocked glutamate actions with AP-5 (100 µM) and CNQX (10 µM) and studied spontaneous IPSCs (sIPSCs)
in voltage-clamped neurons. Hypocretin caused an increase in the
frequency of IPSCs in three of five cells (mean increase, 56%; range,
36-79%; paired t test, p < 0.05; n = 5) (Fig.
1A,D),
measured 1 min after application of the peptide. Bicuculline (20 µM) blocked the IPSCs (data not shown). In parallel, we
tested the effect on glutamatergic neurons in the presence of the
GABAA antagonist bicuculline (20 µM) and
found that hypocretin increased the frequency of spontaneous EPSCs
(sEPSCs) in four of six neurons by 83% (range, 35-130%;
p < 0.05; n = 5) (Fig. 1B,C). Glutamate receptor
antagonists AP-5 (100 µM) and CNQX (10 µM)
blocked the EPSCs (data not shown). These data indicate that both
glutamate- and GABA-releasing neurons, which account for almost all
fast synaptic activity, express hypocretin receptors.
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Figure 1.
Both excitatory and inhibitory neurons express
hypocretin receptors. To determine the transmitter identity of neurons
that respond to hypocretin, different transmitter antagonists were
used, and responses to hypocretin were studied with whole-cell
recording in voltage-clamped cells held at 60 mV. A,
In the presence of glutamate receptor antagonists AP-5 (100 µM) and CNQX (10 µM), only sIPSPs were
found, and these were completely blocked with the GABAA
antagonist bicuculline (20 µM) (data not shown). When
hypocretin (1 µM) was bath-applied, the frequency of
sIPSCs increased dramatically, and after peptide washout, the frequency
of sIPSCs decreased to its normal baseline. B, In the
presence of the GABAA antagonist bicuculline (20 µM), sEPSCs were found, and these were completely blocked
by AP-5 (100 µM) and CNQX (10 µM) (data not
shown). Hypocretin (1 µM) increased the frequency of
sEPSCs, and these returned to normal after peptide washout. Both GABA
and glutamate evoked inward currents (downward deflection) attributable
to the composition of the pipette solution. C,
D, Mean increase in sEPSCs and sIPSCs evoked by
hypocretin and the return toward control levels after peptide
washout.
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Hypocretin enhances evoked IPSP in ARC slices
A high density of immunoreactive axons was found in the lateral
and medial hypothalamus (Fig.
2A-D); many of the
fibers had large endings suggestive of presynaptic boutons. Many
terminals were found in the ARC (Fig. 2C,D) but
not in the median eminence (Fig. 2E). We therefore
tested the hypothesis that hypocretin regulates the activity of
neuroendocrine neurons in hypothalamic slices containing the ARC. A
bipolar electrode was used to stimulate the lateral edge of the ARC
(Fig. 3A). Hypocretin was
applied by microdrop as described in detail elsewhere (Christian and
Dudek,1988; Belousov and van den Pol, 1997). Six of seven
current-clamped neurons showed an increase in the amplitude of the
evoked IPSP in response to microdrop application of hypocretin, with a
mean increase of 48% from 7.1 ± 0.9 mV to 10.6 ± 1.5 mV
(range, 40-61%) based on the responding cells (Fig. 3B).
This was a significant increase in the evoked IPSP (paired t
test, p < 0.003). No cells showed a decrease in the
IPSP.
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Figure 2.
Hypocretin immunoreactivity. A,
Immunocytochemical staining revealed high densities of axons in both
the lateral hypothalamus (A, B,
LH) and arcuate nucleus (C,
D, ARC) of the hypothalamus. Around
immunoreactive cells in the LH, a high density of immunoreactive axons
was found. E, Immunoreactive axons were not found in the
external zone of the median eminence (ME), in which
neurosecretory axons release pituitary tropins that are carried to the
anterior pituitary by the portal blood system. Scale bars:
A, 25 µm; B, 12 µm; C,
25 µm; D, E, 12 µm.
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Figure 3.
Hypothalamic slice electrophysiology.
A, Experimental paradigm for recording hypothalamic
slices that included the arcuate nucleus (ARC).
B, In the presence of AP-5 (100 µM) and
CNQX (10 µM), IPSPs were evoked by orthodromic electrical
stimulation (asterisk), as shown in A.
