Food hoarding, but not food intake, is attenuated by acute diazepam treatment in female Mongolian gerbils (Meriones unguiculatus)

YHBEH-03694; No. of pages: 10; 4C: Hormones and Behavior xxx (2014) xxx–xxx

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Food hoarding, but not food intake, is attenuated by acute diazepam treatment in female Mongolian gerbils (Meriones unguiculatus) Hui-Di Yang a,b,1, Qian Wang a,c,1, De-Hua Wang a,⁎ a b c

State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Science, Beijing 100101, China Department of Physiology, Basic Medical College, Inner Mongolia Medical University, Huhehaote 010110, China Graduate School of the Chinese Academy of Sciences, Yuquan Lu, Beijing 100049, China

a r t i c l e

i n f o

Article history: Received 8 November 2013 Revised 26 February 2014 Accepted 18 March 2014 Available online xxxx Keywords: GABA Diazepam Food hoarding Hippocampus Reward circuitry

a b s t r a c t This article is part of a Special Issue “Energy Balance”. Effects of γ-aminobutyric acid (GABA) on food hoarding are unknown in rodents, and the effects of energy balance and GABA have not been evaluated in females. To evaluate the role of food deprivation and GABA on food hoarding, female Mongolian gerbils were given i.p. injection of diazepam (1 mg/kg and 3 mg/kg, respectively), a GABA A receptor agonist. Among fooddeprived females, there was a bimodal pattern in the frequency of gerbils with different levels of food hoarding. High food hoarding (HFH) and low food hoarding (LFH) gerbils were analyzed. Diazepam blocked food deprivation-induced food hoarding in HFH gerbils, but not in LFH gerbils. This blockade was associated with increased cellular activation in selected brain areas, such as the nucleus accumbens (NAcc), caudate putamen (CP) and ventral tegmental area (VTA), which suggested that direct activation of GABA in the brain reward circuitry decreased food hoarding in HFH females. Moreover, diazepam increased Fos expression in field CA2 and CA3 of the hippocampus, but had no significant effect on Fos expression in field CA1 and dentate gyrus (DG) of the hippocampus, indicating that the hippocampus has area-specific effects on food hoarding in HFH gerbils. Diazepam did not alter food intake in both HFH and LFH gerbils. In addition, serum corticosterone concentrations were higher in the HFH than in the LFH ones. Together, these data indicated that food deprivation increased food hoarding in female gerbils, diazepam reduced food deprivationinduced food hoarding in HFH gerbils, and that GABA might influence food hoarding via classical reward circuitry via the mesolimbic dopamine system and specific hippocampal areas. © 2014 Elsevier Inc. All rights reserved.

Introduction Small mammals living in the temperate zones will face seasonal fluctuations in food availability (reviewed by Bronson, 1989). Thus, to improve chances of survival, some animals have evolved to utilize food hoarding behavior to control the availability of food in space and time (Vander Wall, 1990). Food hoarding is influenced by energy balance, and the neuroendocrine factors, and is increased or decreased by neurotransmitters and hormones such as neuropeptide Y, agoutirelated protein, leptin and ghrelin (Buckley and Schneider, 2003; reviewed by Bartness et al., 2011; Yang et al., 2011; Teubner et al., 2013). In rodents, the coordination of all these systems and factors toward food hoarding and food intake appears to be controlled by a distributed neural network that includes the hypothalamus, hindbrain, ⁎ Corresponding author at: Institute of Zoology, Chinese Academy of Sciences, 1 Beichen West Road, Chaoyang, Beijing 100101, PR China. Fax: +86 10 64807099. E-mail address: [email protected] (D.-H. Wang). 1 Hui-di Yang and Qian Wang contributed equally to this work.

and areas of the forebrain including the mesolimbic dopamine system (reviewed by Keen-Rhinehart et al., 2010; Aja et al., 2001; Grill, 2006; Volkow et al., 2011). Food deprivation is a potent stimulator of food hoarding and food intake and is accompanied by increased γ-aminobutyric acid (GABA), a major inhibitory neurotransmitter, in the hypothalamus (Kamatchi and Rathanaswami, 2012) and nucleus accumbens (Meena et al., 2009). This suggests that GABA may be a link between some brain regions, such as the nucleus accumbens (Söderpalm and Berridge, 2000) and the hypothalamus, and the regulation of ingestive behaviors. The functional relationship between GABA and food hoarding was substantiated as the injection of GABA into the subpallidal region decreased food hoarding in the male rats (Mogenson and Wu, 1988). In addition, evidence has implicated that GABA controlled food intake by acting at two pharmacologically distinct receptor subtypes, namely GABAA and GABAB receptors (Freese et al., 2012). Some studies indicate that microinjections of the GABAA receptor agonist, muscimal, into the ventromedial or paraventricular nucleus of the hypothalamus increase feeding (Freese et al., 2012). Further evidence to support this view comes

http://dx.doi.org/10.1016/j.yhbeh.2014.03.010 0018-506X/© 2014 Elsevier Inc. All rights reserved.

