Download Nicotine excites hypothalamic arcuate anorexigenic

J Neurophysiol 106: 1191–1202, 2011.
First published June 8, 2011; doi:10.1152/jn.00740.2010.
Nicotine excites hypothalamic arcuate anorexigenic proopiomelanocortin
neurons and orexigenic neuropeptide Y neurons: similarities and differences
Hao Huang, Youfen Xu, and Anthony N. van den Pol
Department of Neurosurgery, Yale University School of Medicine, New Haven, Connecticut
Submitted 25 August 2010; accepted in final form 5 June 2011
acetylcholine; hypothalamus; feeding; arcuate nucleus
SMOKING AND OBESITY are two leading causes of morbidity and
mortality worldwide (Haslam and James 2005; Mokdad et al.
2004). Obesity increases the risk for secondary health complications (Peeters et al. 2003). Smokers tend to have a lower
body weight than nonsmokers; people who quit smoking are
likely to gain weight (Ward et al. 2001; Williamson et al.
1991). Smoking cessation leads to increased food intake, decreased resting metabolic rate, and decreased physical activity
(Carney and Goldberg 1984; Ferrara et al. 2001; Hofstetter et
al. 1986). Nicotine, the major addictive component of tobacco,
increases energy expenditure, reduces appetite, and alters feeding patterns (Grunberg 1986; Grunberg et al. 1986; Miyata et
al. 2001), which typically results in reduced body weight. The
mechanism by which nicotine reduces body weight is unclear.
Neuronal nicotine acetylcholine (ACh) receptors (nAChRs) are
transmitter-gated ion channels (Karlin and Akabas 1995). A
Address for reprint requests and other correspondence: A. N. van den Pol,
Dept. of Neurosurgery, Yale Univ., School of Medicine, 333 Cedar St. New
Haven, CT 06520 (e-mail: [email protected]).
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number of different subtypes of nAChRs exist, each with an
individual pharmacological and physiological profile and distinct anatomic distribution in the brain (McGehee and Role
1995; Paterson and Nordberg 2000).
Neurons of the hypothalamic arcuate nucleus that synthesize
and release proopiomelanocortin (POMC) peptides play a key
role in reducing food intake (Wisse and Schwartz 2001). In
contrast, the nearby neurons that synthesize neuropeptide Y
(NPY) and agouti-related protein are thought to enhance food
intake and positive caloric balance by orexigenic actions within
the hypothalamus (Elmquist et al. 1999; Saper et al. 2002;
Schwartz et al. 2000; Seeley and Woods 2003; Spiegelman and
Flier 2001). The hypothalamus receives a rich cholinergic
innervation and has a diverse expression of nicotinic ␣ and ␤
nAChR subunits (Britto et al. 1992; Davila-Garcia et al. 1999;
Harfstrand et al. 1988; Hatton and Yang 2002; O’Hara et al.
1998; Okuda et al. 1993; Pabreza et al. 1991; Shioda et al.
1997). The physiological actions of nicotine on arcuate POMC
and NPY neuronal activity are unknown. Nicotine may have an
effect on NPY expression, but the data are inconsistent, with
nicotine reducing food intake and lowering arcuate nucleus
NPY and NPY mRNA levels (Jang et al. 2003), raising expression (Li et al. 2000a, 2000b), or raising mRNA but decreasing
the peptide (Frankish et al. 1995); duration of nicotine exposure may be one factor that may explain these seemingly
contradictory results.
Hypocretin/orexin cells in the perifornical/lateral hypothalamic area (de Lecea et al. 1998) have also been reported to
modulate food intake (Sakurai et al. 1998). Hypocretin neurons
enhance the wake state and cognitive arousal (Hagan et al.
1999). As smoking enhances cognitive arousal, it is possible
that one site of nicotine action is on the hypocretin cell.
Nicotine has been suggested to modulate hypocretin neurons or
neurons innervated by hypocretin axons (Hollander et al. 2008;
Plaza-Zabala et al. 2010), but little electrophysiological analysis of this in hypocretin neurons has been done. Interestingly,
hypocretin cells may play an important proaddiction role for a
number of unrelated drugs including opiates, cocaine, alcohol,
and nicotine, each of which acts at different receptors (Boutrel
2008; España et al. 2010; Georgescu et al. 2003; Harris et al.
2005; Hollander et al. 2008).
On the basis of an immunocytochemical study presented
here that reveals an abundant cholinergic innervation of the
hypothalamic POMC and NPY neurons, we tested the hypothesis that nicotine might inhibit food intake by activating the
anorexigenic POMC cells. For control purposes, we also studied the effect on NPY neurons. In this study, we used whole
cell voltage- and current-clamp recordings to study the cellular
effects of nicotine on the activity of identified POMC and NPY
0022-3077/11 Copyright © 2011 the American Physiological Society
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Huang H, Xu Y, van den Pol AN. Nicotine excites hypothalamic arcuate
anorexigenic proopiomelanocortin neurons and orexigenic neuropeptide Y
neurons: similarities and differences. J Neurophysiol 106: 1191–1202, 2011.
First published June 8, 2011; doi:10.1152/jn.00740.2010.—Two of the
biggest health problems facing us today are addiction to nicotine and
the increased prevalence of obesity. Interestingly, nicotine attenuates
obesity, but the underlying mechanism is not clear. Here we address
the hypothesis that if weight-reducing actions of nicotine are mediated
by anorexigenic proopiomelanocortin (POMC) neurons of the hypothalamic arcuate nucleus, nicotine should excite these cells. Nicotine
at concentrations similar to those found in smokers, 100 –1,000 nM,
excited POMC cells by mechanisms based on increased spike frequency, depolarization of membrane potential, and opening of ion
channels. This was mediated by activation of both ␣7 and ␣4␤2
nicotinic receptors; by itself, this nicotine-mediated excitation could
explain weight loss caused by nicotine. However, in control experiments nicotine also excited the orexigenic arcuate nucleus neuropeptide Y (NPY) cells. Nicotine exerted similar actions on POMC and
NPY cells, with a slightly greater depolarizing action on POMC cells.
Immunocytochemistry revealed cholinergic axons terminating on both
cell types. Nicotine actions were direct in both cell types, with
nicotine depolarizing the membrane potentials and reducing input
resistance. We found no differences in the relative desensitization to
nicotine between POMC and NPY neurons. Nicotine inhibited excitatory synaptic activity recorded in NPY, but not POMC, cells. Nicotine also excited hypocretin/orexin neurons that enhance cognitive
arousal, but the responses were smaller than in NPY or POMC cells.
