Neuropeptides 48 (2014) 313318

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Neuropeptides
journal homepage: www.elsevier.com/locate/npep

Short communication

The effects of serotonin1A receptor on female mice body weight and food
intake are associated with the differential expression of hypothalamic
neuropeptides and the GABAA receptor q
Isma Butt a,1, Andrew Hong a,1, Jing Di a,1, Sonia Aracena c, Probal Banerjee b,c, Chang-Hui Shen a,b,
a
b
c

Department of Biology, College of Staten Island, City University of New York, Staten Island, NY 10314, USA
Institute for Macromolecular Assemblies, City University of New York, Staten Island, NY 10314, USA
Department of Chemistry, College of Staten Island, City University of New York, Staten Island, NY 10314, USA

a r t i c l e

i n f o

Article history:
Received 26 January 2014
Accepted 23 July 2014
Available online 2 August 2014
Keywords:
Serotonin
Neuropeptides
GABAA b subunits
Food intake
Body weight
Gene expression
qRT-PCR

a b s t r a c t
Both common eating disorders anorexia nervosa and bulimia nervosa are characteristically diseases of
women. To characterize the role of the 5-HT1A receptor (5-HT1A-R) in these eating disorders in females,
we investigated the effect of saline or 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) treatment
on feeding behavior and body weight in adult WT female mice and in adult 5-HT1A-R knockout (KO)
female mice. Our results showed that KO female mice have lower food intake and body weight than
WT female mice. Administration of 8-OH-DPAT decreased food intake but not body weight in WT
female mice. Furthermore, qRT-PCR was employed to analyze the expression levels of neuropeptides,
c-aminobutyric acid A receptor subunit b (GABAA b subunits) and glutamic acid decarboxylase in the
hypothalamic area. The results showed the difference in food intake between WT and KO mice was
accompanied by differential expression of POMC, CART and GABAA b2, and the difference in body weight
between WT and KO mice was associated with signicantly different expression levels of CART and
GABAA b2. As such, our data provide new insight into the role of 5-HT1A-R in both feeding behavior
and the associated expression of neuropeptides and the GABAA receptor.
2014 Elsevier Ltd. All rights reserved.

1. Introduction
People suffering from eating disorders usually exhibit serious
disturbances in eating habits. These disturbances include an alteration of food intake as well as altered psychological perception
towards food, body weight, and self-image. Two common eating
disorders are anorexia nervosa (AN) and bulimia nervosa (BN).
AN is characterized by chronic food refusal, excessive weight loss,
fear of weight gain and a distorted self-image (Attia, 2010;
Weiselberg et al., 2011). On the other hand, BN is characterized
by frequent episodes of binge eating. Self-vomiting, abuse of laxatives and excessive exercise are common compensatory purging
mechanisms used in patients with BN (American Psychiatric
Association, 1994). The gender difference in both AN and BN is
q
Source of support: This work was supported by an NSF Grant (MCB-0919218) to
C.-H. S.
Corresponding author at: Department of Biology, College of Staten Island, City
University of New York, 2800 Victory Blvd, Staten Island, NY 10314, USA. Tel.: +1
(718) 982 3998; fax: +1 (718) 982 3852.
E-mail address: ChangHui.Shen@csi.cuny.edu (C.-H. Shen).
1
Contribute equally to this work.

http://dx.doi.org/10.1016/j.npep.2014.07.003
0143-4179/ 2014 Elsevier Ltd. All rights reserved.

similar with a marked female predominance (Chisuwa and ODea,

2010; Lee et al., 2010; Marques et al., 2011; Chandra et al.,
2012). Although the etiology of eating disorders has yet to be
examined, it is evident that the causes are multifactorial, and are
inuenced by multiple developmental, social, and biological processes (Kim, 2012; Sdersten et al., 2006; Weiselberg et al., 2011).
It is generally believed that 5-Hydroxytryptamine (5-HT; serotonin) plays an important role in the control of feeding behavior
(Blundell, 1977, 1984; Simansky, 1996). It has been shown that
the administration of one of the 5-HT subtype 5-HT1A receptor agonists, including 8-hydroxy-2-(di-n-propylamino) tetralin (8-OHDPAT), gepirone, buspirone and ipsapirone, increased food intake
signicantly in non-deprived or satiated male rats and male mice.
(Dourish et al., 1985; Gilbert and Dourish, 1987; Fletcher and
Davies, 1990; Ebenezer, 1992; Ebenezer and Tite, 1994; Ebenezer
and Surujbally, 2007). Furthermore, imaging studies have shown
that 5-HT1A receptor expression increased signicantly in recovered AN patients brains compared to the ill AN patients brains,
while 5-HT2A receptor expression is unchanged (Audenaert et al.,
2003; Bailer et al., 2007; Galusca et al., 2008). As such, the perturbation of 5-HT1A receptor activity is important in eating disorders.

