selective green fluorescent protein expression in mouse hypothalamic slices. NPY reduced spike frequency and hyperpolarized the
membrane potential of hypocretin neurons. The NPY hyperpolarizing action persisted in tetrodotoxin (TTX), was mimicked by Y1
receptor-selective agonists [Pro34]-NPY and [D-Arg25]-NPY, and was abolished by the Y1-specific antagonist BIBP3226 [(R)-N2-
induced a current that was dependent on extracellular potassium, reversed near the potassium equilibrium potential, showed inward
rectification, was blocked by extracellular barium, and was abolished by GDP-?S in the recording pipette, consistent with a G-protein-
increased spontaneous spike frequency, suggesting an ongoing Y1 receptor-mediated NPY inhibition. In TTX, miniature EPSCs were
reduced in frequency but not amplitude by NPY, NPY13–36, and [D-Trp32]-NPY, but not by [Pro34]-NPY, suggesting the presynaptic
Together, these data support the view that NPY reduces the activity of hypocretin neurons by multiple presynaptic and postsynaptic
The hypocretin/orexin neurons of the brain are found scattered
in the perifornical area/lateral hypothalamus (LH), and their ax-
ons project throughout the brain and spinal cord (Peyron et al.,
1998; van den Pol, 1999). These neurons have been postulated to
play a key role in arousal. CNS administration of hypocretin
increases arousal, and mice, dogs, or humans lacking hypocretin
et al., 2000; Thannickal et al., 2000; Lin et al., 2001). Hypocretin
neurons have also been postulated to play a role in food intake,
increase food intake (Sakurai et al., 1998; Edwards et al., 1999;
in hypocretin mRNA levels (Cai et al., 1999), and hypocretin
knock-out mice are hypophagic (Hara et al., 2001). Arousal reg-
ulated by the hypocretin cells might be important in regulating
energy homeostasis (Yamanaka et al., 2003).
A current view of hypothalamic regulation of food intake is
based on two sets of neurons in the arcuate nucleus, the orexi-
genic neurons that produce neuropeptide Y (NPY) and the an-
orexigenic proopiomelanocortin neurons. Anatomical studies
have shown that leptin-sensitive NPY neurons in the arcuate nu-
cleus project axons to the perifornical hypothalamus (Elias et al.,
1999). NPY axons contact hypocretin neurons (Broberger et al.,
of NPY greatly enhances food intake and body weight (Williams
et al., 2001), and the lateral/perifornical area appears to be a
sensitive hypothalamic site for NPY-induced eating (Stanley et
al., 1993). NPY receptors have been identified in perifornical
hypocretin cells, and direct administration of NPY agonists into
the LH increases Fos-like immunoreactivity in these hypotha-
lamic neurons (Campbell et al., 2003), leading to the hypothesis
that NPY may regulate caloric homeostasis by exciting the neu-
rons of the LH that produce hypocretin (Schwartz et al., 2000;
amus where hypocretin cells are located enhances the soporific
effect of anesthetics (Naveilhan et al., 2001); NPY also has anxi-
raising the question whether hypocretin cells may be involved in
these effects. Medullary neurons send NPY projections to the
TheJournalofNeuroscience,October6,2004 • 24(40):8741–8751 • 8741
hypothalamus potentially conveying sensory and baroreceptive
information from the gut and heart (Stornetta et al., 1999; Ver-
berne et al., 1999).
In this study, using whole-cell voltage- and current-clamp
hypocretin cells by selective expression of green fluorescent pro-
tein (GFP), we tested the hypothesis that NPY excites hypocretin
actions by three different mechanisms, including a Y1 receptor-
mediated enhancement of a G-protein-activated inwardly recti-
axons terminating on hypocretin cells. A possible way in which
NPY might increase the activity of hypocretin neurons is by re-
ducing the synaptic GABA tone, but this attenuation failed to
enhance the activity of hypocretin neurons.
Preparation of hypothalamic slices. Hypothalamic slices were prepared
from transgenic mice (obtained from Dr. T. Sakurai, University of
Tsukuba, Tsukuba, Japan) that expressed enhanced GFP selectively in
hypocretin neurons but not in other cells, as described previously (Li et
al., 2002; Yamanaka et al., 2003). Briefly, 14- to 21-d-old mice main-
tained in a 12 hr light/dark cycle were given an overdose of sodium
pentobarbital (100 mg/kg) during the light part of the cycle (11:00 A.M.