Together, these results indicate that nicotine has a number of similar
actions, but also a few different actions, on POMC and NPY neurons
that could contribute to the weight loss associated with smoking.
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NICOTINE EXCITES POMC AND NPY NEURONS
neurons in hypothalamic arcuate nucleus slices from green
fluorescent protein (GFP)-expressing transgenic mice.
MATERIALS AND METHODS
J Neurophysiol • VOL
RESULTS
Cholinergic axons innervate arcuate nucleus. The present
study focuses on the physiological response to nicotine, a
substance that selectively activates the nicotinic type of ACh
receptor. We first examined the possibility that ACh was found
in axons in the arcuate nucleus with antisera against the ACh
vesicular transporter (VAChT), a marker for cholinergic neurons and axons. A high density of VAChT-immunoreactive
axons was found throughout the arcuate nucleus (Fig. 1).
POMC (Fig. 1A) and NPY (Fig. 1B) neurons, visualized by
GFP expression, were surrounded by red immunoreactive axons that appeared to be in contact with green fluorescent cell
bodies or proximal dendrites, suggesting that arcuate POMC
and NPY neurons may receive cholinergic innervation.
Nicotine excites POMC neurons. Whole cell recording was
then used to study the effect of nicotine on anorexigenic
POMC neuronal activity in hypothalamic slices from 2- to
3-wk-old transgenic mice. The effect of nicotine on POMC
neurons was tested in transgenic mice that expressed GFP
selectively in POMC neurons. Application of nicotine at 1 ␮M,
roughly equivalent to the arterial blood nicotine concentration
shortly after smoking a few cigarettes (Henningfield et al.
1993), excited POMC neurons. As shown in Fig. 2A, nicotine
(1 ␮M) depolarized the membrane potential (change by 6.5 ⫾
0.9 mV; n ⫽ 7, P ⬍ 0.05) and increased the spike frequency by
93.2 ⫾ 30.0% (n ⫽ 7, P ⬍ 0.05). With these low concentrations of nicotine, responses peaked between 30 and 90 s after
initiation of application. After nicotine application, six of seven
cells studied showed an increase ⬎20% in spike frequency;
one cell showed no effect. All cells tested are included in the
statistical analysis. After nicotine washout, the membrane potential and spike frequency returned toward prenicotine control
levels (Fig. 2, A and B). These results indicate that activation of
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Preparation of hypothalamic slices. Experiments were performed
on hypothalamic slices (250 –350 ␮m) obtained from NPY-GFP (van
den Pol et al. 2009), hypocretin-GFP (Li et al. 2002), or POMC-GFP
transgenic mice. POMC-GFP mice have been described elsewhere
(Cowley et al. 2001), and were kindly provided by Dr. Malcolm Low.
Young 14- to 21-day-old or adult 6- to 7-wk-old mice maintained in
a 12:12-h light-dark cycle were given an overdose of pentobarbital
sodium (100 mg/kg) during the light part of the cycle (11:00 AM to
4:00 PM). Brains were then removed rapidly and placed in an
ice-cold, oxygenated (95% O2-5% CO2) high-sucrose solution that
contained (in mM) 220 sucrose, 2.5 KCl, 6 MgCl2, 1 CaCl2, 1.23
NaH2PO4, 26 NaHCO3, and 10 D-glucose, pH 7.4 (with an osmolarity
of 300 –305 mosM). A block of tissue containing the hypothalamus
was isolated, and coronal slices were cut on a vibratome. After a 1- to
2-h recovery period, slices were moved to a recording 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
flow of gassed artificial cerebrospinal fluid (ACSF; 95% O2 and 5%
CO2) that contained (in mM) 124 NaCl, 2.5 KCl, 2 MgCl2, 2 CaCl2,
1.23 NaH2PO4, 26 NaHCO3, and 10 glucose, pH 7.4. Bath temperature in the recording chamber was maintained at 33 ⫾ 1°C with a
dual-channel heat controller (Warner Instruments, Hamden, CT).
Neurons were visualized with an Olympus Optical ⫻40 water-immersion lens. The Yale University Committee on Animal Care and Use
approved all procedures used in this study.
Patch-clamp recording. Whole cell current- and voltage-clamp
recordings were performed with pipettes with 4- to 6-M⍀ resistance
after being filled with pipette solution. The pipettes were made of
borosilicate glass (World Precision Instruments, Sarasota, FL) with a
PP-83 vertical puller (Narishige, Tokyo, Japan). For most recordings,
the composition of the pipette solution was as follows (in mM): 145
KMeSO4 [or KCl for inhibitory postsynaptic currents (IPSCs)], 1
MgCl2, 10 HEPES, 1.1 EGTA, 2 Mg-ATP, 0.5 Na2-GTP, 5 Na2phosphocreatine, pH 7.3 with KOH (with an osmolarity of 290 –295
mosM). Residual series resistance was 3–5 m⍀ after electronic
compensation. Potentials were corrected for liquid junction potential,
which in our system is ⫺9.5 mV when calculated with JPCalc
software (Barry 1994). Slow and fast capacitance compensation was
automatically performed with Pulse software (HEKA Elektronik,
Lambrecht/Pfalz, Germany). Access resistance was continuously
monitored during the experiments. Only those cells in which access
resistance was stable (changes ⬍10%) were included in the analysis.
As unhealthy neurons can show a positive shift in resting membrane
potential (RMP) (Ceranik et al. 1997; Manuel et al. 2009; Stucky and
Lewin 1999), we did not experiment on any neurons with an RMP
positive to ⫺45 mV. An EPC10 amplifier and Pulse software were
used for data acquisition (HEKA Elektronik). PulseFit (HEKA Elektronik), Axograph (Axon instruments, Foster City, CA), and Igor Pro
(WaveMetrics, Lake Oswego, OR) software were used for analysis.
Both excitatory and inhibitory spontaneous postsynaptic currents were
detected and measured with an algorithm in Axograph, and only those
events with amplitude ⬎5 pA were used, as described in detail
previously (Gao and van den Pol 2001). The frequency of action
potentials was measured with Axograph as well. Data are expressed as
means ⫾ SE. Group statistical significance was assessed with Student’s t-test for comparison of two groups and with one-way ANOVA
followed by a Bonferroni post hoc test for three or more groups. P ⬍
0.05 was considered statistically significant.