Please cite this article as: Yang, H.-D., et al., Food hoarding, but not food intake, is attenuated by acute diazepam treatment in female Mongolian gerbils (Meriones unguiculatus), Horm. Behav. (2014), http://dx.doi.org/10.1016/j.yhbeh.2014.03.010

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from studies showing that diazepam, which acts on regulatory sites on the GABAA receptor enhances food intake in rats (Meena et al., 2009). In addition, food deprivation is also a physical stressor (Bowers et al., 2008). It is widely believed that long-term chronic stress enhances the secretion of glucocorticoids, such as corticosterone (Tsigos and Chrousos, 2002). It is possible that corticosterone suppresses the GABAergic transmission in the paraventricular nucleus of the hypothalamus in rats (Verkuyl et al., 2004). Mongolian gerbils and Syrian hamsters are similar to each other and are both different from rats in that food-deprived gerbils and hamsters fail to overeat but instead overhoard food (Buckley and Schneider, 2003; Cabanac and Swiergiel, 1989; Schneider et al., 2007). They hoard food in nature, and are an excellent model to study the mechanisms that control food hoarding and feeding. This investigation provides details about the neuronal populations and brain regions affected by GABA in the control of food hoarding behavior in Mongolian gerbils. We hypothesize that food deprivation-induced increases in food hoarding are associated with increases in glucocorticoids and decreased GABAergic tone. Thus, we predict that gerbils that show food deprivation-induced increases in food hoarding will have higher circulating levels of glucocorticoids, and that increases in food hoarding will be reversed by treatment with the GABA agonist, diazepam.

Materials and methods Animals and experimental design Eighty female Mongolian gerbils, aged 8–9 months were from our laboratory breeding colony. After weaning, animals were housed in same-sex groups, consisting of 2–3 individuals, in plastic cages (300 × 150 × 200 mm) which contained wood shaving bedding, and were under a 16L:8D photoperiod (lights on at 0400 h). The temperature was maintained at 23 ± 1 °C. All animals had ad libitum access to water and commercial standard rat pellets (Beijing KeAo Feed Co.). At about 8–9 months of age, animals were housed individually for 4 weeks, followed by 2 weeks of acclimation in specially designed food hoarding apparatus as previously described (Yang et al., 2011). Briefly, two cages were connected with the convoluted polyvinylchloride tubing system (50 mm ID and 900 mm long). The big or home cage was 320 × 210 × 160 mm (length × width × height) and was equipped with a water bottle, bedding and cotton nesting material. The small or food cage was 300 × 200 × 150 mm. At the beginning of the acclimation period, food pellets were provided on the lid of the food cage. Animals were free to move around within the apparatus to have access to food and water. All experimental procedures complied with the guidelines for animal

Fig. 1. Diagrams showing the location of the brain areas selected (shaded areas) for quantification of Fos-ir. Diagrams were modified from the atlas of Paxinos and Watson (1986). CP: caudate putamen; NAcc: nucleus accumbens; PVN: paraventricular nuclei of the hypothalamus; LH: lateral nuclei of the hypothalamus; VMH: ventromedial hypothalamus. ARC: arcuate nucleus; hippocampus (Hipp) including field of CA1, CA2 and CA3 of hippocampus as well as dentate gyrus (DG); VTA: ventral tegmental area; MM: medial mammillary nucleus, medial part.

Please cite this article as: Yang, H.-D., et al., Food hoarding, but not food intake, is attenuated by acute diazepam treatment in female Mongolian gerbils (Meriones unguiculatus), Horm. Behav. (2014), http://dx.doi.org/10.1016/j.yhbeh.2014.03.010

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care and use as stipulated by the Institute of Zoology of the Chinese Academy of Sciences.