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I. Butt et al. / Neuropeptides 48 (2014) 313318

On the other hand, many studies have shown that expression

levels of both hypothalamic neuropeptides, including both orexigenic and anorexigenic peptides, and GABAA receptor might regulate the food intake and body weight (Simpson et al., 2009; Stanley
et al., 2011; Wu and Palmiter, 2011). Furthermore, it has also been
demonstrated that 5-HT1A receptor might have effects on the
expression of GABAA receptor (Sibille et al., 2000). However, the
interplay among the GABAergic system, neuropeptides and the 5HT1A receptor in the regulation of body weight and food intake
has not been examined. The present study employed an eating
behavioral study to examine the effects of 5-HT1AR in body weight
and food intake for both Wild type (WT) and 5-HT1A-R (/)
knockout (KO) mice. Furthermore, qRT-PCR was used to evaluate
the expression levels of hypothalamic neuropeptides, GABAA b subunits and glutamic acid decarboxylase (GAD). We also evaluated
the effect of a 5-HT1A-R agonist, 8-OH-DPAT, on body weight, food
intake, and the expression levels of hypothalamic neuropeptides
and neurotransmitters.
2. Materials and methods
2.1. Animals and animal care
Both C57BL Wild type (WT) and C57BL 5-HT1A-R (/) (KO)
female mice were used in this study (Bailey and Toth, 2004;
Gleason et al., 2010; Parks et al., 1998; Sarnyai et al., 2000). Mice
were acquired, cared for and handled in conformity with the U.S.
Public Health Services Guide for the Care and Use of Laboratory
Animals. Female mice were housed at 22 C24 C with a 12-h
light, 12-h dark cycle. Regular care was provided by qualied and
experienced staff of the Animal Care Facility, Center for Developmental Neuroscience, College of Staten Island.

primers used were listed in Table 1. All real-time PCR reactions

were done in triplicate. Target DNA sequence quantities were estimated from the threshold amplication cycle number (CT) using
Sequence Detection System software (Applied Biosystems). A DCT
value was calculated for each sample by subtracting their CT value
from the CT value for the corresponding GAPDH to normalize the
differences in cDNA aliquots. Each cDNA quantity was then calculated with the following formula: 2DC T .
2.5. Statistical analysis
Regression analyses were used to test for association between
gene expression and food intake or body weight. Where necessary,
the food intake and/or gene expression variables were transformed
to standardize the variance.
3. Result
3.1. Effect of 5-HT1AR on body weight and food intake
It has been suggested that alterations of 5-HT1AR activity may
affect feeding behavior (Ebenezer, 1992). To further understand
the relationship between 5-HT1AR and feeding behavior, we rst
examined the effect of 5-HT1AR on body weight. Three female mice
for each treatment group of either WT or KO were used for this

Table 1
Oligonucleotides used in qRT-PCR reaction.

2.2. Drug administration

The 5-HT1A-R agonist 8-OH-DPAT (1 mg of 8-OH-DPAT per kg
mouse body weight) was administered subcutaneously to both
WT and KO 90-day old female mice every day at 12:00 pm for
3 weeks (Dourish et al., 1985; Ebenezer, 1992; Ebenezer and Tite,
1994; Ebenezer and Surujbally, 2007). Before the beginning of
the drug administration, adult female mice were observed for
3 weeks to make sure that the mice with similar feeding behavior
were in the same group. A parallel experiment was performed by
administering saline.
2.3. Food intake measurements and body weight measurements
Standard chow and water was available ad libitum. Adult female
mice were housed individually. The food intake was monitored at
5 pm every day throughout the study (Ebenezer and Surujbally,
2007). To monitor the amount of the food intake, each mouse
was given 100 g of chow. The amount of food was quantied by
weighing the remaining chow the next day. The difference was calculated and recorded as food intake for each day. Body weight was
monitored weekly. Food was relled to their original amount every
other day.
2.4. Brain dissection and qRT-PCR assay
Adult female mice were sacriced in accordance with the principles and procedures of the National Institutes of Health Guideline
for the Care and Use of Laboratory Animals to minimize pain or discomfort. Hypothalamic tissues were dissected from brains within
3 min of the sacrice and frozen on dry ice. RNA was prepared from
tissue samples as described previously (Zhang et al., 2009). The