Drugs and drug application. (⫺)-Nicotine hydrogen tartrate, D-tubocurarine (d-TC), dihydro-␤-erythroidine hydrobromide (DHBE),
methyllycaconitine citrate (MLA), and mecamylamine hydrochloride
(MEC) were purchased from Tocris Bioscience (Ellisville, MO);
6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), DL-2-amino-5-phosphonopentanoic acid (APV), bicuculline (BIC), and acetylcholine
chloride (ACh) were purchased from Sigma (St. Louis, MO). Tetrodotoxin (TTX) was obtained from Alomone Labs (Jerusalem, Israel),
and ipratropium bromide (atropine) was obtained from Tocris Bioscience. All drugs were given by large-diameter (500 ␮m) flow pipette,
directed at the recorded cell, unless otherwise noted. When a drug was
not being administered, normal ACSF continuously flowed from the
flow pipe. Drug solutions were prepared by diluting the appropriate
stock solution with ACSF.
Immunocytochemistry. To identify cholinergic axons, we used an
antibody against the cholinergic vesicular transporter VAChT (ACh
vesicular transporter). Transgenic mice expressing GFP selectively in
POMC and NPY neurons were heavily anesthetized with pentobarbital and then perfused transcardially with saline followed by 4%
paraformaldehyde. Fifteen- to twenty-five-micrometer-thick coronal
sections were cut on a cryostat, immersed in normal PBS, and then
placed in guinea pig anti-VAChT (Chemicon) at a dilution of 1:2,500
overnight (Gras et al. 2002); this antibody labeled a single band on a
Western blot of the expected size for VACh, and this band was absent
with VAChT antigen incubation. After washing five times in normal
buffer, sections were placed in secondary antiserum of donkey antiguinea pig conjugated to CY5 at a dilution of 1:200 for 1–2 h, washed,
and mounted on glass slides. Sections were studied on an Olympus
IX70 inverted fluorescence microscope. Micrographs were recorded
on a Spot digital camera (Diagnostic Imaging), and contrast and
brightness were corrected with Photoshop CS2.
NICOTINE EXCITES POMC AND NPY NEURONS
nicotine receptors has a strong excitatory action on POMC
neurons.
Nicotine excites NPY neurons. Actions of nicotine on nearby
orexigenic NPY neurons were also studied. Nicotine (1 ␮M)
depolarized GFP-expressing NPY neurons (membrane depolarization: 4.7 ⫾ 0.4 mV; n ⫽ 11) and increased the frequency
of action potentials by 63.2 ⫾ 18.5% (from 1.1 ⫾ 0.2 to 1.7 ⫾
0.3 Hz; n ⫽ 11, P ⬍ 0.05; Fig. 2F). In these 11 cells studied,
9 cells showed an increase ⬎20% in spike frequency following
nicotine application; the remaining 2 cells showed little or no
effect. After nicotine washout, the NPY neurons returned
toward control values (Fig. 2, F and G).
Postsynaptic actions of nicotine on POMC and NPY
neurons. To investigate whether nicotine had a direct postsynaptic effect on POMC neurons, we examined the actions of
nicotine on the membrane potential of POMC neurons in the
presence of the sodium channel blocker TTX. In the presence
of TTX (0.5 ␮M), nicotine depolarized the membrane potential
by 5.2 ⫾ 0.7 mV (n ⫽ 7, P ⬍ 0.05; Fig. 2C), suggesting that
nicotine has a direct effect on POMC neurons.
J Neurophysiol • VOL
To determine whether the excitatory actions of nicotine on
POMC neurons were accompanied by changes in the whole
cell input resistance, we delivered negative current steps (from
⫺10 to ⫺60 pA during 500 ms; increments of 10 pA) through
the recording pipette and evaluated changes in the membrane
potential before and after nicotine application in the presence
of TTX (0.5 ␮M). In the presence of nicotine (1 ␮M), the
hyperpolarizing shifts in the membrane potential in response to
the injection of negative current steps were reduced (Fig. 2D),
and the current-voltage relationship showed a consistent alteration compared with control prenicotine conditions (Fig. 2E). A
linear function was fitted to the current-voltage relationship,
and a significant decrease in the slope was observed after
nicotine application, consistent with a reduction in the whole
cell input resistance from 1.4 ⫾ 0.2 to 1.1 ⫾ 0.1 G⍀ (n ⫽ 6,
P ⬍ 0.05; Fig. 2E), suggesting that the mechanism of nicotine
excitation is due to opening of ion channels.
Similar nicotine actions were also observed on NPY neurons. In the presence of TTX, nicotine (1 ␮M) depolarized the
membrane potential by 4.6 ⫾ 0.5 mV (n ⫽ 7, P ⬍ 0.05; Fig.
2H). As in POMC neurons, the whole cell input resistance was
also significantly reduced in NPY neurons from 1.2 ⫾ 0.08 to
0.9 ⫾ 0.09 G⍀ (n ⫽ 9; P ⬍ 0.05; Fig. 2, I and J) after nicotine
(1 ␮M) application.
Nicotine-induced depolarization in POMC and NPY neurons is mediated via both ␣4␤2 and ␣7 nicotinic receptors.
Nicotine acts on cholinergic receptors. We next tested whether
ACh would directly excite POMC neurons. In the presence of
5 ␮M atropine, an irreversible muscarinic receptor antagonist,
1 ␮M ACh evoked a membrane depolarization of 3.2 ⫾ 0.6
mV (n ⫽ 6; Fig. 3A, left) and 100 ␮M ACh evoked a
depolarization of 8.8 ⫾ 0.6 mV (n ⫽ 6; Fig. 3A, right).
We then studied the pharmacology of the nicotine receptors
in POMC neurons. The experiments were conducted in the
presence of TTX (0.5 ␮M). In control conditions, the depolarization by nicotine was 5.2 ⫾ 0.7 mV (n ⫽ 7, P ⬍ 0.05; Fig.