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Body weight and tissue preparation Animals were weighed at 0900 h each morning before the food hoarding experiment. After completion of the food hoarding test, animals were immediately sacrificed by an overdose of CO2. Trunk blood was collected by decollation for corticosterone in experiment 1. In experiment 2, animals were administered a lethal dose of sodium pentobarbital (0.2 ml, i.p.) following food hoarding test. They were transcardially perfused with 200 ml of 0.01 M phosphate buffered saline (PBS) followed by 200 ml of 4% paraformaldehyde in PBS. Brains were rapidly removed and post-fixed in 4% paraformaldehyde in PBS overnight followed by 20% sucrose in PBS for 72 h. Coronal sections (40 μm) were taken using a cryostat and stored in cryoprotectant at 4 °C until processed for Fos.

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Experiment 2: does the GABA agonist, diazepam, regulate food deprivationinduced food hoarding in female Mongolian gerbils?

The food hoarding experiment started on the day immediately following acclimation. At 0900 h, food pellets were put directly into the food cage. During the next 2 h, animals were allowed to eat and carry food pellets from the food cage to the home cage. At 1100 h, any remaining food pellets were taken out from both the food and the home cages and were weighed. Food hoarding was defined as the amount of pellets found in the home cage. Food intake was determined by subtracting the food hoarded and the food remaining in the food cage from the total amount of food placed in the food cage.

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Thirty HFH and thirty LFH gerbils were food deprived for 22 h, tested for food hoarding and identified as described in Experiment 1. At 0900 h on the fourth day of food deprivation, HFH and LFH gerbils were, respectively, randomly assigned to one of three groups that received intraperitoneal (i.p.) injections of 200 μl of saline vehicle (n = 10), or the GABA agonist, diazepam, at either 1 mg/kg (n = 10) or 3 mg/kg (n = 10). Food hoarding was quantified over following 2 h. Animals were sacrificed after the food hoarding test; brain tissues were processed for Fos-ir. Because post hoc Bonferroni tests indicated that gerbils given two doses of diazepam (1 mg/kg or 3 mg/kg) did not show significant differences in food hoarding, these two groups were combined into one group for Fos-ir straining in gerbils.

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Experiment 1: does food deprivation induce food hoarding in female Mongolian gerbils? Twenty female gerbils were food deprived. Food deprivation began at 1100 h on day 7 and lasted until 0900 h of the following day (22 hour fast), followed by 2 h during which food was freely available and food hoarding measurements were obtained. This procedure was repeated for 3 consecutive days, and the individual's body weight, food intake, and food hoarded mass were recorded daily. Females had no opportunities to eat, except during their daily 2 h of food hoarding period. Immediately after the food-hoarding test, they were sacrificed. Those that hoarded more than 40 g of food pellets per day during the food deprivation experiment were referred as “high food deprivationinduced hoarding” (HFH, n = 20), while those that hoarded less than 3 g of food pellets per day were designated as “low food deprivationinduced hoarding” (LFH, n = 20).

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Day Fig. 2. Differences in the amount of food hoarded, food intake, and body weight in female Mongolian gerbils that were undergoing 4 days of food deprivation. (A) No differences were found in basal levels of food hoarded. However, female gerbils showed discrete patterns of food hoarding in response to food deprivation (indicted by the arrow). Some females displayed a significant increase in food hoarding (HFH), others continued to display basal levels of food hoarding (LFH), and such differences persisted throughout the entire course of food deprivation. **: p b 0.01. (B) A significant decrease in food intake was found in both HFH and LFH females. **: p b 0.01 compared to the baseline. (c) HFH and LFH females did not differ in basal levels of body weight. However, food deprivation significantly decreased body weight in both HFH and LFH females during food deprivation. **: p b 0.01 compared to the baseline. Data are presented as mean ± SEM.

Please cite this article as: Yang, H.-D., et al., Food hoarding, but not food intake, is attenuated by acute diazepam treatment in female Mongolian gerbils (Meriones unguiculatus), Horm. Behav. (2014), http://dx.doi.org/10.1016/j.yhbeh.2014.03.010

H.-D. Yang et al. / Hormones and Behavior xxx (2014) xxx–xxx

Corticosterone concentration (mg/ml)

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assay was 1.0 n mol/l when using a 125 μl sample. The detailed procedure followed the manufacturer's instructions of the rat corticosterone ELISA kit.

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Fig. 3. The serum corticosterone in female Mongolian gerbils with high level of food hording (HFH) or with low level of food hoarding (LFH) during food deprivation. Data are presented as mean ± SEM. **: p b 0.01.

