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

Neuropeptides 48 (2014) 313–318

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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 significantly 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. Ó 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: [email protected] (C.-H. Shen). 1 Contribute equally to this work.

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

similar with a marked female predominance (Chisuwa and O’Dea, 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 influenced by multiple developmental, social, and biological processes (Kim, 2012; Södersten 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 significantly 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 significantly 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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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 Service’s ‘‘Guide for the Care and Use of Laboratory Animals’’. Female mice were housed at 22 °C–24 °C with a 12-h light, 12-h dark cycle. Regular care was provided by qualified 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 amplification 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 first 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 quantified 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 refilled to their original amount every other day. 2.4. Brain dissection and qRT-PCR assay Adult female mice were sacrificed 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 sacrifice and frozen on dry ice. RNA was prepared from tissue samples as described previously (Zhang et al., 2009). The

a

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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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 significantly 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 significant decrease of food intake, but the food intake of WT mice was still significantly 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 influence 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 influenced 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 significantly lower in KO mice than in WT mice (Table 2). The levels of GAL, on the other hand, were significantly higher in KO mice. Regression analysis further showed that the difference of body weight between WT and KO mice was accompanied by significantly different expression levels of CART (Supplementary Table 1), and that the difference of food intake between WT and KO mice was associated with significantly 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 significantly in WT mice. For KO mice, only the expression of GAL and PMCH dropped significantly. 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 significantly in KO mice, while the levels of GABAA b3 increased significantly in KO mice (Table 3). Regression analysis further showed that the significantly 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 significantly in WT mice. For KO mice, only the expression of GABAA b1 dropped significantly. On the other hand, both GABAA b2 and GABAA b3 increased significantly. Again regression analysis was employed to further analyze this difference and it was determined that the significantly 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

A

*

50

p=0.00013

B

*

*

30

20

10

*

8

Food Intake (g)

Body Weight (g)

p=0.00654

p=0.00597

40

p=0.02214

*

6

4

2

0

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, Student’s t test.

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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 significantly 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 significantly 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 significantly 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 mice’s 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 findings, 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 influence 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 influenced 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 4–5 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 findings 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 finding that 5-HT1A-R signaling causes hyperpolarization-mediated inhibition of firing 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 findings 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) 313–318 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 first 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 findings 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. References American Psychiatric Association, 1994. Diagnostic and Statistical Manual of Mental Disorders, fourth ed. American Psychiatric Press, Washington, DC. Attia, E., 2010. Anorexia nervosa: current status and future directions. Annu. Rev. Med. 61, 425–435. Audenaert, K., Van, L.K., Dumont, F., Vervaet, M., Goethals, I., Slegers, G., Mertens, J., van Heeringen, C., Dierckx, R.A., 2003. Decreased 5-HT2a receptor binding in patients with anorexia nervosa. J. Nucl. Med. 44, 163–169. Avraham, Y., Katzhendler, J., Vorobeiv, L., Merchavia, S., Listman, C., Kunkes, E., Harfoush, F., Salameh, S., Ezra, A.F., Grigoriadis, N.C., Berry, E.M., Najajreh, Y., 2013. Novel acylethanolamide derivatives that modulate body weight through enhancement of hypothalamic pro-opiomelanocortin (POMC) and/or decreased neuropeptide Y (NPY). J. Med. Chem. 56, 1811–1829. Bailer, U.F., Frank, G.K., Henry, S.E., Price, J.C., Meltzer, C.C., Mathis, C.A., Wagner, A., Thornton, L., Hoge, J., Ziolko, S.K., Becker, C.R., McConaha, C.W., Kaye, W.H., 2007. Exaggerated 5-HT1A but normal 5-HT2A receptor activity in individuals ill with anorexia nervosa. Biol. Psychiatry. 61, 1090–1099.