Oligonucleotides

Sequence

GAPDH ORFa
Forward primer
Reverse primer

50 -ACAGGGTGGTGGACCTCATG-30
50 -GTTGGGATAGGGCCTCTCTTG-30

GABAA b1 ORF
Forward primer
Reverse primer

50 -CTGCATCCTGATGGAACTGTTC-30
50 -CTCATCCAGAGGGTATCTTCGAA-30

GABAA b2 ORF
Forward primer
Reverse primer

50 -GTGGGCACGAGGGTTAGAAC-30
50 -GATCCACCACAGCAGCCATT-30

GABAA b3 ORF
Forward primer
Reverse primer

50 -CCACGGAGTGACAGTGAAAA-30
50 -CACGCTGCTGTCGTAGTGAT-30

GAD65 ORF
Forward primer
Reverse primer

50 -GGCTCTGGCTTTTGGTCCTTC-30
50 -TGCCAATTCCCAATTATACTCTTGA-3

NPY ORF
Forward primer
Reverse primer

50 -AGAGATCCAGCCCTGAGACA-30
50 -TTTCATTTCCCATCACCACA-30

AgRP ORF
Forward primer
Reverse primer

50 -GGCACAAGAGACCAGGACAT-30
50 -ACTTCTTCTGCTCGGTCTGC-30

POMC ORF
Forward primer
Reverse primer

50 -GAACAGCCCCTGACTGAAAA-30
50 -AACGTTGGGGTACACCTTCA-30

CART ORF
Forward primer
Reverse primer

50 -GAGGTCCAGAACCATGGAGA-30
50 -ACTTCTTGCAACGCTTCGAT-30

GAL ORF
Forward primer
Reverse primer

50 -AGCCTTGATCCTGCACTGAC-30
50 -AGGGTCACAACCAACAGGAG-30

GALP ORF
Forward primer
Reverse primer

50 -TGGTCCTCTTCCTCACCATC-30
50 -AGGACCCAGGAGGTAACCAG-30

PMCH ORF
Forward primer
Reverse primer

50 -TGCTGAGTCCACACAGGAAA-30
50 -GCCAACATGGTCGGTAGACT-30

ORF: open reading frame.

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I. Butt et al. / Neuropeptides 48 (2014) 313318

study. Our results showed that the body weight of WT mice was
maintained at 38.31 0.31 g, but the body weight of KO mice
was only 24.43 0.71 g (Fig. 1A). Furthermore, the daily food
intake of KO mice was signicantly lower than WT mice
(Fig. 1B). As such, these results suggested that WT and KO mice
showed differences in body weight and daily food intake, and these
differences were subject to the presence of 5-HT1A-R.
We then examined how the 5-HT1A-R agonist 8-OH-DPAT
affects the body weight and food intake. Our results showed that
the administration of 8-OH-DPAT in WT mice caused the signicant decrease of food intake, but the food intake of WT mice was
still signicantly higher than the food intake of KO mice (Fig. 1B).
Intriguingly, we did not observe a change in body weight during
the 3 weeks period. Furthermore, the administration of 8-OH-DPAT
on KO mice did not have any inuence on body weight and daily
food intake. A similar pattern for both saline and 8-OH-DPAT treatment in KO mice was observed.
3.2. Effect of 5-HT1A-R on the expression of hypothalamic
neuropeptides
Since both body weight and food intake can be inuenced by 5HT1A-R, we examined the associated neuropeptides expression levels. We used quantitative real-time PCR to examine mRNA levels of
neuropeptides in the hypothalamus of WT and KO mice. These
neuropeptides included neuropeptide Y (NPY), agouti-gene related
protein (AgRP), pro-opiomelanocortin (POMC), cocaine- and
amphetamine-related transcript (CART), galanin (GAL), galaninlike peptide (GALP) and pro-melanin-concentrating hormone
(PMCH).
In the absence of the 8-OH-DPAT, the expression levels of
POMC, AgRP and CART were signicantly lower in KO mice than
in WT mice (Table 2). The levels of GAL, on the other hand, were
signicantly higher in KO mice. Regression analysis further showed
that the difference of body weight between WT and KO mice was
accompanied by signicantly different expression levels of CART
(Supplementary Table 1), and that the difference of food intake
between WT and KO mice was associated with signicantly different levels of POMC and CART (Supplementary Tables 2 and 3).
In the presence of the 8-OH-DPAT, expression levels of all neuropeptides decreased signicantly in WT mice. For KO mice, only
the expression of GAL and PMCH dropped signicantly. Again