3B, 1st trace). In all the neurons tested (n ⫽ 8), the excitatory
response to nicotine was significantly blocked by d-TC, a
broad-spectrum nicotinic receptor antagonist (Fig. 3B, 2nd
trace). When the slice was pretreated for 10 –15 min with d-TC,
the depolarization evoked by nicotine was blocked (change by
0.4 ⫾ 0.4 mV; P ⬍ 0.05 compared with control, n ⫽ 8). To
determine the specific nicotinic receptor subtype(s) involved,
we tested the effect of subtype-selective receptor antagonists.
As shown in Fig. 3B (3rd trace), when the slice was pretreated
for 10 –15 min with DHBE, an ␣4␤2 nicotine receptor antagonist, the depolarization by nicotine was significantly reduced.
In the presence of DHBE (1 ␮M), the depolarization by nicotine
was 1.9 ⫾ 0.6 mV (n ⫽ 5, P ⬍ 0.05 compared with control,
ANOVA; Fig. 3C). Similarly, when the slice was pretreated with
the ␣7-preferring antagonist MLA, the depolarization by nicotine
was also significantly reduced (Fig. 3B, 4th trace). With MLA (20
nM) in the bath for 10 –15 min, the depolarization by nicotine was
3.5 ⫾ 0.4 mV (n ⫽ 6, P ⬍ 0.05 compared with control, ANOVA;
Fig. 3C). When comparing the effect on the depolarization of
d-TC with either DHBE or MLA, a statistically significant difference was detected (Fig. 3C).
We also investigated the pharmacology of the nicotine
receptors in NPY neurons. Ten micromolar d-TC almost completely blocked the depolarization evoked by nicotine in NPY
neurons (Fig. 3D, 2nd trace). In control conditions, the depo-
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Fig. 1. Cholinergic axons innervate arcuate nucleus. Cholinergic axons were
identified with immunostaining against the vesicular acetylcholine (ACh)
transporter VAChT. Red VAChT immunoreactive axons and boutons (arrows)
surround a neuron expressing green fluorescent protein (GFP) under regulation
of the proopiomelanocortin (POMC; A) and neuropeptide Y (NPY; B) promoter. Scale bar, 6 ␮m.
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NICOTINE EXCITES POMC AND NPY NEURONS
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Fig. 2. Nicotine excites POMC and NPY
neurons. A: nicotine (1 ␮M) increases the
spike frequency of a POMC neuron. B: time
course of the nicotine effect on spike frequency in the above POMC neuron; 100%
was defined as the mean spike frequency in
the minute preceding nicotine application.
C: in the presence of tetrodotoxin (TTX),
nicotine depolarizes the membrane potential
of a POMC neuron. Resting membrane potential (RMP), ⫺61.5 mV. D: nicotine decreases the voltage response of POMC neurons to hyperpolarizing current steps (shown
below response). E: current-voltage (I-V) relationship in the absence () and presence
(Œ) of nicotine in POMC neurons. F: nicotine (1 ␮M) increases the spike frequency of
a NPY neuron. G: time-course of effect of
nicotine on spike frequency in the above
NPY neuron. H: in the presence of TTX,
nicotine depolarizes the membrane potential
of a NPY neuron. RMP, ⫺60.5 mV. I: nicotine decreases the voltage response of NPY
neurons after hyperpolarizing current steps
(shown below response). J: current-voltage
relationship in the absence () and presence
(Œ) of nicotine in NPY neurons.
larization by nicotine was 4.9 ⫾ 0.1 mV (n ⫽ 12; Fig. 3D, 1st
trace) in NPY neurons. In the presence of d-TC, the depolarization evoked by nicotine was 0.8 ⫾ 0.2 mV (n ⫽ 5, P ⬍ 0.05
compared with control; Fig. 3E). When the slice was pretreated
with the ␣4␤2 nicotine receptor antagonist DHBE for 10 –15
min, the depolarization by nicotine was significantly reduced
(Fig. 3D, 3rd trace). In 1 ␮M DHBE, the depolarization by
nicotine was 2.6 ⫾ 0.1 mV (n ⫽ 6; P ⬍ 0.05 compared with
control; ANOVA, Fig. 3E). MLA (20 nM) also significantly
reduced the response induced by nicotine (Fig. 3D, 4th trace).
The depolarization by nicotine in MLA was 1.7 ⫾ 0.2 mV (n ⫽
6; P ⬍ 0.05 compared with control, ANOVA; Fig. 3E).
These data provide pharmacological evidence that nicotine
receptors containing ␣4␤2 and ␣7 subunits are functionally
expressed in both POMC and NPY neurons and both ␣4␤2 and
␣7 subunits of nicotine receptors are involved in nicotineinduced membrane depolarization.
J Neurophysiol • VOL
Nicotine excites immature and adult POMC and NPY
neurons. NPY and POMC neurons have opposite actions on
food intake and energy metabolism (Elmquist et al. 1999;
Wisse and Schwartz 2001). To investigate the net effect of
nicotine on NPY and POMC neurons, we compared the effect
of nicotine on these two types of neurons in 2- to 3-wk-old
mice. As shown in Fig. 4, A and B, 1 ␮M nicotine evoked a
greater effect on spike frequency and membrane potential (P ⬍
0.05) in POMC than in NPY neurons. In addition, we studied
the effect of lower concentrations of nicotine (100 nM and 300
nM) on both POMC and NPY neurons. Nicotine at 100 nM and
300 nM depolarized the membrane potential of POMC neurons
by 3.0 ⫾ 0.3 mV (n ⫽ 7) and 4.1 ⫾ 0.4 mV (n ⫽ 6) and
increased the spike frequency by 29.6 ⫾ 8.0% and 56.2 ⫾
15.5%, respectively (Fig. 4, A and B). Similarly, nicotine at 100
nM and 300 nM depolarized the membrane potential of NPY
neurons by 3.2 ⫾ 0.4 mV (n ⫽ 7) and 3.6 ⫾ 0.3 mV (n ⫽ 5)
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NICOTINE EXCITES POMC AND NPY NEURONS
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and increased the spike frequency by 24.5 ⫾ 8.4% and 37.0 ⫾
12.1%, respectively (Fig. 4, A and B). When data from all
concentrations (100 nM to 1 ␮M) were pooled (n ⫽ 43),
nicotine’s depolarizing action on POMC cells was significantly
greater than on NPY cells (P ⬍ 0.05).