Serum corticosterone concentrations Serum corticosterone concentrations for stress were determined by rat corticosterone ELISA kit (Cat. No. HR083, RapidBio Lab. Calabasas, CA, USA). The lowest level of corticosterone that could be detected by this

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Coronal brain sections of 40 μm thickness were cut on a cryostat. Sections were stored in 0.01 M PBS at 4 °C. Floating brain sections at 160 μm intervals were processed for Fos immunohistochemistry using previously established methods (Yang et al., 2011). Briefly, brain sections were washed three times in 0.01 M PBS (pH 7.4) for 5 min. Sections were incubated in 1% sodium borohydride for 20 min, 3% H2 O2 –methanol for 10 min, 1% Triton X-100 for 10 min, and 10% normal goat serum for 1 h in 0.01 M PBS. For Fos immunoreactive (Fos-ir) staining, sections were incubated with rabbit anti-Fos antibody (Fos [4]-G: sc-52; 1:8000; Santa Cruz Biotechnology, Santa Cruz, CA) in 0.01 M PBS with 0.1% Triton X-100 and 2% normal goat serum for 48 h at 4 °C. Thereafter, sections were incubated in biotinylated goat anti-rabbit IgG (BA-1000; 1:300; Vector, Burlingame, CA) for 2 h at room temperature and avidin–biotin complex (Vectastain Elite, Vector, Burlingame, CA) in 0.01 M PBS for 90 min. Staining was detected using 3′-diaminobenzidine (DAB/H2O2 tablet; Sigma, St. Louis, MO) with NiCl powder and rinsed in 0.01 M PBS for

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Fig. 4. GABA agonist diazepam influenced food hoarding and food intake in female Mongolian gerbils with high level of food hording (HFH) or with low level of food hoarding (LFH) during food deprivation. Diazepam significantly blocked food hoarding (A) but not food intake (B) in HFH females. But diazepam failed to alter food hoarding (C) and food intake (D) in LFH females. Data are presented as mean ± SEM. **: p b 0.01.

Please cite this article as: Yang, H.-D., et al., Food hoarding, but not food intake, is attenuated by acute diazepam treatment in female Mongolian gerbils (Meriones unguiculatus), Horm. Behav. (2014), http://dx.doi.org/10.1016/j.yhbeh.2014.03.010

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20 min. Finally, sections were then mounted, dehydrated a series of three separate washes in 95% EtOH (2 × 5 min), 100% EtOH (2 × 5 min), and citrosolve (2 × 5 min), and coverslipped. To reduce variability in the staining, brain sections from all animals were processed concurrently.

a fast, while others continued to display minimal food hoarding behavior (LFH). These differences persisted throughout the entire course of the experiment (two-way ANOVA, group, F(1,90) = 32.103, p b 0.001, day, F(4,90) = 9.982, P b 0.01; interaction group × day, F(4,90) = 9.207,

Quantification for Fos-ir cells

Data were analyzed using the SPSS 17.0 software (SPSS Inc., Chicago, IL, USA). Prior to statistical analysis, data were examined for normality and homogeneity of variance, using Kolmogorov– Smirnov and Levene tests, respectively. Because food hoarded mass was not normally distributed, we took a natural logarithm transformation, so that they are more likely to have a normal distribution, which will reduce the number of clusters found in a model-based clustering. Body weight, food intake, and food hoarded mass from experiment 1 were analyzed by two-way repeated measure ANOVA with hoarding group (high vs low hoarding) and day, followed by Bonferroni post hoc tests. The numbers of Fos-ir cells were analyzed by two-way ANOVA with these main effects: drug treatment (diazepam or saline) and hoarding tendency (HFH or LFH). These tests were followed by post hoc analyses by Bonferroni and when the main effects were significant. Corticosterone concentrations from experiment 1 were analyzed by Student's t-test. Body weight, food intake, and food hoarded mass from experiment 2 were analyzed using one-way analysis of variance (ANOVA) followed by Bonferroni's post-hoc test. In addition, the significance of the linear relationship between the number of Fos cells in each brain area and the mass of food hoarded in saline-treated gerbils was determined using Pearson's product–moment correlations. All results are presented as means ± SE, and p b 0.05 was considered to be statistically significant. Results Experiment 1: food deprivation and food hoarding in female gerbils Female gerbils showed a bimodal food hoarding response to food deprivation, similar to those seen in male gerbils (Yang et al., 2011). Some animals increased the amount of food hoarded (HFH) following