(in mM) 220 sucrose, 2.5 KCl, 6 MgCl2, 1 CaCl2, 1.23 NaH2PO4, 26
prepared, and coronal slices (220–350 ?m thick) were cut on a vi-
chamber mounted on a BX51WI upright microscope (Olympus, Tokyo,
Japan) equipped with video-enhanced infrared-differential interference
contrast (DIC) and fluorescence. Slices were perfused with a continuous
that contained (in mM) 124 NaCl, 3 KCl, 2 MgCl2, 2 CaCl2, 1.23
NaH2PO4, 26 NaHCO3, and 10 glucose, pH 7.4, with NaOH. Neurons
were visualized with a blue excitation light and an Olympus 40? water-
immersion lens. The use of mice for these experiments was approved by
the Yale University Committee on Animal Use.
Patch-clamp recording and synaptic stimulation. Whole-cell current-
and voltage-clamp recordings were performed using pipettes with 4–6
borosilicate glass (World Precision Instruments, Sarasota, FL) using a
[or KCl for IPSCs and miniature IPSCs (mIPSCs)], 1 MgCl2, 10 HEPES,
1.1 EGTA, 2 Mg-ATP, 0.5 Na2-GTP, and 10 Na2-phosphocreatine, pH
7.3, with KOH. Values for membrane potential are uncompensated for
junction potential. The pipette solution for IBarecording contained (in
10 HEPES, pH 7.3, with CsOH. The bath solution for IBarecording
contains (in mM) 79.5 NaCl, 40 TEA-Cl, 3 KCl, 2 MgCl2, 5 BaCl2, 1.23
with 95% O2and 5% CO2. Hypocretin neurons under direct visual ob-
servation of GFP fluorescence and DIC were recorded. After a gigaohm
seal was obtained, a gentle negative pressure was applied to break
through to the whole-cell configuration. Seal resistance was at least 800
M?. An EPC9 amplifier and Pulse software (HEKA Elektronik, Lam-
brecht/Pfalz, Germany) were used for data acquisition. Capacitance was
compensated automatically using Pulse software. Input resistance was
monitored continuously, and only those cells with stable access resis-
tance (change ?10%) were used for analysis. The recordings were made
[(4-hydroxyphenyl)methyl]-D-arginine-amide] were applied focally to
the recorded neurons via a flow pipette. Some neurons were filled with
lysine fixable; Molecular Probes, Eugene, OR) from the pipette to verify
that GFP-expressing hypocretin cells were recorded (see Fig. 1).
To evoke excitatory potentials, bipolar electrodes (World Precision
cell as described previously (Acuna-Goycolea et al., 2004). The electrical
(A320; World Precision Instruments). The stimulating current was 50–
added to the bath perfusion to block GABAAreceptor-mediated
Pulsefit (HEKA Electronik), Axograph (Axon Instruments, Foster
(sPSCs) were detected and measured with an algorithm in Axograph
(Bekkers and Stevens, 1995), and only those events with amplitude ?5
pA were used, as described in detail previously (Gao and van den Pol,
1999). The frequency of action potentials was measured using Axograph
post hoc test, and Kolmogorov–Smirnov statistical tests were used. p ?
0.05 was considered statistically significant.
Chemicals and reagents. BIC, DL-2-amino-5-phosphonovaleric acid
(AP-5), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), CsMeSO3, tet-
raethylammonium hydrochloride (TEA-Cl), and GDP-?S were pur-
chased from Sigma (St. Louis, MO); tetrodotoxin (TTX) was obtained
from Tocris Cookson (Ballwin, MO). NPY (human, rat) and analogs
NPY13–36 (porcine), [D-Trp32]-NPY (human), pancreatic polypeptide
(PP) (human), [Pro34]-NPY (human), and [D-Arg25-NPY] (human,
rat) were purchased from Phoenix Pharmaceuticals (Belmont, CA),
American Peptide (Sunnydale, CA), and AnaSpec (San Jose, CA).
BIBP3226 was from Bachem (San Carlos, CA).
to study the physiological actions of NPY agonists and antago-
nists. To ensure that the recorded cell did contain GFP, we used
microruby in the pipette to verify that the recorded cell was the
same as the GFP-containing cell (n ? 6 of 6). Figure 1, A–C,
shows the confirmation that the hypocretin cell that expressed
GFP was also filled with microruby and could be identified with
infrared-DIC imaging. NPY is synthesized in several brain re-
gions, including the hypothalamus (Chronwall et al., 1985;
Bleakman et al., 1993). A high density of NPY-containing fibers
(Broberger et al., 1998; Elias et al., 1998; Horvath et al., 1999),
raising the possibility that NPY might exert an effect on these
cells. To test this hypothesis, we first evaluated the NPY actions
on the frequency of spontaneous spikes in identified hypocretin
neurons in hypothalamic slices under current clamp (Fig. 2).