We also studied the effect of nicotine in 6- to 7-wk-old adult
NPY and POMC transgenic mice. Nicotine at 1 ␮M significantly increased the spike frequency of POMC neurons by
62.4 ⫾ 19.2% and depolarized the membrane potential by
6.1 ⫾ 0.4 mV (from an initial membrane potential of ⫺59.0 ⫾
2.2 mV) (n ⫽ 9, P ⬍ 0.05; Fig. 4, C and D). In NPY neurons,
nicotine (1 ␮M) significantly increased the spike frequency by
44.2 ⫾ 13.2% and depolarized the membrane potential by
4.2 ⫾ 0.4 mV (from an initial membrane potential of ⫺59.6 ⫾
3.1 mV) (n ⫽ 9, P ⬍ 0.05; Fig. 4, C and D). Nicotine evoked
a significantly greater effect on membrane depolarization in
POMC neurons than in NPY neurons (P ⬍ 0.05; Fig. 4D).
These results indicate that nicotine has a slightly greater
excitatory action on POMC neurons than on NPY neurons at a
J Neurophysiol • VOL
1 ␮M concentration. Although the nicotine responses appeared
slightly greater in the younger mice, this difference was not
statistically significant (P ⬎ 0.05, unpaired t-test).
POMC and NPY neurons show similar desensitization to
nicotine. The response to nicotine can desensitize with repeated exposure (Quick and Lester 2002). One possibility that
could underlie different effects of nicotine on the two cell types
is different levels of desensitization. In the first experiments
testing both POMC and NPY neurons, after repetitive nicotine
microapplications (10 mM for 1 s) from a micropipette, a
similar progressive decrease in the amplitude of the nicotineinduced current was recorded (Fig. 5, A and B), suggesting
similar receptor desensitization in both cell types after repetitive activation.
The properties of desensitization were then studied by a
somewhat different second approach in which a two-pulse
protocol of identical nicotine pulses (10 mM, 1-s duration) was
delivered at increasing time intervals (from 1 s to 60 s) from a
micropipette. The desensitization was assessed by calculating
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Fig. 3. Both ␣4␤2 and ␣7 nicotinic receptors
mediate nicotine-induced membrane depolarization in POMC and NPY neurons.
A: action of ACh (1 ␮M, left and 100 ␮M,
right) on membrane potentials of POMC
neurons in the presence of atropine (5 ␮M),
TTX (0.5 ␮M), 6-cyano-7-nitroquinoxaline2,3-dione (CNQX, 10 ␮M), AP-5 (50 ␮M),
and bicuculline (BIC, 30 ␮M). B: actions of
nicotine (1 ␮M) on POMC neuron membrane potential in the absence (control;
RMP, ⫺62.5 mV) or presence of broadspectrum nicotine receptor antagonist D-tubocurarine (d-TC, 10 ␮M; RMP, ⫺59.0
mV), ␣4␤2 nicotine receptor antagonist dihydro-␤-erythroidine hydrobromide (DHBE, 1
␮M; RMP, ⫺60.5 mV), or ␣7 nicotine receptor
antagonist methyllycaconitine (MLA, 20 nM;
RMP, ⫺61.5 mV). C: mean effect of nicotine on membrane potential obtained from B.
Number of cells is shown in parentheses.
Error bars indicate SE. D: action of nicotine
(1 ␮M) on NPY neuron membrane potential
in the absence (control; RMP, ⫺65 mV) or
presence of broad-spectrum nicotine receptor antagonist d-TC (10 ␮M; RMP, ⫺66
mV), ␣4␤2 nicotine receptor antagonist
DHBE (1 ␮M; RMP, ⫺70 mV), or ␣7 nicotine receptor antagonist MLA (20 nM;
RMP, ⫺60 mV). E: mean effect of nicotine
on membrane potential obtained from D.
Number of cells is shown in parentheses.
Error bars indicate SE. *Each of the 3 test
groups was significantly different from control. #Response to nicotine in the presence of
either DHBE or MLA alone was significantly different from that in the presence of
d-TC.
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NICOTINE EXCITES POMC AND NPY NEURONS
the ratio of the amplitude of the second nicotine-induced
current divided by that of the first one. As shown in Fig. 5, C
and D, both POMC and NPY neurons developed quick desensitization and mostly recovered by 60 s after the first nicotine
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Fig. 4. Nicotine exerts slightly greater magnitude depolarizing actions in
POMC neurons than in NPY neurons. A: mean spike frequency after nicotine
application at different concentrations, including 100 nM, 300 nM, and 1 ␮M,
in NPY and POMC neurons from young mice. B: mean depolarization of
membrane potential evoked by nicotine at concentrations of 100 nM, 300 nM,
and 1 ␮M in NPY and POMC neurons from young mice. C: action of nicotine
(1 ␮M) on spike frequency in NPY and POMC neurons from adult mice.
D: action of nicotine (1 ␮M) on depolarization of membrane potential in NPY
and POMC neurons from adult mice. Numbers of cells are shown in parentheses. Error bars indicate SE. *Each of the test groups was significantly
different from the control (A–D). #Membrane depolarization induced by
nicotine in POMC neurons was significantly higher than in NPY neurons (B
and D).
application. When the desensitization ratios at 1 s, 2 s, 3 s, 4 s,
8 s, 16 s, 32 s, and 60 s after the first nicotine application were
compared, there was no significant difference between POMC
and NPY neurons (P ⬎ 0.05; Fig. 5E). These results suggest
that nicotine has similar desensitizing actions in both POMC
and NPY neurons.
Nicotine depresses excitatory postsynaptic currents in NPY
neurons but not in POMC neurons. We next investigated the
effect of nicotine on synaptic transmission. First we tested
nicotine actions on excitatory postsynaptic currents (EPSCs) in
POMC neurons. In these experiments, BIC (30 ␮M) was added
to the bath to block GABAA receptors. Nicotine showed no
detectable effect on frequency (change by 0.2 ⫾ 4.8% of
control) or amplitude (change by 3.1 ⫾ 2.9% of control) of
spontaneous EPSCs (sEPSCs) (n ⫽ 7, P ⬎ 0.05; Fig. 6, A and
C). Figure 6B shows the time course of the nicotine effect on
the frequency of sEPSCs. Further application of the glutamate
receptor antagonists CNQX (10 ␮M) and APV (50 ␮M)
completely suppressed the synaptic currents, confirming the
glutamatergic nature of these currents (n ⫽ 3; data not shown).