regression analysis was employed for further analysis. We found

that none of the neuropeptides expression levels are associated
with the difference of body weight and food intake between WT
and KO mice in the presence of agonist.
3.3. Effect of 5-HT1A-R on the expression of hypothalamic
neurotransmitters
It has been shown that mice whose AgRP neurons are unable to
release c-aminobutyric acid (GABA) show decreased inhibitory
input on POMC neurons, suggesting a possible physiological role
of GABA in feeding behavior (Tong et al., 2008). As such, it is
instructed to examine the expression levels of both GABAA b subunits and glutamic acid decarboxylase (GAD) to understand the
importance of the GABAergic system in body weight and food
intake.
In the absence of the 8-OH-DPAT, expression levels of GABAA b2
and GAD65 dropped signicantly in KO mice, while the levels of
GABAA b3 increased signicantly in KO mice (Table 3). Regression
analysis further showed that the signicantly different expression
levels of GABAA b2 was associated with the difference of both body
weight and food intake between WT and KO mice (Supplementary
Tables 4 and 5). In the presence of the 8-OH-DPAT, the expression
levels of all GABAAb subunits decreased signicantly in WT mice.
For KO mice, only the expression of GABAA b1 dropped signicantly. On the other hand, both GABAA b2 and GABAA b3 increased
signicantly. Again regression analysis was employed to further
analyze this difference and it was determined that the signicantly
different expression levels of GABAA b3 corresponded with the difference of body weight between WT and KO mice in the presence
of the agonist (Supplementary Table 6).
4. Discussion
Patients with eating disorders, such as AN and BN, usually have
disturbed attitude towards body weight and shape. Furthermore,
neurotransmitter, neuropeptide, and neuroendocrine systems are
also usually aberrant in these patients (Kaye et al., 2000). Several
lines of evidence indicated that the serotonergic system plays
important roles in body weight regulation and eating disorders
(Jimerson et al., 1992; Blundell et al., 1995; Wurtman and
Wurtman, 1996; Brewerton and Jimerson, 1996). In this study,

p=0.02092

50

p=0.00013

30

20

10

Food Intake (g)

Body Weight (g)

p=0.00654

p=0.00597

40

p=0.02214

0
Saline

8-OH-DPAT

WT

Saline 8-OH-DPAT

KO

Saline

8-OH-DPAT Saline

WT

8-OH-DPAT

KO

Fig. 1. The role of 5-HT1A-R in food intake and body weight. (A) The body weight of WT mice and KO mice after 3 weeks of drug delivery. (B) The daily average food intake
under freely feeding scheme for both WT and KO mice during the 3 weeks period of drug delivery. p < 0.05, Students t test.

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I. Butt et al. / Neuropeptides 48 (2014) 313318

Table 2
The mRNA levels of hypothalamic neuropeptides.
WT

POMC
CART
AgRP
NPY
GAL
GALP
PMCH

KO

PBS

8OH-DPAT

PBS

8OH-DPAT

0.1675 0.0062
0.2355 0.0240
0.0115 0.0001
1.9884 1.4556
7.1020 2.2543
0.0055 0.0036
191.8407 75.8636

0.0014 0.0006
0.0353 0.0024
0.0002 0.0001
0.0769 0.0128
0.1872 0.1173
0.00004 0.00002
0.0332 0.0126

0.0035 0.0002
0.0592 0.0164
0.0004 0.0001
3.9509 1.0919
16.7854 4.6389
0.0078 0.0071
124.2015 34.3247