Application of NPY (1 ?M) for 1 min hyperpolarized the mem-
brane potential and strongly depressed the spontaneous firing of
hypocretin neurons in a partly reversible manner (Fig. 2A,C,D).
In 12 cells tested, 1 ?M NPY depressed the spike frequency by
91.4 ? 7.4% (Fig. 2D). This effect was highly significant ( p ?
0.01; ANOVA). The NPY effects were dose dependent, because
lower NPY concentrations evoked smaller changes in the action
potential frequency (Fig. 2B,E). Ten and 100 nM NPY decreased
and 50.4 ? 10.6% ( p ? 0.05; n ? 8; ANOVA), respectively. The
effect of NPY was mediated by direct action on hypocretin neu-
rons; in the presence of 0.5 ?M TTX, NPY (1 ?M) significantly
hyperpolarized hypocretin neurons (Fig. 3A) (mean effect,
?6.6 ? 1.9 mV; p ? 0.01; n ? 7; ANOVA).
direct postsynaptic inhibitory actions of NPY. All these experi-
8742 • J.Neurosci.,October6,2004 • 24(40):8741–8751Fuetal.•NPYInhibitionofHypocretinNeurons
indirect spike-dependent synaptic effects of NPY on hypocretin
neurons. Four NPY receptor-selective agonists were tested. The
Y1R agonist [Pro34]-NPY (Potter et al., 1991) (1 ?M) exhibited a
strong inhibitory effect on hypocretin neurons (Fig. 3B), hyper-
polarizing their membrane potential by 8.0 ? 2.2 mV (Fig. 3D).
These [Pro34]-NPY actions were reversible and statistically sig-
nificant ( p ? 0.05; n ? 6; ANOVA). [D-Arg25]-NPY (1 ?M),
another selective Y1R agonist (Mullins et al., 2001), also revers-
ibly hyperpolarized the membrane potential of hypocretin cells
contrast, the Y2R agonist NPY13–36 (Guo et al., 2002) and Y5R
agonist [D-Trp32]-NPY (Guo et al., 2002) did not change the
of [D-Trp32]-NPY: ?0.8 ? 1.8 mV, p ? 0.05, n ? 4; ANOVA),
suggesting that the NPY-induced hyperpolarization was mainly
experiments using the specific Y1 receptor antagonist BIBP3226
(Doods et al., 1996; Sun et al., 2003) were performed to confirm
not hyperpolarize hypocretin neurons (Fig. 3C,D) (mean effect,
2.22 ? 2.23 mV; p ? 0.05; n ? 6; ANOVA), supporting the view
that Y1 receptors are mediating the direct hyperpolarizing ac-
tions of NPY on membrane potential of hypocretin neurons.
One mechanism of peptide inhibition is the activation of potas-
sium currents (Sun et al., 2001a). To explore this possibility in
hypocretin neurons, voltage-ramp commands (from ?140 to
?20 mV for 600 msec) were delivered to these neurons, and the
compared. These experiments were done in the presence of 0.5
?M TTX and 200 ?M CdCl2in the bath to eliminate voltage-
components were subtracted, net NPY-induced currents were
obtained (Fig. 4B). Under normal extracellular K?(3 mM), the
NPY-sensitive current reversed at ?90.9 ? 1.6 mV (n ? 14),
consistent with the idea that it was caused by the activation of a
K?conductance. We then increased the extracellular K?con-
centration from 3 to 16 mM and evaluated the actions of NPY on
the current in high K?. In 16 mM external K?, NPY (1 ?M)
evoked a consistent current that showed a robust increase in its
8), showing a 40.2 mV positive shift of the reversal potential
in this montage from different depths of the section. C, DIC imaging with infrared (IR) light
ing the effect of 1 ?M (A) and 0.1 ?M (B) NPY on spontaneous spike frequency in typical
(*p ? 0.01; n ? 12). This effect was partially reversible. Ctrl, Control; Wash, washout. E,
Fuetal.•NPYInhibitionofHypocretinNeurons J.Neurosci.,October6,2004 • 24(40):8741–8751 • 8743
Nernst equation (41.5 mV). The NPY-induced current showed
inward rectification at positive potentials and showed saturation
at very negative voltages (??140 mV) (Fig. 4B). Furthermore,
the NPY effects were sensitive to the presence of barium (1 mM),
which blocks inwardly rectifying potassium currents (Sodickson
and Bean, 1996). In slices treated with barium, NPY application
did not alter the current response to ramp-voltage commands
the NPY-induced current was caused by the activation of an in-
wardly rectifying K?conductance (Sodickson and Bean, 1996;
Sun et al., 2001a).