In addition, the effect of nicotine on spontaneous IPSCs (sIPSCs) in POMC neurons was studied. Nicotine at 1 ␮M exerted
no significant effect on the frequency (change by ⫺2.9 ⫾
10.5%; n ⫽ 9, P ⬎ 0.5) or amplitude (change by ⫺4.9 ⫾ 5.2%;
n ⫽ 9, P ⬎ 0.5) of sIPSCs in POMC neurons (data not shown).
The GABAA receptor antagonist BIC completely suppressed
the inhibitory synaptic currents, confirming that they are attributable to the activation of GABAA receptors (n ⫽ 3; data
not shown).
The effect of nicotine on sEPSCs in NPY neurons was then
studied. As shown in Fig. 6, D and F, nicotine (1 ␮M)
significantly decreased the frequency of sEPSCs by 37.9 ⫾
6.1% (from 3.6 ⫾ 0.6 Hz to 2.4 ⫾ 0.5 Hz; n ⫽ 14, P ⬍ 0.05).
Furthermore, nicotine also decreased the amplitude of sEPSCs
by 11.6 ⫾ 2.7% (n ⫽ 14, P ⬍ 0.05; Fig. 6, D and F). The time
course of the nicotine effect is shown in Fig. 6E. In 12 of 14
cells tested, the decrease was ⬎20%; 2 cells showed no effect.
In a follow-up experiment with TTX (0.5 ␮M) in the bath,
nicotine (1.0 ␮M) showed no significant effect on miniature
EPSCs (to 117.9 ⫾ 7.1% and 103.9 ⫾ 2.3% of control
frequency and amplitude, respectively; n ⫽ 9; P ⬎ 0.05) (not
shown). These results suggest that nicotine reduces glutamate
release through actions on presynaptic cell bodies or dendrites,
and probably not directly on the presynaptic axons.
We next asked whether the reduction in EPSCs in NPY
neurons might be due to an initial increase and then a desensitization-mediated longer term decrease. However, we found
no initial increase in EPSC frequency, but rather a continued
reduction. During a 10-min application of 1 ␮M nicotine, by
the 3rd minute EPSC frequency was decreased by 26.2 ⫾
5.0%, by the 6th minute 32.6 ⫾ 5.6%, and by the 10th minute,
EPSC frequency was reduced by 31.3 ⫾ 5.4% (n ⫽ 4; data not
shown). No significant difference in EPSC frequency reduction
was found between the three intervals (P ⬎ 0.05).
To corroborate that the nicotine-induced inhibition of sEPSCs in
NPY neurons was mediated by the activation of nicotinic
receptors, we investigated the effect of nicotine on sEPSCs in
the presence of the noncompetitive nicotinic receptor antagonist MEC (Rabenstein et al. 2006). Nicotine (1 ␮M) induced
a significant decrease in sEPSC frequency and amplitude
(Fig. 6H) with a mean reduction of 31.3 ⫾ 0.6% and 27.2 ⫾
NICOTINE EXCITES POMC AND NPY NEURONS
1197
1.6%, respectively (n ⫽ 5, P ⬍ 0.05 compared with control,
ANOVA). When the slice was pretreated with MEC (1 ␮M)
for 10 min, this inhibition by nicotine was significantly reduced
to 14.9 ⫾ 0.4% in frequency and 16.2 ⫾ 0.4% in amplitude
(n ⫽ 7, P ⬍ 0.05 nicotine vs. nicotine ⫹ MEC, ANOVA; Fig.
6H). MEC may not block all nicotine receptors, for instance,
␣7 nAChR (Gao et al. 2010; Ishibashi et al. 2009), potentially
explaining the absence of complete block by MEC. We therefore employed a cocktail of three nAChR antagonists (MEC,
MLA, and DHBE). Treatment for 10 min with MEC (1 ␮M),
MLA (20 nM), and DHBE (1 ␮M) blocked the nicotine (1
␮M)-mediated reduction in sEPSC frequency and amplitude
(n ⫽ 6, P ⬎ 0.5 control vs. nicotine ⫹ all 3 nAChR antagonists
and P ⬍ 0.05 nicotine vs. nicotine ⫹ all 3 nAChR antagonists,
ANOVA; Fig. 6, G and H). These data are consistent with the
view that the reduction of glutamate release by nicotine application is modulated via nAChRs.
As we earlier found immunocytochemical evidence for cholinergic innervation of NPY and POMC neurons, to determine
whether the nicotine effect on sEPSCs described above in NPY
neurons might be modulated by ongoing ACh release, d-TC
(10 ␮M), a broad-spectrum nicotine receptor antagonist, was
applied to brain slices. d-TC had little effect on sEPSCs (n ⫽
5, not shown) and no effect on baseline current. Similarly, we
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found no effect of d-TC (10 ␮M) on baseline current in voltage
clamp in POMC neurons (n ⫽ 5). These data suggest that
under our conditions, there was little ongoing intrinsic action
of ACh on nicotinic receptors expressed by NPY or POMC
neurons.
We also investigated the effect of nicotine on IPSCs in NPY
neurons in the presence of the ionotropic glutamate receptor
blockers APV (50 ␮M) and CNQX (10 ␮M). Nicotine (1 ␮M)
showed no clear effect on the frequency or amplitude of
sIPSCs (change by 6.0 ⫾ 6.8% and 3.9 ⫾ 5.6% of control
frequency and amplitude, respectively; n ⫽ 6, P ⬎ 0.05) (data
not shown).
Hypocretin cells. POMC and NPY cells showed a somewhat
similar sensitivity to nicotine; we also compared a third cell
type, the hypocretin neuron from the lateral hypothalamus. The
hypocretin cells play a role in enhancing cognitive arousal, and
have also been suggested to modulate arousal related to energy
homeostasis. Hypocretin cells project to, and excite, both
POMC and NPY neurons (Acuna-Goycolea and van den Pol
2009; Horvath et al. 1999; van den Top et al. 2004). Nicotine
(1 ␮M) significantly increased the spike frequency by 19.1 ⫾
3.3% (n ⫽ 10, P ⬍ 0.05). In the 10 hypocretin cells tested here,
6 cells showed an increase ⬎20% of control spike frequency
following nicotine application; the remaining 4 cells showed
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Fig. 5. Similar rates of desensitization to nicotine application exist between POMC and NPY neurons. A and
B: nicotine-induced currents desensitize during repeated
application of nicotine (10 mM, 1-s duration) in POMC (A)
and NPY (B) neurons. C: traces show the currents induced
by equimolar applications of nicotine at different intervals,
including 1 s, 2 s, 3 s, 4 s, 8 s, 16 s, 32 s, and 60s, in POMC
neurons. D: traces show the currents induced by equimolar
nicotine applications at different intervals, including 1 s, 2
s, 3 s, 4 s, 8 s, 16 s, 32 s, and 60 s, in NPY neurons. E: ratio
of the amplitude of the 2nd nicotine-induced current divided by that of the 1st one with increasing intervals as
shown in C. There was no significant difference in the rate
of desensitization between POMC and NPY neurons.