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A single researcher blind to the experimental condition of the slides counted black punctate nuclear staining for Fos (Fos-ir) cells. Photomicrographs were captured by using a Nikon Eclipse 80i microscope with a SPOT RTKE 7.4 Slider (Diagnostic Instruments) camera using × 10 objective lens. Photomicrographs of representative tissue labeling were processed using Adobe Photoshop CS8 (Adobe, San Jose, CA) for brightness, contrast, and tone. Fos-ir cells were found in various brain areas. Profile counting for Fos-ir cells was conducted bilaterally in selected brain areas, including the nucleus accumbens (NAcc) and caudate putamen (CP); paraventricular (PVN), ventromedial (VMH), and lateral (LH) nuclei of the hypothalamus; arcuate nucleus (ARC); hippocampus (field CA1, field CA2, field CA3 and DG) and ventral tegmental area (VTA) (Fig. 1). Brain areas were defined according to a rat brain atlas (Paxinos and Watson, 1986). For each brain area, data were quantified from 4 representative sections that were anatomically matched between animals. In a given area from distinct treatments, the images were thresholded to the same value by means of eliminating background and noise staining to ensure that Fos-ir cells were selected. Counts of Fos-ir cells were summed from unilateral sites for each brain area, and then divided by the total area of the counting domains in order to generate cell number (labeled cells per mm2). The values from ten gerbils of each treatment group were averaged to obtain the final mean ± SE.

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Fig. 5. Differences in the number of cells labeled for Fos-ir in selected brain areas in female food-deprived Mongolian gerbils with receiving diazepam or saline. Diazepam treated gerbils had higher number of Fos-ir cells in the nucleus accumbens (NAcc) (A), caudate putamen (CP) (A), ventral tegmental area (VTA) (A), the field CA2 and (B) CA3 of hippocampus (B), than saline treated gerbils. But diazepam failed to alter Fos expression in the field CA1 (B) and dentate gyrus (DG) (B) of hippocampus, paraventricular (PVN) (C), ventromedial (VMH) (C), and lateral (LH) (C) nuclei of the hypothalamus; arcuate nucleus (ARC) (C) in four groups. Different letters (a, b or c) above hatched bars and solid bars indicate significant differences (P b 0.05). Data are presented as mean ± SEM.

Please cite this article as: Yang, H.-D., et al., Food hoarding, but not food intake, is attenuated by acute diazepam treatment in female Mongolian gerbils (Meriones unguiculatus), Horm. Behav. (2014), http://dx.doi.org/10.1016/j.yhbeh.2014.03.010

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Fig. 6. A Photomicrographs displaying cells labeled for Fos-ir (black punctate nuclear staining) in the nucleus accumbens (NAcc; a & b), caudate putamen (CP; c & d) and ventral tegmental area (VTA; e & f) in HFH gerbils with receiving diazepam or saline. ac: anterior commissure, ec: external capsule. MM: medial mammillary nucleus, medial part. Scale bar = 100 μm. B Photomicrographs displaying cells labeled for Fos-ir (black punctate nuclear staining) in the field CA1 (a & b), CA2 (c & d), CA3 (e & f) of hippocampus. Scale bar = 100 μm. C Photomicrographs displaying cells labeled for Fos-ir (black punctate nuclear staining) in the paraventricular (PVN) (a & b) and lateral (LH) (c & d) nuclei of the hypothalamus; arcuate nucleus (ARC) (e & f). f: fornix. 3v: 3rd ventricular. Scale bar = 100 μm.

P b 0.01; Fig. 2A). There were no gerbils that hoarded in the intermediate range. There were no differences in the basal levels of food intake, food hoarding or body weight between HFH and LFH females. Food intake of both groups was decreased from day 1 to day 4 of food deprivation (two-way ANOVA, group, F(1,90) = 13.519, P b 0.01; day, F(4,90) = 2.135, P N 0.01; interaction group × day, F(4,90) = 0.327, P N 0.01; Fig. 2B). There were also significant decreases in body weight both HFH and LFH females during food deprivation (two-way ANOVA, group, F(1,90) = 1.613, P N 0.01; day, F(4,90) = 5.131, P b 0.01; interaction group × day, F(4,90) = 4.427, P N 0.01, Fig. 2C). In addition, HFH females had significantly higher serum corticosterone concentrations than the LFH ones (t(18) = 4.017, P b 0.01, Fig. 3).