We hypothesized that the NPY-induced inwardly rectifying
K?current was dependent on the activation of G-proteins in
hypocretin cells. To address this issue, we tested the GTP depen-
dence of the NPY-induced current using the nonhydrolyzable
GDP analog GDP-?S (800 ?M) (Sodickson and Bean, 1996) in
the recording pipette. Under these conditions, after 3–8 min of
dialysis with GDP-?S in the pipette solution, NPY did not alter
nels is dependent on GTP acting via G-proteins. These experi-
ments support the idea that NPY directly depresses hypocretin
neurons by the activation of GIRK currents in the postsynaptic
K?current, then the Y1R agonist [Pro34]-NPY should generate
the same effect as NPY. To evaluate this, voltage-ramp com-
mands were delivered to hypocretin neurons, and the current
responses were evaluated with application of the selective Y1 re-
ceptor agonist [Pro34]-NPY (1 ?M). With a 16 mM extracellular
reversal potential at ?51.5 ? 2.5 mV (n ? 4) (Fig. 4E). This
current was attenuated by 1 mM extracellular barium. Further-
slices treated with BIBP3226 (1 ?M; n ? 5) (Fig. 4F). Together,
postsynaptic inwardly rectifying potassium currents in hypocre-
tin cells. BIBP3226 (1 ?M) also reduced the current response of
hypocretin cells to the ramp-voltage commands from ?140 to
?M) mimics the hyperpolarizing actions of NPY. C, In the presence of 1 ?M BIBP3226 (Y1
onist: NPY (*p ? 0.01), Y1R agonist [Pro34]-NPY (Pro;*p ? 0.05), Y2R agonist NPY13–36
showing the currents elicited by 600 msec voltage ramps from ?140 to ?20 mV before,
during, and after the application of NPY (1 ?M) in a typical hypocretin cell. High (16 mM)
and was blocked by external barium (1 mM). F, The NPY-induced current was blocked with
NPY activates a GIRK current in hypocretin neurons. A, Representative traces
8744 • J.Neurosci.,October6,2004 • 24(40):8741–8751Fuetal.•NPYInhibitionofHypocretinNeurons
paired t test; n ? 5), suggesting a Y1R-mediated, tonically
activated inwardly rectifying K?current is present in hypocretin
excitability is the modulation of voltage-dependent calcium cur-
rents (Dolphin, 2003). To test whether NPY can alter voltage-
dependent calcium currents in hypocretin neurons, BaCl2was
tance of the calcium channels. All the experiments were done
block voltage-dependent sodium channels. Additionally, 40 mM
TEA-Cl-containing ACSF (TEA-Cl replaced an equimolar con-
centration of NaCl to preserve the osmolarity of the bath solu-
tion) was used as the extracellular solution to inhibit potassium
currents. IBawas activated by a voltage step from ?80 to 0 mV
with a duration of 150 msec under voltage-clamp conditions
(Gao and van den Pol, 2002) (Fig. 5A, bottom trace). The appli-
cation of NPY (1 ?M) resulted in a substantial inhibition of IBa
amplitude (Fig. 5A). A time course graph showing the reversible
NPY actions on the IBaamplitude is presented in Figure 5B. The
(range, 12.6–64.5; p ? 0.01; n ? 7; ANOVA) (Fig. 5D), and the
inhibition fully recovered within 2 min of washout (Fig. 5B).
CdCl2(200 ?M) was applied to four neurons and blocked the IBa
in all cells tested. The current response in CdCl2was used as the
baseline from which to compare the other calcium responses.
voltage-dependent calcium currents in hypocretin cells.
To determine whether Y1 receptors were involved in the in-
hibitory actions of NPY on voltage-gated calcium channels in
hypocretin neurons, additional experiments were performed in
the presence of the Y1R antagonist BIBP3226 (1 ?M) in the bath
solution. Under this condition, NPY (1 ?M) evoked a small re-
duction in the amplitude of whole-cell barium current (Fig. 5C);
we compared the actions of NPY in the presence and the absence
of the Y1 antagonist, a statistically significant difference was de-
involved in the NPY depression of calcium channels in hypocre-
tin cells. Because of the finding that even in the presence of
BIBP3226 NPY can still reduce the amplitude of IBa, other recep-
tor subtypes may also be involved in the modulation of the
from ?60 to ?40 mV). The current response of hypocretin cells
to the voltage-ramp commands was significantly depressed by
results indicate that NPY can modulate voltage-dependent cal-
tor in hypocretin cells.