The IPSP was blocked in the presence of bicuculline (20 µM), indicating that it was attributable to GABA release.
Hypocretin (HCRT) applied by microdrop (30 µM) caused a substantial increase in the amplitude of the
IPSP. This recovered to baseline levels after washout of the peptide.
Each trace is the mean of eight sweeps. C, After testing
hypocretin in B, the axon of the same cell was
antidromically stimulated from the median eminence. That median
eminence stimulation evoked an action potential even in the presence of
control application of Co2+ (1 mM)
indicated that the action potential response was not attributable to
recurrent collaterals releasing transmitters on the recorded cell. A
fixed latency of 3-5 mS between stimulus artifact and antidromic spike
was routinely found. These data indicate that hypocretin enhances the
action of transmitters released onto identified neuroendocrine
neurons.
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Neuroendocrine neurons that secrete factors that control the anterior
pituitary gland send efferent axons to the median eminence in which the
factors are released from axon terminals. Antidromic stimulation of
axons in the median eminence was used to detect neurosecretory neurons
of the ARC. Neurons (n = 3) that were antidromically identified showed an increase in the amplitude of the IPSP in the
presence of hypocretin. To ensure that the response to stimulation of
the median eminence was attributable to antidromic activation of ARC
neurons, at the end of the experiments, control bath application of
Co2+ was used to block orthodromic activation of
recurrent collaterals (Fig. 3C). These data indicate that
hypocretin modulates the afferent input that controls putative
neuroendocrine neurons.
Calcium response to hypocretin
Electrical stimulation of the LH leads to increased feeding
(Miller, 1960); hypocretin, made in LH cell bodies, also increases feeding (Sakurai et al., 1998). Lesions of the LH cause a long-lasting reduction in body weight and food intake in both male and female rodents (Powley and Keesey, 1970; van den Pol, 1982). In addition to
the cell bodies (Gautvik et al., 1996; de Lecea et al., 1998; Sakurai
et al., 1998) (Fig. 2A,B), large
numbers of hypocretin immunoreactive axons and their terminals are
found in the LH (Fig. 2A,B),
suggesting that hypocretin could have a direct effect on neurons in the
LH. In our past work on hypothalamic cultures, calcium digital imaging
with fura-2 proved to be sensitive in detecting responses to both
transmitters and a number of peptide and nonpeptide neuromodulators
(Obrietan et al., 1995; van den Pol et al., 1996a,b). Because
hypocretin immunoreactive axons terminate in both lateral (Fig.
2B) and medial (Fig. 2D)
hypothalamus, we tested the hypothesis that each of these areas would
express hypocretin receptors that alter cytoplasmic calcium. Both
lateral (23% of 180 neurons) (Fig.
4A) and medial (Fig.
4B) hypothalamic neurons showed substantial
Ca2+ rises in response to hypocretin (1 µM) stimulation. In 1260 medial hypothalamic neurons from
21 experiments, a mean of 29.7% of the neurons showed a response to 1 µM hypocretin, using a criterion Ca2+
rise of at least 20 nM. The mean (± SE)
Ca2+ rise was 106 ± 7 nM, with a
range of 20-930 nM.
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Figure 4.
Calcium responses to hypocretin. A,
Based on fura-2 calcium recordings, neurons from the lateral
hypothalamus (LH), the site of the hypocretin
immunoreactive cell bodies, showed strong responses to hypocretin
(HCRT) (1 µM) and clear recovery
after two bath applications of the peptide. Horizontal lines
above Ca2+ trace indicate time of drug
application. Except for Figure 3C, all other experiments
used hypocretin-2. B, Medial hypothalamus cells also
showed strong Ca2+ elevations in response to
hypocretin but no response to the C-terminal 1-17 (1-17) peptide of
preprohypocretin (1 µM). C, Hypocretin-1
(HCRT: 1), using the 33 amino acid structure with the
double disulfide bonds found for orexin A, and hypocretin-2
(HCRT: 2) were compared. Each evoked a
Ca2+ rise in this typical medial hypothalamic
neuron. D, Hypothalamic neuron that showed an increasing
peak response to increasing concentrations of the peptide, with no
response to 1 nM hypocretin, a response to 10 nM hypocretin, and a bigger response to 100 nM
hypocretin.