Experiment 2: role of diazepam in food deprivation-induced food hoarding in female Mongolian gerbils Peripheral diazepam treatment significantly blocked the effects of food deprivation on food hoarding, in a non-dose dependent manner in HFH gerbils. Both doses of diazepam (l mg/kg and 3 mg/kg) decreased the number of pellets hoarded compared with saline injection (one-way ANOVA, F(2,27) = 11.752, P b 0.01, Fig. 4a). It was important to note that this inhibition was not due to sickness in the gerbils which could be seen in the decrease of food hoarding. An animal that has consumed too much diazepam typically displays one or more of the following symptoms: drowsiness, dizziness, and impaired balance

Please cite this article as: Yang, H.-D., et al., Food hoarding, but not food intake, is attenuated by acute diazepam treatment in female Mongolian gerbils (Meriones unguiculatus), Horm. Behav. (2014), http://dx.doi.org/10.1016/j.yhbeh.2014.03.010

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Fig. 6 (continued).

(Kaur and Kulkarni, 2002). However, we did not observe these symptoms when gerbils received diazepam. Diazepam failed to significantly alter food intake in HFH gerbils in either doses (Fig. 4b). There were also no significant differences in body weight before or during drug treatment for the three treatment groups in HFH gerbils. Within LFH gerbils, the effect of diazepam on food hoarding was not significant, regardless of drug doses (l mg/kg or 3 mg/kg) (one-way ANOVA, F(2,27) = 0.227, P N 0.05 Fig. 4c). Furthermore, before treatment or during drug treatment period, diazepam also failed to alter food intake in LFH gerbils in either doses (Fig. 4d). There were no significant differences in body weight before or during drug treatment for the three treatment groups in LFH gerbils. Cellular activation was significantly increased by food hoarding after food deprivation in the NAcc (drug, F(1,36) = 4.931, P b 0.05, hoarding, F1,36 = 11.89, P b 0.01, interaction drug × hoarding, F1,36 = 37.821, P b 0.01), CP (drug, F1,36 = 4.045, P b 0.05, hoarding, F1,36 = 9.89, P b 0.01, interaction drug × hoarding, F1,36 = 30.021, P b 0.01), CA3 (drug, F1,36 = 3.781, P N 0.05, hoarding, F1,36 = 6.87, P b 0.01, interaction drug × hoarding, F1,36 = 19.621, P b 0.01), and VTA (drug, F1,36 = 4.461, P b 0.05, hoarding, F1,36 = 8.89, P b 0.01, interaction drug × hoarding, F1,36 = 18.821, P b 0.01) (Fig. 6). Higher neural activation was observed in DZ-HFH in the NAcc (F1,36 = 4.021, P b 0.01), CP (F1,36 = 4.722, P b 0.01) and VTA (F1,36 = 3.487, P b 0.01) (Figs. 5A and 6A), and CA2 (F1,36 = 4.208, P b 0.01), CA3 (F1,36 = 3.421, P b 0.05) (Figs. 5B and 6B) after diazepam administration, than other groups. While no group differences were detected in the number of Fos-ir cells in four groups in the CA1 (F1,36 = 2.540, P N 0.05), DG (F1,36 = 6.299, P N 0.05), PVN (F1,36 = 0.102, P N 0.05), LH (F1,36 = 2.220, P N 0.05), VMH (F1,36 = 8.832, P N 0.05) and ARC (F1,36) =1.062, P N 0.05) (Figs. 5C and 6C). Moreover, there were significant positive correlations between the number of Fos-ir cells and the food hoarded