on hypocretin neurons, we evaluated the effect of the Y1R antag-
onist BIBP3226 on spontaneous spike frequency and membrane
potential of hypocretin neurons. BIBP3226 is a highly specific
Fuetal.•NPYInhibitionofHypocretinNeurons J.Neurosci.,October6,2004 • 24(40):8741–8751 • 8745
min resulted in a robust and reversible increase in spike fre-
quency in six cells tested. The spike frequency was increased by
30.7 ? 10.4% (range, 6.8–73.1%) and showed recovery after
4–13 min of BIBP3226 washout ( p ? 0.05; ANOVA) (Fig. 6A–
C). The membrane potential of hypocretin neurons was revers-
ibly depolarized by 2.5 ? 0.8 mV (Fig. 6A), a statistically signif-
icant effect ( p ? 0.05; n ? 6; ANOVA). These results are
current in hypocretin neurons and suggest that a spontaneous
NPY release activates Y1 receptors in the postsynaptic sites,
thereby causing ongoing direct postsynaptic depression of hypo-
In addition to the direct postsynaptic actions, NPY might also
indirectly affect the excitability of hypocretin neurons by modu-
lating their excitatory and/or inhibitory synaptic inputs. To ex-
amine the possible effect of NPY on synaptic input to hypocretin
neurons, sPSCs were recorded at ?60 mV (holding potential)
under whole-cell voltage-clamp configuration in normal ACSF.
NPY (1 ?M) decreased the frequency of sPSCs by 30.1 ? 4.2%, a
ANOVA; data not shown).
To study the NPY effects on the excitatory synaptic inputs in
included in the bath solution, and EPSCs were detected using
KMeSO4pipettes. NPY (1 ?M) depressed the EPSC frequency by
35.3 ? 6.9%. This effect was statistically significant ( p ? 0.01;
n ? 9; ANOVA; data not shown). Similar to a previous report
(Rhim et al., 1997), partial recovery was observed after 5–20 min
of peptide washout. When AP-5 (50 ?M) and CNQX (10 ?M)
were added to the bath, a complete suppression of the excitatory
currents was observed, confirming that they were attributable to
the activation of ionotropic glutamate receptors.
In addition, we studied NPY actions on the inhibitory synap-
tic transmission in hypocretin cells. These experiments were
done in the presence of AP-5 (50 ?M) and CNQX (10 ?M) using
KCl pipettes. BIC (30 ?M) completely abolished the IPSCs. NPY
(1 ?M) depressed the frequency of IPSCs by 45.7 ? 7.4%, an
effect that was statistically significant ( p ? 0.01; n ? 6; ANOVA;
data not shown). Taken together, these experiments suggest that
NPY depresses both glutamatergic and GABAergic synaptic
transmission to hypocretin neurons.
aptic transmission in hypocretin cells, we evaluated the NPY ac-
tions on excitatory, glutamate-mediated, evoked potentials re-
electrical stimuli (50–100 ?A, 0.5 msec, 0.2 Hz) were generated
using bipolar stimulation electrodes placed within the LH, me-
dial or ventral to the recorded cell, as described previously
(Acuna-Goycolea et al., 2004). Bath application of NMDA and
AMPA receptor antagonists AP-5 (50 ?M) and CNQX (10 ?M)
completely blocked the evoked responses (Fig. 7B) (n ? 4), con-
firming that the evoked potentials recorded in hypocretin cells
were attributable to glutamate actions. NPY (1 ?M) caused a
substantial depression in the amplitude of the eEPSPs (Fig. 7A).
When the time integral of the eEPSPs (time ? ?V) before, dur-
ing, and after NPY application was compared (Fig. 7C), a signif-
icant decrease (32.3 ? 4.9%) was detected ( p ? 0.01; n ? 8;
ANOVA). These inhibitory actions of NPY on eEPSPs partially
recovered after 5–20 min of peptide washout (Fig. 7C).
located in presynaptic or postsynaptic sites in different brain re-
of NPY in hypocretin neurons, we further studied its effects on
the amplitude and the frequency of miniature EPSCs (mEPSCs)
under voltage clamp. These experiments were done in the pres-
ence of TTX (0.5 ?M) and BIC (30 ?M) in the bath. The mEPSCs
were completely blocked by AP-5 (50 ?M) and CNQX (10 ?M),
consistent with their glutamatergic nature (n ? 5; data not
shown). NPY (1 ?M) significantly inhibited the frequency of
was reversible because mEPSC frequency returned to 91.0 ?