0.0031 0.0001
0.0562 0.0034
0.0024 0.0009
3.2804 1.2674
0.1022 0.0395
0.0121 0.0109
0.0012 0.0004

Summary of the real-time PCR analysis of neuropeptides mRNA relative to GAPDH mRNA. All real-time PCR reactions were done in triplicate.

we examined the effects of a 5-HT1A-R/5-HT7-R agonist, 8-OHDPAT, in food intake, body weight and neuropeptide expression
of female WT and KO mice. We demonstrated that the body weight
and food intake of KO mice were signicantly lower than the WT
mice (Fig. 1). Further analysis showed that such weight loss and
reduced food intake were associated with altered expression levels
of hypothalamic POMC, CART and GABAA b2 (Tables 2 and 3).
Finally, the body weight of KO mice was still signicantly lower
than that of the WT mice in the presence of 8-OH-DPAT, but only
the expression levels of hypothalamic GABAA b3 was associated
with this observation (Tables 2 and 3). As such, we have demonstrated how expression levels of neuropeptides and neurotransmitters are associated with food intake and body weight through
the presence of 5-HT1A-R.
Our results showed that the daily food intake and the body
weight of KO mice were signicantly lower than the daily food
intake and the body weight of WT mice (Fig. 1). This strongly suggested that 5-HT1AR might play an important role in food intake
and body weight. Previously, it has been demonstrated that KO
mice have an increased tendency to avoid a novel and fearful environment and to escape a stressful situation. As such, KO mice
showed behaviors consistent with an increased anxiety and stress
response (Parks et al., 1998). Furthermore, KO mice were impaired
in hippocampal-dependent spatial learning and memory tasks
such as the hidden platform version of Morris water maze and
the delayed version of the Y maze (Sarnyai et al., 2000). Clearly,
5-HT1A receptors are required for maintaining normal hippocampal
functions and this implicates a role for the 5-HT1A receptor in hippocampal-related symptoms, such as cognitive disturbances, in
stress-related disorders. Therefore, these abnormal behaviors
might affect KO mices food intake and subsequent change in body
weight.
We observed that administration of 8-OH-DPAT caused a significant decrease of food intake for WT female mice, which suggested
a satiety effect from 8-OH-DPAT in WT female mice (Fig. 1B). It has
been shown that the administration of 5-HT1A-R agonists increased
food intake in non-deprived or satiated rats (Dourish et al., 1985;
Gilbert and Dourish, 1987; Fletcher and Davis, 1990; Ebenezer,
1992; Ebenezer and Tite, 1994). Furthermore, a recent study
showed that the administration of 8-OH-DPAT to non-deprived
C57BL6 male mice increases food intake (Ebenezer and
Surujbally, 2007). Here, C57BL6 female mice were used in the test,
and our observations are not completely consistent with previous
ndings, suggesting that the discrepancy might arise from the
sex difference. Indeed, similar observations were also reported
for the female adult Wistar rats (Steffens et al., 2008). As such,
the gender difference indeed plays a role in 5-HT1A-R action.
Since female mice were used in this study, one might ask the
inuence of estrous cycle in the pharmacokinetic action of 8-OHDPAT. In general, the estrous cycle for a mouse is about 4.5 days
(Sullivan et al., 2002). Recent studies have demonstrated that the
pharmacokinetic action of 8-OH-DPAT is not inuenced by the