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As a control, a 17 amino acid peptide based on amino acids 1-17 of the
C terminal of preprohypocretin was also tested. No response to the
1-17 amino acid sequence was found in 194 cells, and no response was
found in 29 neurons that responded to hypocretin (Fig.
4B). Preprohypocretin is cleaved to form two
peptides, hypocretin-1 and hypocretin-2. The actions of hypocretin-1
have not been examined previously in single neurons. In three
experiments, all 28 neurons that showed a response to one peptide also
responded to the other with similar amplitude Ca2+
rises (Fig. 4C). Thus, most hypothalamic neurons express
receptors that are sensitive to both hypocretin-1 and hypocretin-2.
Neurons responded in a dose-dependent manner (Fig.
4D) to  5 nM hypocretin.
Hypocretin could generate a Ca2+ rise by increasing
the release of an excitatory transmitter or by increasing the frequency of action potentials. To test these hypotheses, we examined the effect
of hypocretin in the presence of tetrodotoxin (TTX) (1 µM). Even in the presence of TTX, hypocretin evoked a
Ca2+ rise of at least 30 nM in 27% of
248 neurons (Fig. 5A). In the responding cells, hypocretin evoked a mean rise of 120 ± 16 nM Ca2+. These results support the
concept that functional hypocretin receptors are found on or near the
cell body of approximately one-third of hypothalamic neurons and that
the Ca2+ rise is not dependent on synaptic
modulation or action potentials.
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Figure 5.
Mechanisms of hypocretin elevation of calcium.
A, To demonstrate that hypocretin can evoke
Ca2+ elevations directly at the cell body rather
than by altering synaptic interactions, hypocretin (1 µM)
was added in the presence of tetrodotoxin (TTX)
(1 µM), used to block action potential-mediated release
of transmitters. B, Depleting intracellular stores of
Ca2+ with thapsigargin (2 µM)
pretreatment did not affect the action of hypocretin in elevating
Ca2+ in the presence of 1 µM TTX.
C, Eliminating extracellular Ca2+ and
adding the Ca2+ chelator EGTA (1 mM)
completely blocked the Ca2+ rise evoked by 1 µM hypocretin (HCRT). When
bath Ca2+ levels were returned to normal, the action
of hypocretin returned. D, Cd2+ (100 µM) blocked the Ca2+ rise, indicating
an extracellular origin of Ca2+. E,
Pretreatment of hypothalamic neurons with bisindolylmaleide (1 µM for 12 hr), a PKC inhibitor, completely blocked the
actions of hypocretin (1 µM). Glutamate
(GLU) (10 µM), applied as a
control, evoked a large Ca2+ rise. F,
Most cortical cells showed no response to hypocretin, as shown by the
typical cell in f-1. A small percent of cortical cells
did show a small Ca2+ rise in response to
hypocretin, as shown in f-2. A control application of
glutamate (GLU) (10 µM) evoked a
Ca2+ rise.
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A hypocretin-evoked increase in cytoplasmic Ca2+
could result from release from intracellular stores or could come from
outside the cell via Ca2+ channels in the plasma
membrane. To test whether the hypocretin-mediated increase in
Ca2+ is dependent on intracellular stores, we
depleted the intracellular stores with thapsigargin (2 µM). In 24 neurons that responded to the peptide,
hypocretin induced a 113 ± 15 nM
Ca2+ rise; 45 min after thapsigargin treatment,
hypocretin evoked virtually the same amplitude Ca2+
rise in these cells (110 ± 12 nM). This absence of a
thapsigargin effect (Fig. 5B) suggests that hypocretin does
not act primarily by a mechanism dependent on release of
Ca2+ from intracellular stores.
The thapsigargin experiments suggest that hypocretin acts by increasing
Ca2+ influx from outside the cell. To test this
hypothesis further, we used a zero Ca2+ buffer with
1 mM EGTA. In normal Ca2+- containing
HEPES buffer, hypocretin evoked a 130 ± 12 nM
Ca2+ rise in 38 neurons. In contrast, in the
Ca2+-free buffer, hypocretin evoked a negligible
Ca2+ rise of 7 ± 3 nM in the same
neurons (Fig. 5C). In parallel, adding
Cd2+ to the buffer blocked any hypocretin-evoked
Ca2+ rise (Fig. 5D). These data support
the view that hypocretin raises intracellular Ca2+
by opening plasma membrane Ca2+ channels that allow
Ca2+ entry from extracellular space.