A3). No substantial effect of NPY on the amplitude of mEPSCs
was detected when the cumulative probability of mEPSC ampli-
tudes were compared before, during, and after NPY application
( p ? 0.05; n ? 6; Kolmogorov–Smirnov test) (Fig. 8A4), sug-
gesting a presynaptic inhibition of NPY on excitatory synaptic
transmission to hypocretin neurons.
To study NPY actions on mIPSCs, TTX (0.5 ?M), AP-5 (50
?M), and CNQX (10 ?M) were added to the bath (Fig. 8B1). A
KCl pipette solution was used to identify the inhibitory currents
in hypocretin cells; under these conditions, the inhibitory cur-
rents were detected as downward deflections when the cells were
held at ?60 mV (Fig. 8B1). The miniature inhibitory activity in
hypocretin cells was mainly attributable to GABA actions on
GABAAreceptors, because 30 ?M BIC completely blocked
the frequency ( p ? 0.05; n ? 7; ANOVA) or the cumulative
distribution of the amplitude ( p ? 0.05; n ? 7; Kolmogorov–
Smirnov test) of mIPSCs (Fig. 8B1–B4). Taken together, our
potentials recorded before, during, and after application of 1 ?M BIBP3226, Y1R antagonist.
on a typical neuron. C, Averaged effect of 1 ?M BIBP3226 on spontaneous spike frequency
Tonic activation of Y1 receptors in hypocretin neurons. A, Spontaneous action
8746 • J.Neurosci.,October6,2004 • 24(40):8741–8751 Fuetal.•NPYInhibitionofHypocretinNeurons
results support the view that NPY may indirectly depress the
activity of hypocretin cells by selective suppression of glutamate,
To determine the receptor subtypes that account for the pre-
synaptic NPY actions in the hypocretin neurons glutamate syn-
aptic transmission, the effect of Y1, Y2, and Y5 receptor selective
agonists on the frequency of the mEPSC was tested. The Y1 re-
ceptor agonist [Pro34]-NPY (1 ?M) did not modify the mEPSC
?M [Pro34]-NPY depressed the frequency of mEPSCs by 10.1 ?
5.5%, a nonsignificant effect (Fig. 9A1) ( p ? 0.05; n ? 9;
ANOVA). In contrast, the Y2R agonist NPY13–36 and Y5R ago-
nist [D-Trp32]-NPY (1 ?M) consistently inhibited mEPSC fre-
in a partially reversible manner (recovery for NPY 13–36, 78.2 ?
NPY, 71.5 ? 2.8% after 10–20 min peptide washout; control,
100%). When the cumulative distribution of the mEPSC ampli-
tudes in the presence and the absence of NPY receptor subtype
was detected, suggesting that none of these analogs has a detect-
able effect on the amplitude of mEPSCs ( p ? 0.05; n ? 9 for
[Pro34]-NPY, n ? 8 for NPY13–36, and n ? 6 for [D-Trp32]-
NPY; Kolmogorov–Smirnov test) (Fig. 9A2–C2). These results
tative traces showing the NPY actions on the amplitude of eEPSCs. B, The eEPSPs were com-
NPY attenuates eEPSPs in hypocretin neurons. eEPSPs were recorded under
mEPSCs were recorded at holding potentials of ?60 mV with 0.5 ?M TTX in the bath. A1,
effect on the mEPSC amplitude ( p ? 0.05; n ? 6; Kolmogorov–Smirnov test). B1–B4, No
amplitude ( p ? 0.05; n ? 7; Kolmogorov–Smirnov test) of mIPSCs. mIPSCs were recorded
Fuetal.•NPYInhibitionofHypocretinNeuronsJ.Neurosci.,October6,2004 • 24(40):8741–8751 • 8747
tors on the presynaptic glutamatergic terminals innervating
Hypocretin neurons have been suggested to express Y4 receptor
immunoreactivity in the rat (Campbell et al., 2003). Previous
studies have suggested that the Y4 receptor may be relatively
ceptors in the mouse, we evaluated the actions of the Y4 receptor
agonist PP (Sun and Miller, 1999) on hypocretin cells. PP (0.1 or
1 ?M) reversibly hyperpolarized the membrane potential of
hypocretin neurons; 0.1 ?M PP shifted the membrane potential
by ?6.6 ? 1.8 mV (n ? 6; p ? 0.05; ANOVA) (Fig. 10A). In
addition, PP (0.1 ?M) activated an inwardly rectifying K?cur-
rent with a reversal potential of ?50.9 ? 1.4 mV (16 mM extra-
cellular K?concentration; n ? 8)(Fig. 10B). Similar PP actions
on K?currents have been observed in other hypothalamic neu-
on the frequency of mEPSCs (control, 100%; PP, 109.4 ? 7.9%;
functional Y4 receptors were expressed in hypocretin cell bodies
innervating those neurons.