Bailey, S.J., Toth, M., 2004. Variability in the benzodiazepine response of serotonin 5-HT1A receptor null mice displaying anxiety-like phenotype: evidence for genetic modifiers in the 5-HT-mediated regulation of GABAA receptors. J. Neurosci. 24, 6343–6351. Blundell, J.E., 1977. Is there a role for serotonin (5-hydroxytryptamine) in feeding? Int. J. Obes. 1, 15–42. Blundell, J.E., 1984. Serotonin and appetite. Neuropharmacology 23, 1537–1551. Blundell, J.E., Lawton, C.L., Halford, J.C., 1995. Serotonin, eating behavior, and fat intake. Obes. Res. 3, S471–S476. Brewerton, T.D., Jimerson, D.C., 1996. Studies of serotonin function in anorexia nervosa. Psychiatry Res. 62, 31–42. Chandra, P.S., Abbas, S., Palmer, R., 2012. Are eating disorders a significant clinical issue in urban India? A survey among psychiatrists in Bangalore. Int. J. Eat. Disord. 45, 443–446. Chisuwa, N., O’Dea, J.A., 2010. Body image and eating disorders amongst Japanese adolescents. A review of the literature. Appetite 54, 5–15. Dourish, C.T., Hutson, P.H., Curzon, G., 1985. Characteristics of feeding induced by the serotonin agonist 2-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT). Brain Res. Bull. 15, 377–384. Ebenezer, I.S., 1992. Effects of the 5-HT1A agonist 8-OH-DPAT on operant food intake in non-deprived rats. Neuro. Report 3, 62–64. Ebenezer, I.S., Tite, R., 1994. Sex difference in the feeding responses of non-deprived rats to the 5-HT1A agonists 8-OH-DPAT and gepirone. Methods Find Exp. Clin. Pharmacol. 16, 91–96. Ebenezer, I.S., Surujbally, A., 2007. The effects of 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) on food intake in non-deprived C57BL6 mice. Eur. J. Pharmacol. 559, 184–188. Fedotova, Y.O., Ordyan, N.E., 2010. Effects of 8-OH-DPAT and NAN-190 on anxiousdepressive-like behavior of female rats during the estrous cycle. Bull. Exp. Biol. Med. 150, 165–167. Fletcher, P.J., Davies, M., 1990. The involvement of 5-hydroxytryptaminergic and dopaminergic mechanisms in the eating induced by buspirone, gepirone and ipsapirone. Br. J. Pharmacol. 99, 519–525. Galusca, B., Costes, N., Zito, N.G., Peyron, R., Bossu, C., Lang, F., Le, B.D., Estour, B., 2008. Organic background of restrictive-type anorexia nervosa suggested by increased serotonin 1A receptor binding in right frontotemporal cortex of both lean and recovered patients: [18F]MPPF PET scan study. Biol. Psychiatry 64, 1009–1013. Germano, C.M., de Castro, M., Rorato, R., Laguna, M.T., Antunes-Rodrigues, J., Elias, C.F., Elias, L.L., 2007. Time course effects of adrenalectomy and food intake on cocaine- and amphetamine-regulated transcript expression in the hypothalamus. Brain Res. 1166, 55–64. Gilbert, F., Dourish, C.T., 1987. Effects of the novel anxiolytics gepirone, buspirone and ipsapirone on free feeding and on feeding induced by 8-OH-DPAT. Psychopharmacology 93, 349–352. Gleason, G., Liu, B., Bruening, S., Zupan, B., Auerbach, A., Mark, W., Oh, J.-E., Gal-Toth, J., Lee, F., Toth, M., 2010. The serotonin1A receptor gene as a genetic and prenatal maternal environmental factor in anxiety. Proc. Natl. Acad. Sci. U.S.A. 107, 7592–7597. Jimerson, D.C., Lesem, M.D., Kaye, W.H., Brewerton, T.D., 1992. Low serotonin and dopamine metabolite concentrations in cerebrospinal fluid from bulimic patients with frequent binge episodes. Arch. Gen. Psychiatry 49, 132–138. Kaye, W.H., Klump, K.L., Frank, G.K., Strober, M., 2000. Anorexia and bulimia nervosa. Annu. Rev. Med. 51, 299–313. Kim, S.F., 2012. Animal models of eating disorders. Neuroscience 211, 2–12. Lee, S., Ng, K.L., Kwok, K., Fung, C., 2010. The changing profile of eating disorders at a tertiary psychiatric clinic in Hong Kong (1987–2007). Int. J. Eat. Disord. 43, 307– 314. Marques, L., Alegria, M., Becker, A.E., Chen, C., Fang, A., Chosak, A., Diniz, J.B., 2011. Comparative prevalence, correlates of impairment, and service utilization for eating disorders across U.S. ethnic groups: implications for reducing ethnic disparities in health care access for eating disorders. Int. J. Eat. Disord. 44, 412– 420. Morrison, S.F., Nakamura, K., Madden, C.J., 2008. Central control of thermogenesis in mammals. Exp. Physiol. 93, 773–797. Muraki, Y., Yamanaka, A., Tsujino, N., Kilduff, T.S., Goto, K., Sakurai, T., 2004. Serotonergic regulation of the orexin/hypocretin neurons through the 5-HT1A receptor. J. Neurosci. 24, 7159–7166. Parks, C., Robinson, P., Sibille, E., Shenk, T., Toth, M., 1998. Increased anxiety of mice lacking the serotonin1A receptor. Proc. Natl. Acad. Sci. U.S.A. 95, 10734–10739.