mass for NAcc, CP and VTA in saline treated HFH gerbils. Likewise, a number of Fos-ir cells in CA2 were also significantly and positively correlated with food hoarded mass in saline treated HFH gerbils (Fig. 7). Discussion Forging for, hoarding and eating food are critical for survival and are therefore among the most important behaviors of the lives of animals (Vander Wall, 1990). In the present experiments, female Mongolian gerbils showed remarkable individual differences in food hoarding in response to food deprivation. Although there was no identifiable difference in the expression of food hoarding behavior during ad libitum feeding, some animals displayed a tendency toward high levels of hoarding (HFH) whereas the other showed a tendency toward low levels of hoarding (LFH), specifically after food deprivation. This is consistent with previous research on male Mongolian gerbils under similar conditions (Yang et al., 2011). Mongolian gerbils show a bimodal food hoarding after food deprivation. One possible reason is that HFH gerbils have different responses to stress. Stress induced by food deprivation markedly activates the hypothalamic–pituitary–adrenal axis (HPAA). Glucocorticoids are the final effectors of the HPA axis (Tsigos and Chrousos, 2002). Food deprivation increases circulating corticosterone in the rodents and birds (McGhee et al., 2009; Pravosudov, 2003). Our data shows that food-deprived HFH females had higher corticosterone concentrations than fooddeprived LFH females, suggesting that corticosterone could be involved in facilitating food hoarding in gerbils. On the other hand, some studies, suggest that specific stressors can induce differential physiological responses, including: brain activation patterns, Fos immunoreactivity and receptor expression (Bowers et al., 2008). Some nuclei or regions, such as the NAcc, CP, CA2 and VTA, were activated that control food hoarding.

Please cite this article as: Yang, H.-D., et al., Food hoarding, but not food intake, is attenuated by acute diazepam treatment in female Mongolian gerbils (Meriones unguiculatus), Horm. Behav. (2014), http://dx.doi.org/10.1016/j.yhbeh.2014.03.010

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Fig. 6 (continued).

After food deprivation, i.p. treatment with the GABA A agonist diazepam treatment blocked food deprivation-induced increases in food hoarding in HFH gerbils but, diazepam failed to affect food intake regardless of hoarding tendency. Furthermore, food hoarding in response to food deprivation when gerbils were treated with diazepam was associated with high cellular activation in brain areas important for reward and learning, such as the NAcc, CP, CA2, CA3 and VTA. In addition, serum corticosterones were higher in food-deprived HFH than in food-deprived LFH gerbils. Together, these data indicate that activation of the GABAA receptor inhibits food-deprivation-induced increases in food hoarding in fooddeprived HFH gerbils. GABA is the major inhibitory neurotransmitter in the mamalian central nervous system (Panhelainen and Korpi, 2012; Salminen et al., 1996; Yong and Chu, 1990). Other data, however, suggest that GABA might motivate animals to procure food (Pulman et al., 2012). In the present experiment, the GABAA receptor agonist diazepam inhibited food deprivation-induced increases in food hoarding in food-deprived HFH gerbils, consistent with the earlier reports in rats, in which injection of diazepam reduced food hoarding (McNamara and Whishaw, 1990). Interestingly, diazepam failed to disrupt food hoarding in fooddeprived LFH gerbils; one possible reason is that the food hoarding in food-deprived LFH gerbils after food deprivation is a floor effect that it might be almost impossible to detect a further decrease in the behavior. Dragunow and Faull (1989) stated that cellular activation can occur without increases in Fos-immunoreactivity. Other immediate-early