In the present study, we recorded from mouse hypocretin neu-
strong inhibition on spontaneous spike frequency, which would
inhibition were found, including activation of a GIRK current
and depression of voltage-dependent calcium currents. Presyn-
aptically, NPY attenuated release of glutamate, but not GABA,
from axons terminating on hypocretin neurons. Although some
previous models of energy homeostasis relating to NPY activity
have suggested that the peptide might be involved in the excita-
tion of hypocretin neurons, we found that hypocretin neurons
were consistently depressed.
Application of NPY consistently depressed the activity of hypo-
cretin neurons in a dose-dependent manner. In the presence of
TTX to block spike-mediated synaptic activity, NPY hyperpolar-
ized the membrane potential, demonstrating a direct action on
the somatodendritic complex. A parallel hyperpolarization was
or [D-Trp32]-NPY, suggesting that this direct effect was mainly
mediated by a Y1 receptor; this was further substantiated by the
block of the NPY hyperpolarizing actions by BIBP3226, a Y1R-
ramp protocols revealed an inwardly rectifying current that was
hyperpolarized the membrane potential in the presence of TTX (0.5 ?M) in the bath. B, PP
abolished by 1 mM Ba2?in the bath (gray trace). This experiment was done with 16 mM
The Y4 receptor agonist PP inhibits hypocretin cells. A, PP (0.1 ?M) reversibly
8748 • J.Neurosci.,October6,2004 • 24(40):8741–8751Fuetal.•NPYInhibitionofHypocretinNeurons
inclusion of the nonhydrolyzable GDP analog GDP-?S in the
recording pipette eliminated the NPY effect on the inwardly rec-
a GIRK-type current. NPY may exert similar actions on potas-
des et al., 2003). Previous studies in the hypothalamus and other
brain regions have shown that the Y1 receptor subtype can ac-
count for a substantial part of the postsynaptic action of NPY
(Zhang et al., 1994; Chen and van den Pol, 1996; Sun et al.,
2001b). Consistent with this idea, the Y1R agonist [Pro34]-NPY
stimulated a similar K?current, and in the presence of the Y1R
K?current, suggesting that NPY acting through a Y1 receptor,
activates a GIRK-type conductance in hypocretin neurons.
increase the conductance of calcium channels. NPY consistently
depressed whole-cell barium currents in hypocretin neurons.
The NPY depression of barium currents was significantly inhib-
ited by a Y1R antagonist, suggesting that the Y1 receptor subtype
contributes to the NPY modulation of calcium channels. Other
NPY receptors may also modulate calcium channels in hypocre-
tin cells, because even in the presence of the Y1 antagonist, NPY
this, in other regions of the brain, multiple NPY receptors have
been shown to modulate calcium channels (Chen and van den
Pol, 1996; Sun and Miller, 1999). The NPY effects appear to be
voltage independent because no obvious differences were de-
tected when the effects of NPY at voltages between ?60 to ?40
were compared, similar to thalamic neurons (Sun et al., 2001a).
axons presynaptic to hypocretin neurons (Li et al., 2002). NPY
reduced the glutamate release onto hypocretin neurons. In the
of mEPSCs, suggesting activation of presynaptic receptors lo-
could also be equally induced by the Y2R agonist NPY13–36 and
by the Y5R agonist [D-Trp32]-NPY, suggesting both receptors
might be expressed on presynaptic axons. Whether single axons
express both receptors or different populations of axons express
only one type of presynaptic receptor remains to be determined.
In previous studies of autaptic hypothalamic suprachiasmatic
neurons, single axons were identified that expressed multiple
types of the NPY receptor (Chen and van den Pol, 1996).
The Y4 receptor agonist PP hyperpolarized the membrane
potential and activated an inwardly rectifying K?current in
in these cells, consistent with immunocytochemical findings
(Campbell et al., 2003). Because Y4 receptor possesses a high
affinity for PP but low affinity to NPY (Sun and Miller, 1999;
Mullin et al., 2001), the Y4 receptor may not play an important
role in mediating NPY actions on hypocretin neurons.