phase of the estrous cycle (Steffens et al., 2008; Fedotova and

Ordyan, 2010). Since we administered the agonist every day for
3 weeks, all female mice went through 45 estrous cycles throughout the study. Furthermore, we examined the long-term cumulative effects of agonist on the food intake and body weight. As
such, the effect of estrous phase on 8-OH-DPAT pharmacokinetic
action would not have any impact on our observation.
In the analysis of neuropeptide expression, we observed that
low expression levels of POMC and CART were associated with
low food intake in KO mice. Furthermore, low levels of CART were
associated with low body weight in KO mice. However, both POMC
and CART are anorexigenic neuropeptides. Their expression levels
are believed to be inversely related to food intake (Weston-Green
et al., 2012; Avraham et al., 2013). Our ndings are not consistent
with these results. Intriguingly, our observations are very similar to
the results from the study of the effects of adrenalectomy. Those
studies showed that adrenalectomy decreases food intake and
body weight and is associated with the decreased expression of
hypothalamic AgRP, NPY, POMC and CART (Savontaus et al.,
2002; Germano et al., 2007). It is possible that the 5-HT1A-R knockout mice behave similar to mice treated with adrenalectomy.
Although the mechanism is not clear, it is possible that the
decreases in POMC and CART, in the setting of low food intake
and low body weight, may serve to counteract the decline in AgRP
and NPY, and protect from weight loss.
In the absence of the 5-HT1A-R, 8-OH-DPAT is expected to function primarily through the 5-HT7-R, which is positively coupled to
adenylyl cyclase. Here, we observed that the mRNA level of the
orexigenic peptide NPY undergoes a dramatic decrease upon 8OH-DPAT treatment of the WT but not KO mice (Table 2). We also
observed a trend of increase in NPY mRNA expression in the KO
mice compared to the WT mice. Taken together, it suggests that
5-HT1A-R signaling causes suppression of the orexigenic protein
NPY, thereby causing reduced food intake in the presence of
8-OH-DPAT (Fig. 1). This is consistent with the nding that
5-HT1A-R signaling causes hyperpolarization-mediated inhibition
of ring by orexin neurons (Muraki et al., 2004). This would
explain the suppression of food intake but no weight loss for the
WT mice following 8-OH-DPAT treatment (Fig. 1). On the other
hand, the induction of 5-HT7 receptor by 8-OH-DPAT in the
absence of 5-HT1A-R could elicit an increase in brown adipose
tissue thermogenesis (Morrison et al., 2008), and it should result
in the weight loss. We did observe that the body weight of KO mice
was lower than WT mice under 8-OH-DPAT treatment. Mood disorders are known to be linked to the 5-HT1A and 5-HT7 receptors,
and eating disorders and aberrance in body weight often co-occur
with mood disorders. Therefore, our ndings emphasize the necessity of further studies to understand the role of these two receptors
in food intake and body weight.
We observed that the expression levels of GABAA b2 and GABAA
b3 were associated with low food intake and low body weight of
KO mice. It has been demonstrated that the administration of

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I. Butt et al. / Neuropeptides 48 (2014) 313318

Table 3
The mRNA levels of hypothalamic neurotransmitter peptides.
WT

GABAA b1
GABAA b2
GABAA b3
GAD65

KO

PBS

8OH-DPAT

PBS

8OH-DPAT

345.13 75.42
2022.01 339.78
750.31 700.16
3791.44 540.60

155.35 46.65
482.51 92.85
24.12 21.03
283.55 144.24

360.96 265.32
64.95 17.95
1884.33 520.76
321.87 88.95

4.67 1.81
647.56 77.06
6198.68 1718.16
413.70 159.84

Summary of the real-time PCR analysis of neurotransmitters mRNA relative to GAPDH mRNA. All real-time PCR reactions were done in triplicate.

GABAA receptor agonists can reduce food intake and cause substantial weight loss (Turenius et al., 2009a,b). Here, we further
examined the transcripts levels of each subtype. Our results indicated that low food intake and low body weight are associated
with low GABAA b2 and high GABAA b3 (Fig. 1; Table 3). This is
the rst report to examine how different GABAA b subtypes affect
food intake and body weight. As such, we have provided a new
possible view to understand the relationship between GABAergic
system and food intake.
5. Conclusions
In this report, we examined the effects of a 5-HT1A-R/5-HT7-R
agonist, 8-OH-DPAT, in eating behavior at the molecular level.
Our ndings suggest that 5-HT1A-R plays an important role in
female mice feeding behavior and body weight. Our observations
have also demonstrated the effect of 5-HT1A-R on the expression
levels of neuropeptide and the peptide neurotransmitters. It is possible that 5-HT1A-R regulates eating behavior through its effects on
the levels of those peptides. A better understanding of the molecular mechanism underlying the unique features of 5-HT1A-R in
the expression of neuropeptide and GABAA receptor can facilitate
a better understanding of the neural regulation of feeding
behavior.
Acknowledgments
We are grateful to Dr. Lisa Manne for statistical analysis and to
Michelle Esposito for helpful discussion and comments on the
manuscript and to our laboratory colleagues for technical assistance. This work was supported by an National Science Foundation
Grant (MCB-0919218) to C.-H. S.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the
online
version,
at
http://dx.doi.org/10.1016/
j.npep.2014.07.003.
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