Intracellular signaling
Next, we assessed possible intracellular signaling pathways
that mediate the excitatory effects of hypocretin. cAMP levels in
[3H]adenine-labeled cells were measured by
determining the ratio of [3H]ATP,
[3H]ADP and [3H]AMP to
[3H]cAMP on cultures done in triplicate (Wong et
al., 1991). Hypocretin (10 nM to 10 µM) did
not increase cAMP levels in primary cultures of hypothalamic neurons.
In sister cultures, a 12 ± 0.82 (mean ± SE) and
25.4 ± 3.6-fold increase was triggered by vasoactive intestinal
peptide (VIP) (1 µM) and forskolin (1 µM),
respectively. The VIP-evoked cAMP rise was not affected by the
coadministration of hypocretin (data not shown). These results suggest
that the hypocretin receptor does not act via heteromeric
Gi or Gs proteins.
Another possibility is that the hypocretin receptor couples to a
Gq protein, as suggested in transfected non-neuronal cells (Sakurai et al., 1998). Activation of Gq triggers the
stimulation of phospholipase C, which, via the hydrolysis of
phosphatidylinositol and the generation of inositol 1,4,5-trisphosphate
and diacylglycerol, can activate protein kinase C (PKC) (Exton, 1994).
To test whether hypocretin triggers PKC activation, hypothalamic
neurons were pretreated with the PKC-specific inhibitor
bisindolylmaleide (1 µM). Bisindolylmaleide completely
blocked hypocretin responses (Fig. 5E), and no
hypocretin-mediated Ca2+ rise was found in 128 neurons. These results suggest that hypocretin may work via
Gq-mediated PKC, resulting in phosphorylation of Ca2+ channels that has been reported to increase
Ca2+ conductance (Yang and Tsien, 1993; Stea
et al., 1995).
Small cortical response
Hypocretin immunoreactive axons are found in the cortex but in
small numbers, suggesting that a minor response to the peptide might be
found in cortical neurons. Consistent with this hypothesis, only 5% of
122 cultured cortical neurons showed a hypocretin-evoked (1 µM) Ca2+ rise >30 nM
(Fig. 5F). In the six responding cells, the mean Ca2+ rise was 65 ± 6 nM
Ca2+, approximately half the amplitude of that found
in hypothalamic neurons. No Ca2+ responses were
found in 128 hippocampal neurons, and in parallel, no effect of
hypocretin (1 µM) on synaptic activity was found in
synaptically coupled hippocampal neurons (n = 7).
Presynaptic actions of hypocretin
Although hypocretin evoked a substantial increase in synaptic
activity, little direct effect on membrane potential was found in 28 neurons tested, even in cells in which increases in synaptic activity
were detected. To test the hypothesis that hypocretin exerted an effect
directly on presynaptic axons to alter release of neurotransmitter, we
examined the effect of the peptide on miniature postsynaptic currents
(mPSCs) in the presence of TTX in vitro. To study modulation
of GABA-mediated mIPSCs, glutamate receptors were blocked with AP-5
(100 µM) and CNQX (10 µM). The miniature
events that were found in this buffer were caused by the release of
GABA and were blocked by bicuculline (20 µM) (data not
shown). In three of five neurons, hypocretin generated a substantial increase in the frequency of mIPSCs, with a mean increase of 54.7 ± 12.3% (range, 30.2-67.4%; paired t test,
p < 0.05; n = 5) (Fig. 6A,D).
To determine whether hypocretin also might increase the release of
glutamate-secreting cells, we did parallel experiments in TTX (1 µM) and a GABA receptor antagonist. Hypocretin caused an
increase in the frequency of glutamate-mediated mEPSCs by 48.2 ± 9.7% (range, 40.6- 65.1%) in three of five synaptically
coupled neurons (p < 0.05; n = 5) (Fig. 6B,C); AP-5 and CNQX
blocked the mEPSCs (data not shown). We found no change in the
amplitude of the mEPSC in cells that showed an increase in frequency
(Fig. 6E), suggesting that the increase in frequency
of mPSCs was mediated at a presynaptic site of hypocretin action.
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Figure 6.
Presynaptic mechanism for hypocretin to enhance
GABA or glutamate secretion. In the presence of TTX (1 µM), mPSCs were studied with whole-cell voltage-clamp
recording. A, In the presence of the glutamate receptor
antagonists AP-5 (100 µM) and CNQX (10 µM),
hypocretin (1 µM) increased the frequency of mIPSCs.