NPY neurons have been suggested to increase food intake by
exciting the orexigenic melanin concentrating hormone (MCH)
and hypocretin neurons (Elias et al., 1999; Schwartz et al., 2000;
Saper et al., 2002; van den Pol, 2003). This would be consistent
with the finding that CNS application of both NPY and hypo-
cretin may increase food intake (Stanley et al., 1993; Sakurai
et al., 1998) and the NPY innervation of hypocretin neurons
(Broberger et al., 1998; Elias et al., 1998). However, our electro-
physiological data do not support that view, and NPY induced
robust inhibitory actions on hypocretin neurons, as shown here.
In addition, NPY also evokes substantive inhibitory actions on
the nearby MCH neurons (van den Pol et al., 2004), suggesting a
direct activation of either of these LH cell types by NPY-
containing axons is unlikely. That arcuate nucleus NPY cells
would be inhibitory is further corroborated by the fact that NPY
Hypocretin has been reported to enhance feeding (Sakurai et
al., 1998). In the presence of a Y1-specific antagonist, the orexi-
genic effect of intracerebroventricular injections of hypocretin
was reduced, suggesting interactions between hypocretin and
jection from hypocretin neurons to the arcuate nucleus (Peyron
et al., 1998; van den Pol et al., 1998). Hypocretin application to
arcuate nucleus slices enhances evoked transmitter release (van
den Pol et al., 1998), hypocretin-containing axons make direct
synaptic contact with NPY neurons, as shown with dual ultra-
structural immunostaining (Horvath et al., 1999), and hypocre-
tin excites a subset of medial arcuate neurons that contain NPY
(van den Top et al., 2004). Because we show here that NPY is
consistently inhibitory on hypocretin neurons, it seems unlikely
An alternate possibility whereby arcuate nucleus NPY neurons
could influence hypocretin cells is that NPY neurons could
project to inhibitory neurons outside the LH, perhaps in the
paraventricular nucleus region (Cowley et al., 1999), and inhibi-
tion of the hypocretin neurons. Consistent with this idea, we
found that NPY decreased the release of GABA onto hypocretin
cells, probably by actions on the somatodendritic field of GABA
cells because NPY showed no effect on mIPSCs. Although NPY
inhibition of GABA neurons might be expected to lead to disin-
hibition, we consistently found that NPY reduced spike fre-
quency in hypocretin cells.
In addition to a role in energy homeostasis, arcuate nucleus
NPY cells have also been reported to modulate the endocrine
glucocorticoid receptors and coexist with somatostatin and
growth hormone-releasing hormone. NPY is released into the
pituitary portal system from arcuate axons in the median emi-
nence and inhibits release of pituitary hormones, including lu-
teinizing hormone (McDonald and Koenig, 1993). Inhibitory
signaling to the hypocretin neurons could be related to synchro-
nization of the endocrine state with arousal.
hypocretin neurons (Broberger et al., 1998; Elias et al., 1998;
other NPY terminals could arise from NPY neurons in the tha-
lamic intergeniculate leaflet that project to the LH among other
ceives retinal input and may be involved in temporal synchroni-
zation of circadian rhythms to environmental light. NPY axons
majority of epinephrine-containing C1 neurons that project to
the hypothalamus (Stornetta et al., 1999) and also colocalizes
with norepinephrine neurons innervating the hypothalamus
(Sawchenko et al., 1985). These medullary cells may be part of a
pathway sending enteroceptive and baroreceptive information
from the viscera and heart to the hypothalamus (Stornetta et al.,
Fuetal.•NPYInhibitionofHypocretinNeuronsJ.Neurosci.,October6,2004 • 24(40):8741–8751 • 8749
1999; Verberne et al., 1999). Finally, neurons in the LH near the
hypocretin cells may synthesize NPY (Hendry, 1993) and could
be involved in local circuit inhibition of hypocretin cells.
An interesting, but unexpected, finding of the present study
was that hypocretin neurons were under tonic inhibition by en-
dogenous NPY, suggested by the increase in spike frequency and
depression of an inwardly rectifying K?current when a selective
Y1R antagonist was applied. NPY release is consistent with the
high density of NPY axons observed near hypocretin neurons. If
the output of hypocretin neurons enhances arousal, then the
Consistent with this, NPY has been postulated to enhance seda-
tion after application to the LH, the region where hypocretin
neurons are found. This action involved a decrease of wakeful-
ness in pentobarbital- or avertin-induced sedation (Naveilhan et
al., 2001), suggesting that this effect may be explained in part by
NPY inhibition of the arousal-enhancing hypocretin cells. Simi-
et al., 1997). Because high levels of arousal may correlate with
a reduction in arousal through inhibition of the hypocretin neu-
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