1198
NICOTINE EXCITES POMC AND NPY NEURONS
no effect. Nicotine at 300 nM evoked only a modest increase in
spike frequency (increase by 11.3 ⫾ 6.8%; n ⫽ 4). Because of
the continuous spiking of hypocretin cells and the consequent
variable membrane potential (Li et al. 2002), we did not
examine depolarization in the absence of TTX. In the presence
of TTX (0.5 ␮M), nicotine (1 ␮M) evoked a 1.8 ⫾ 0.8 mV
(n ⫽ 5) depolarization. The increase in spike frequency evoked
by 1 ␮M nicotine and the depolarization in TTX were significantly less in hypocretin neurons than in NPY or POMC
neurons (P ⬍ 0.05).
DISCUSSION
In the present study, we used voltage- and current-clamp
whole cell recording to study the actions of nicotine on GFPexpressing POMC and NPY neurons in hypothalamic slices in
vitro. Nicotine evoked a slightly greater depolarization in
POMC cells than in NPY cells, but with TTX no difference
was found between these two cell types. POMC and NPY
neurons showed similar desensitization rates upon repeated
exposure to nicotine. Nicotine exerted an indirect inhibitory
effect on the synaptic release of glutamate onto NPY, but not
J Neurophysiol • VOL
POMC, neurons. The modestly bigger direct excitatory action
on anorexigenic POMC neurons and indirect inhibitory actions
on orexigenic NPY neurons suggest that nicotine actions on
these cells may contribute to multiple mechanisms by which
body weight and food intake are reduced by nicotine.
Actions of nicotine on POMC and NPY neurons. POMC and
NPY neurons both appear quite sensitive to even low (nanomolar) concentrations of nicotine. Low concentrations (100
and 300 nM) of nicotine evoked a similar membrane depolarization and spike frequency increase in the two cell types.
Furthermore, the desensitization profiles of these two cell
types to prolonged or repeated nicotine application were
similar. The depolarizing action on membrane potential
continues in the presence of the sodium channel blocker
TTX, suggesting that this is a direct postsynaptic effect. In
addition, the nicotine-mediated decrease in input resistance
in both cell types suggests that the excitatory effect of
nicotine on these cells probably results from the opening of
ion channels. The depolarizing actions on membrane potential in POMC cells are significantly decreased by MLA, an
␣7 nAChR antagonist, and by DHBE, a selective ␣4␤2
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Fig. 6. Nicotine attenuates excitatory synaptic input to NPY cells but not POMC cells.
A: traces show that nicotine (1 ␮M) evokes
little effect on spontaneous excitatory postsynaptic currents (sEPSCs) in a POMC neuron. B: time-course effect of nicotine on the
frequency of sEPSCs as shown in A. C: mean
effect of nicotine on the frequency and amplitude
of sEPSCs in POMC neurons. D: traces show
that nicotine (1 ␮M) decreases sEPSCs in a NPY
neuron. E: time-course effect of nicotine on the
frequency of sEPSCs as shown in D. F: mean
effect of nicotine on the frequency and amplitude
of sEPSCs in NPY neurons. Nicotine significantly decreases the frequency and amplitude of
sEPSCs in NPY neurons. G: traces show sEPSC
before (control) and during nicotine (1 ␮M) in
the presence of 3 nAChR antagonists: MEC (1
␮M), the broad-spectrum nicotinic receptor antagonist, MLA (20 nM), an ␣7 nicotinic receptor
antagonist, and DHBE (1 ␮M), an ␣4␤2 nicotinic receptor antagonist. H: effectiveness of
nAChR antagonists in blocking the nicotine-induced inhibition in sEPSC frequency and amplitude in NPY neurons. sEPSCs were significantly
decreased by nicotine both in frequency and in
amplitude, and this nicotine-mediated reduction
was partially blocked by MEC and almost
blocked by a combination of MEC, MLA, and
DHBE. Number of cells is shown in parentheses.
Error bars indicate SE. *Each of the test groups
was significantly different from the control (F
and H). #Statistical significance between nicotine
and nicotine⫹MEC or between nicotine and
nicotine⫹MEC⫹MLA⫹DHBE (H). Ctrl, control; Nic, nicotine; W, washout.
NICOTINE EXCITES POMC AND NPY NEURONS
J Neurophysiol • VOL
neurons was less substantial than that found in either POMC or
NPY neurons. As hypocretin cells are responsible for maintaining a wake state, and are associated with enhanced cognitive arousal, the nicotine-mediated excitation could underlie
some of the behavioral actions of nicotine on arousal (Mineur
and Picciotto 2010; Picciotto et al. 2000). Growing evidence
also suggests a key role for hypocretin cells in supporting
addiction to a number of drugs, including nicotine (Boutrel
2008; España et al. 2010; Georgescu et al. 2003; Harris et al.
2005; Hollander et al. 2008). The modest response to nicotine
suggests that in addition to a direct effect on the hypocretin
cells, indirect effects, including possible presynaptic effects on
hypocretin axon terminals, or nicotine actions on cells postsynaptic to hypocretin axons are involved in the interaction of
hypocretin and nicotine.