After washout of the peptide, the frequency of mIPSCs returned to
baseline levels. B, An example of hypocretin (1 µM) increasing the frequency of mEPSCs in the presence of
bicuculline (20 µM). C,
D, Mean increase in mEPSCs and mIPSCs and the
return toward baseline levels after peptide washout. E,
Cumulative probability for mEPSC amplitude in the presence of
hypocretin (1 µM) or in its absence
(control). Superimposition of the two
lines indicates that although hypocretin increased the
frequency of mEPSCs, it did not change the amplitude, suggesting that
the peptide acted at a presynaptic site to enhance transmitter
release.
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DISCUSSION |
In the present set of experiments, we demonstrate that hypocretin
increases synaptic activity of both glutamatergic and GABAergic neurons, and in part, this is attributable to actions at presynaptic receptors on GABA- and glutamate-secreting axons. In addition, we find
that the afferent input to physiologically identified neuroendocrine
neurons is modulated by hypocretin. Mechanistically, hypocretin
elevates cytosolic calcium levels via plasmalemmal calcium
channels. These actions are consistent with activation of a
Gq protein acting via PKC to phosphorylate
voltage-activated calcium channels.
Neuroendocrine modulation
Most, if not all, fast synaptic activity in the hypothalamus
appears to be mediated by GABA and glutamate release (van den Pol et
al., 1990; Randle and Renaud, 1987; Renaud and Bourque, 1991; van den
Pol and Trombley, 1993), and this is also true of the ARC
(Belousov and van den Pol, 1997), an area of the hypothalamus that
regulates the endocrine system. Immunocytochemical analysis of
hypocretin axons indicates a strong innervation of the ARC. Our finding
that hypocretin modulates the afferent input to physiologically identified neuroendocrine neurons in the hypothalamus suggests that
hypocretin could modulate the final common neuronal pathway regulating
the hormone system. The antidromically identified neuroendocrine neurons probably maintain axon terminals in the external zone of the
median eminence but may also send axons to the neurohypophysis. Pituitary tropins are released into the pituitary portal system in the
median eminence with a dependence on the electrical activity of the
neuroendocrine neurons. That hypocretin-containing axons innervate the
ARC and hypocretin enhances signaling to these neurons suggests that by
influencing the electrical activity of ARC neurons, hypocretin could
play a pivotal role in hormone regulation. A number of different
pituitary tropins are contained in neurons of the ARC, and the identity
of the specific neuroendocrine neurons that respond to hypocretin
remains to be determined. Although many different transmitters and
modulators are found in axon terminals in the external zone of the
median eminence, the striking lack of hypocretin fibers here suggests
that hypocretin-containing cells probably do not directly control
pituitary secretions but may indirectly participate in neuroendocrine
regulation by modulating the activity of neurons that do directly
control the pituitary.
Hypocretin immunoreactive axons are found not only in the ARC but also
in other medial regions of the hypothalamus that may play a role in
endocrine regulation and in other hypothalamic functions. Innervation
of the LH is also found. In parallel, that both lateral and medial
hypothalamic neurons respond to hypocretin suggests that hypocretin may
play a role in the general modulation of a number of hypothalamic
functions. This is consistent with the effects of hypocretin on
hypothalamic neurons that contain either GABA or glutamate.
Presynaptic actions and nonhypothalamic responses
That functional hypocretin receptors appear to exist on cell
bodies and on presynaptic axons suggests that hypocretin could have a
direct effect on cytosolic calcium at the cell body, and in addition,
could modulate incoming axonal input from a wide variety of sources. It
remains to be determined if extrahypothalamic projections to the
hypothalamus express presynaptic hypocretin receptors on their axons.
Because hypocretin can increase cytosolic calcium levels in neurons
independent of action potentials, this suggests that hypocretin may
enhance presynaptic transmitter release by a parallel mechanism of
increasing calcium levels of the presynaptic bouton, which would
enhance calcium-dependent transmitter release. Calcium increases in
response to hypocretin have been described in non-neuronal cells
transfected with hypocretin receptor cDNA (Sakurai et al., 1998). The
lack of a direct effect of hypocretin on membrane potential is
consistent with a neuromodulator function of the peptide.