genes, such as c-Jun, might be expressed in response to some stimuli in some brain areas. Thus, if a particular brain area did not show Fosimmunocreactivity, this does not mean that cells were not activated. Our data clearly also showed that diazepam induced Fos expression in the NAcc, CP, CA2, CA3 and VTA in food-deprived HFH gerbils. It is unlikely that the increases in Fos-ir are secondary to engaging in food hoarding behavior because baseline Fos-ir was higher in hoarders than in nonhoarders, and in the cases where Fos-ir was highest, food hoarding was prevented by diazepam treatment. The increase in Fos-ir indiazepam treated animals occurred in gerbils that hoarded less than saline treated gerbils. This argues against hoarding-induced changes in Fos-immunoreactivity. Furthermore, significantly positive correlations were found between food hoarding and the number of Fos-ir cells in the NAcc, CP, CA2 and VTA in saline-treated HFH gerbils. These data suggested that the GABA mediated the processing of food hoarding in the NAcc, CP, CA2 and VTA in food-deprived HFH gerbils. Although, many studies suggested that GABA robustly stimulate feeding behavior (Pulman et al., 2012). GABAA receptor agonism failed to influence feeding in food-deprived HFH or LFH gerbils in this study. In addition, Fos-ir showed no change in several hypothalamic nuclei, such as, PVN, LH ARC and VMN, which are previously shown to be involved in the control of ingestion behaviors. So, one possible reason is that these regions are disinhibited; food intake of the LFH and HFH gerbils was not affected. It is widely accepted that motivated behaviors that promote fitness, such as food intake and sexual behavior, are regulated by brain reward circuitry, which is implicated in naturally occurring motivated behaviors in prairie voles (Young and Wang, 2004). The VTA contains GABAergic neurons, which project to the NAcc shell (Bubar et al., 2011), and direct activation of VTA GABA projections to the NAcc in mice (van Zessen et al., 2012). In the present study, diazepam injection decreased food hoarding and activated the central reward circuitry, as evidenced by Fos increased expression in the NAcc, CP and VTA, in food-deprived HFH females, similar to our previous study, where cellular activation in the NAcc and VTA was increased in male gerbils (Yang et al., 2011). Therefore, our data provided further evidence to support the notion that GABA and reward circuitry, such as the NAcc, CP and VTA, are important for the regulation of food hoarding. The hippocampus and related neuronal networks are most commonly associated with memory processing (Massa et al., 2010); however, several studies demonstrate a role of the hippocampus in the control of food hoarding in rats (Martin and Wallace, 2007) and birds (László Zsolt Garamszegi and Eens, 2004). For example, the hippocampus volume of black-capped chickadees (Poecile atricapillus), a scatterhoarding bird, varies seasonally (Smulders et al., 2000). Our data indicate that DZ-HFH females increased Fos-ir in the CA2 and CA3, compared with saline treated HFH females, indicating cellular increased activation in the hippocampus associated with high level of food hoarding after food deprivation. However, these data are inconsistent with data from a previous study showing that hippocampal lesions facilitate food hoarding behavior in food-deprived rats (Wishart et al., 1969). Furthermore, rats do not typically hoard food in nature, unlike hamsters and gerbils. So, one possibility is that the hippocampus has species-specific and/or area-specific effects on food hoarding. In a previous study, lesions of dorsal hippocampus significantly increased food hoarding in rats (Borker and Mascarenhas, 1990). Whereas, in rats in which the hippocampal formation was removed, food hoarding was essentially abolished (Vanderwolf et al., 1978). These data, together, support the notion that GABA in the hippocampus is correlated to food hoarding in HFH gerbils, and that the effects of GABA differ according to the subnuclei of the hippocampus. Future experiments should determine whether the effects of diazepam on food deprivation-induced increases in food hoarding are due to diazepam-induced decreases in plasma glucocorticoids. In summary, the current study demonstrates that injection of the GABAA receptor agonist, diazepam blocks food deprivation-induced

Please cite this article as: Yang, H.-D., et al., Food hoarding, but not food intake, is attenuated by acute diazepam treatment in female Mongolian gerbils (Meriones unguiculatus), Horm. Behav. (2014), http://dx.doi.org/10.1016/j.yhbeh.2014.03.010

H.-D. Yang et al. / Hormones and Behavior xxx (2014) xxx–xxx

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Fig. 7. Relationships between the density of Fos-ir cells and the food hoarded mass. When gerbils received saline, the density of Fos-ir cells was positively correlated with the food hoarded mass for the nucleus accumbens (NAcc), the caudate putamen (CP), the field CA2 of hippocampus, and ventral tegmental area (VTA) in saline-HFH gerbils but not in saline-LFH.

increases in food hoarding in female HFH Mongolian gerbils, similar to rats injected with diazepam (McNamara and Whishaw, 1990) or GABA (Mogenson and Wu, 1988). Because of the pattern of observed increases in Fos-ir, GABA activation in the brain reward circuitry appears to be important for the regulation of the behavioral response to fasting in HFH gerbils.

Acknowledgments We would like to thank all the members in the Animal Physiological Ecology Group in the Institute of Zoology of the Chinese Academy of Sciences for their assistance with the experiments and for their helpful discussion. We also thank the anonymous reviewers and Dr. Jill Schneider

Please cite this article as: Yang, H.-D., et al., Food hoarding, but not food intake, is attenuated by acute diazepam treatment in female Mongolian gerbils (Meriones unguiculatus), Horm. Behav. (2014), http://dx.doi.org/10.1016/j.yhbeh.2014.03.010

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for their helpful comments and suggestions. This study was financially supported by grants from the National Scientific Foundation of China (31071930 and 31272312) to DHW and the State Key Laboratory of Integrated Management of Pest Insects and Rodents (Chinese IPM1402) to HDY.

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Please cite this article as: Yang, H.-D., et al., Food hoarding, but not food intake, is attenuated by acute diazepam treatment in female Mongolian gerbils (Meriones unguiculatus), Horm. Behav. (2014), http://dx.doi.org/10.1016/j.yhbeh.2014.03.010