Functional relevance. Nicotine is the primary addictive
agent in tobacco, the most widespread substance of abuse;
tobacco is responsible for a wide variety of health problems. A
second major health problem today is the growing incidence of
obesity and its own secondary health problems. In the context
of the worldwide obesity epidemic and a high prevalence of
smoking, the relation between smoking and obesity thus has
major public health relevance. These two major health risks are
not independent. Nicotine is an appetite suppressant, and reduced smoking often leads to an increase in eating and body
weight (Ward et al. 2001; Williamson et al. 1991). In this
study, we found that nicotine not only excited POMC neurons
but also had an excitatory effect on NPY neurons. The excitatory effect on adult POMC neurons was modestly greater than
on NPY neurons, and this greater excitatory effect on these
anorexigenic cells may contribute to nicotine-mediated weight
loss. The absence of nicotine stimulation as a result of smoking
cessation would tend to reduce the firing rate of anorexigenic
POMC cells and reduce the inhibition of excitatory synaptic
inputs to NPY neurons; the combination of these two effects
may constitute one mechanism for the increase in body weight
occurring when nicotine intake is stopped. A caveat here is that
the difference in POMC and NPY cell responses was blocked
by TTX and was not very large; desensitization profiles for
nicotine were remarkably similar for both cell types. Additionally, our experiments were done on brain slices, sometimes in
the presence of various transmitter receptor antagonists. Study
of nicotine responses by NPY and POMC cells in vivo would
help clarify the complexity of differential actions in the two
cell types. Thus it seems probable that nicotine actions on other
neuron types also contribute to the appetite-suppressant actions
of nicotine.
We show here that nicotine excites hypocretin cells, but this
is unlikely to explain the nicotine-mediated reduced food
intake, since hypocretin cells have been suggested to be orexigenic. On the other hand, the nicotine activation of the hypocretin cell could serve as a partial explanation for the increased
cognitive arousal associated with nicotine. Another cell type
that may play a role in food intake is the orexigenic hypothalamic melanin concentrating hormone (MCH) neuron. Nicotine
increases the inhibitory synaptic activity to MCH cells (Jo et al.
2005), which would be consistent with reduced food intake.
A number of papers have reported opposing actions of
several signaling molecules relevant to energy homeostasis in
POMC and NPY cells. For instance, POMC cells are excited
by leptin and serotonin, whereas NPY cells are inhibited by
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nAChR antagonist, suggesting that both ␣7 and ␣4␤2
nAChRs coexist in these neurons.
Two primary differences in nicotine responses were found in
POMC and NPY neurons. The first was that, with higher
concentrations (1 ␮M), nicotine evoked a slightly greater
depolarizing action on POMC than on NPY cells. The second
difference was that nicotine evoked an inhibitory effect on
synaptic release of glutamate onto NPY neurons, whereas it
had no detectable effect on glutamate release onto POMC
neurons. Whereas nicotine generally enhances synaptic release
(Jo et al. 2005; Kawa 2002; Neff et al. 1998; Wu et al. 2003),
in this case we find an inhibitory effect. Although this inhibitory effect is unusual, it is not without precedent (Fisher and
Dani 2000; Levy et al. 2006; Maggi et al. 2004; Zhu and
Chiappinelli 1999). In most cases, such a decrease has been
attributed to excitation of GABA neurons that then inhibit
glutamate release; this is consistent with the lack of effect of
nicotine on miniature EPSCs in NPY cells, suggesting that
nicotine did not act directly on glutamatergic axon terminals to
inhibit release. In other brain regions, there is some evidence
for a nicotine-mediated reduction in NMDA responses (Fisher
and Dani 2000), and a possible presynaptic inhibition has been
suggested, particularly for high probability-release synapses
(Levy et al. 2006; Maggi et al. 2004).
The inhibitory effect on glutamate release onto NPY neurons
might result from the depolarization-induced release of cannabinoids from the postsynaptic neurons, which in turn could
inhibit the release of glutamate from the presynaptic cells.
However, this mechanism is not likely, since in our previous
study the cannabinoid type 1 receptor agonist WIN55,212-2
produced no effect on NPY neuronal activity, either on membrane potential or frequency of sIPSCs or sEPSCs (van den Pol
et al. 2009). Previous studies have examined nicotine excitatory actions on GABA interneurons (de Rover et al. 2002;
Frazier et al. 1998; Maggi et al. 2001; Porter et al. 1999;
Takeda et al. 2007), and these actions increase inhibitory
synaptic transmitter release (Buhler and Dunwiddie 2002). As
both NPY and POMC cells may also contain GABA, and are
excited by nicotine and maintain local axon collaterals, nicotine would increase GABA release within the arcuate nucleus.
However, activation of GABA interneurons by nicotine is
unlikely to underlie the phenomenon here because it was found
in the presence of GABA receptor antagonists.
If nicotine reduces excitatory synaptic transmission to NPY
cells, it would tend to reduce the activity of the NPY cells,
similar to the reduced spike frequency in arcuate neurons
evoked by glutamate receptor antagonists (Acuna-Goycolea
and van den Pol 2005). Compared with other regions of the
brain, the weak blood-brain barrier in the arcuate nucleus may
allow a faster or greater accumulation of nicotine in the
extracellular space near the POMC and NPY cells than in cells
outside the arcuate nucleus, making these neurons a potential
critical target in nicotine-mediated weight regulation. This,
however, does not imply that cells in other sites within the
hypothalamus or other regions of the brain that respond to
nicotine might not also play a role in modulating energy
homeostasis (Jo et al. 2002, 2005).
Nicotine excites hypocretin neurons. We also studied perifornical/lateral hypothalamic neurons that synthesize hypocretin. Nicotine depolarized the membrane potential and increased
spike frequency in these cells. The response in hypocretin
1199
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NICOTINE EXCITES POMC AND NPY NEURONS
ACKNOWLEDGMENTS
We thank Y. Yang, V. Rogulin, and J. N. Davis for technical assistance.
GRANTS
This work was supported by National Institute of Neurological Disorders
and Stroke Grants NS-41454 and NS-48476.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
J Neurophysiol • VOL
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2006). In addition, POMC cells are inhibited by ghrelin,
whereas NPY neurons are excited (Cowley et al. 2003). In
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The question remains as to the role of nicotine receptors in
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cholinergic axons, abutting both POMC and NPY cells, ACh
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membrane current. This could suggest that synaptically released ACh does not exert a substantive effect on these cells;
however, there are alternate interpretations: Most cholinergic
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that ACh functions as a presynaptic neuromodulator more
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brain (Dani and Bertrand 2007; Lambe et al. 2005). That all
three neuron types examined, POMC, NPY, and hypocretin,
show responses to nicotine at the cell body suggests that the
axon terminals of these same cells may also respond to nicotine, and the presynaptic responses of those axons may be
critical for the endogenous action of axonally released ACh.
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