318

I. Butt et al. / Neuropeptides 48 (2014) 313–318

Sarnyai, Z., Sibille, E.L., Pavlides, C., Fenster, R.J., McEwen, B.S., Toth, M., 2000. Impaired hippocampal-dependent learning and functional abnormalities in the hippocampus in mice lacking serotonin1A receptors. Proc. Natl. Acad. Sci. U.S.A. 97, 14731–14736. Savontaus, E., Conwell, I.M., Wardlaw, S.L., 2002. Effects of adrenalectomy on AGRP, POMC, NPY and CART gene expression in the basal hypothalamus of fed and fasted rats. Brain Res. 958, 130–138. Sibille, E., Pavlides, C., Benke, D., Toth, M., 2000. Genetic inactivation of the Serotonin(1A) receptor in mice results in downregulation of major GABA(A) receptor alpha subunits, reduction of GABA(A) receptor binding, and benzodiazepine-resistant anxiety. J. Neurosci. 20, 2758–2765. Simansky, K.J., 1996. Serotonergic control of the organization of feeding and satiety. Behav. Brain Res. 73, 37–42. Simpson, K.A., Martin, N.M., Bloom, S.R., 2009. Hypothalamic regulation of food intake and clinical therapeutic applications. Arq. Bras. Endocrinol. Metabol. 53, 120–128. Södersten, P., Bergh, C., Zandian, M., 2006. Understanding eating disorders. Horm. Behav. 50, 572–578. Stanley, B.G., Urstadt, K.R., Charles, J.R., Kee, T., 2011. Glutamate and GABA in lateral hypothalamic mechanisms controlling food intake. Physiol. Behav. 104, 40–46. Steffens, S.M., da Cunha, I.C., Beckman, D., Lopes, A.P., Faria, M.S., Marino-Neto, J., Paschoalini, M.A., 2008. The effects of metergoline and 8-OH-DPAT injections into arcuate nucleus and lateral hypothalamic area on feeding in female rats during the estrous cycle. Physiol. Behav. 95, 484–491. Sullivan, S.D., Howard, L.C., Clayton, A.H., Moenter, S.M., 2002. Serotonergic activation rescues reproductive function in fasted mice: does serotonin

mediate the metabolic effects of leptin on reproduction? Biol. Reprod. 66, 1702–1706. Tong, Q., Ye, C.P., Jones, J.E., Elmquist, J.K., Lowell, B.B., 2008. Synaptic release of GABA by AgRP neurons is required for normal regulation of energy balance. Nat. Neurosci. 11, 998–1000. Turenius, C.I., Htut, M.M., Prodon, D.A., Ebersole, P.L., Ngo, P.T., Lara, R.N., Wilczynski, J.L., Stanley, B.G., 2009a. GABA(A) receptors in the lateral hypothalamus as mediators of satiety and body weight regulation. Brain Res. 1262, 16–24. Turenius, C.I., Charles, J.R., Tsai, D.H., Ebersole, P.L., Htut, M.H., Ngo, P.T., Lara, R.N., Stanley, B.G., 2009b. The tuberal lateral hypothalamus is a major target for GABAA- but not GABAB-mediated control of food intake. Brain Res. 1283, 65–72. Weiselberg, E.C., Gonzalez, M., Fisher, M., 2011. Eating disorders in the twenty-first century. Minerva Ginecol. 63, 531–545. Weston-Green, K., Huang, X.F., Deng, C., 2012. Alterations to melanocortinergic, GABAergic and cannabinoid neurotransmission associated with olanzapineinduced weight gain. PLoS One 7, e33548. Wu, Q., Palmiter, R.D., 2011. GABAergic signaling by AgRP neurons prevents anorexia via a melanocortin-independent mechanism. Eur. J. Pharmacol. 660, 21–27. Wurtman, R.J., Wurtman, J.J., 1996. Brain serotonin, carbohydrate-craving, obesity and depression. Adv. Exp. Med. Biol. 398, 35–41. Zhang, A., Shen, C.-H., Ma, S.Y., Ke, Y., El Idrissi, A., 2009. Altered expression of autism-associated genes in the brain of fragile X mouse model. Biochem. Biophys. Res. Commun. 379, 920–923.