Although the focus in the present study is on hypothalamic
actions of hypocretin, there are a small number of fibers that innervate the cerebral cortex, and consistent with that, we find that a
small percentage of cortical cells tested show small responses to
hypocretin. Because hypocretin axons are found in many brain regions,
these data suggest that hypocretin receptors may also have a widespread
distribution and that the receptor density may parallel the level of
innervation. This remains to be substantiated in other brain regions
and with other approaches, including in situ hybridization
and immunostaining with hypocretin receptor antisera. That cells in
some areas of the rat brain, for instance the hippocampus, appear
unresponsive to hypocretin, suggests that functional hypocretin
receptors are expressed in a limited population of CNS neurons.
Hypocretin and energy homeostasis
In light of our data, the role of hypocretin in
physiological homeostasis is not restricted to the enhancement of
feeding (Sakurai et al., 1998) but is also involved in the regulation of the neuroendocrine system in the ARC, perhaps related to endocrine regulation of energy balances. ARC neurons not only synthesize and
release neuropeptide Y, involved in feeding (Leibowitz, 1991; Stanley,
1993) and endocrine (McDonald and Koenig, 1993) regulation, but also
express receptors for leptin (Hakansson et al., 1998), the signal that
adipose tissue releases into the vascular system that reduces food
intake and plays a key role in some forms of obesity (Zhang et al.,
1994); falling leptin levels may initiate mechanisms for energy
conservation (Flier and Maratos-Flier, 1998). Our finding that
hypocretin regulates the synaptic input to these arcuate cells and that
hypocretin-containing axons strongly innervate this area suggests the
hypocretin is in a key position to influence neurons that are the hub
for vascular signals from the periphery and afferent and efferent
axonal signals involved in feeding. Feeding studies have demonstrated
that both GABA and glutamate can regulate hypothalamic control of food
intake (Maldonado-Irizarry et al., 1995; Stanley et al., 1996),
suggesting that one mode of hypocretin action based on the present
paper is to modulate presynaptically GABA and glutamate circuits
involved in energy regulation. The fact that hypocretin can modulate
neurons containing either the primary excitatory or primary inhibitory
transmitter in the hypothalamus suggests that the local microcircuitry,
particularly the proximity of hypocretin axons to other axons, may be
critical in hypocretin function. This raises the general question of
how to best interpret animal responses to mass injections of hypocretin into the hypothalamus or ventricular system that may activate circuits
other than those that would be activated by discrete axonal release of
the peptide.
That approximately one-third of all hypothalamic neurons express
hypocretin receptors and that hypocretin axon terminals are found
throughout the hypothalamus suggests that hypocretin could influence
the general level of activity in many hypothalamic systems. Included
are other recently identified medial and lateral hypothalamic systems
involved in regulating energy balances, including those related to
melanin-concentrating hormone, melanocyte-stimulating hormone,
Agouti-related protein, (Mountjoy et al., 1994; Qu et al., 1996; Huszar
et al., 1997; Ollmann et al., 1997), and neurons that respond to
glucose (Oomura, 1983). These are found in the same hypothalamic
regions as high densities of hypocretin axons, suggesting that many
opportunities exist for hypocretin cells to not only directly
affect postsynaptic neurons, but also, based on data presented here, to
modulate the synaptic input to these areas at the axonal terminal.
Thus, hypocretin may be an important link in the chain of regulatory
cells that orchestrate behavioral, metabolic, and endocrine systems to
maintain energy homeostasis.
| |
FOOTNOTES |
Received May 20, 1998; revised July 8, 1998; accepted July 10, 1998.
This work was supported by National Institutes of Health Grants NS31573
and NS34887, the National Science Foundation, the United States Army
Research Office, and the Air Force Office of Scientific Research. We
thank Y. Yang and J. Belousov for excellent technical assistance and
Dr. C. Heller for help with the facilities.
Correspondence should be addressed to Anthony van den Pol, Department
of Neurosurgery, Yale University School of Medicine, 333 Cedar Street,
New Haven, CT 06520.
| |
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P.-E. Lund, R. Shariatmadari, A. Uustare, M. Detheux, M. Parmentier, J. P. Kukkonen, and K. E. O. Akerman
The Orexin OX1 Receptor Activates a Novel Ca2+ Influx Pathway Necessary for Coupling to Phospholipase C
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Abstract
Introduction
