Stimulation of Lateral Hypothalamic Kainate Receptors Selectively Elicits Feeding Behavior

BRAIN RESEARCH 1184 (2007) 178– 185

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Stimulation of lateral hypothalamic kainate receptors selectively elicits feeding behavior

Stacey R. Hettesa, Theodore W. Heyming, B. Glenn Stanleyb,c,⁎ aDepartment of Biology, Wofford College, Spartanburg, SC 29303, USA bDepartment of Psychology, University of California-Riverside, Riverside, CA 92521, USA cDepartment of Cell Biology and Neuroscience, University of California-Riverside, Riverside, CA 92521, USA

ARTICLE INFO ABSTRACT

Article history: Glutamate and its receptor agonists, NMDA, AMPA, and KA, elicit feeding when Accepted 24 September 2007 microinjected into the lateral hypothalamus (LH) of satiated rats. However, determining Available online 2 October 2007 the relative contributions of AMPA receptors (AMPARs) and KA receptors (KARs) to LH feeding mechanisms has been difficult due to a lack of receptor selective agonists and Keywords: antagonists. Furthermore, LH injection of KA produces behavioral hyperactivity, Lateral hypothalamus questioning a role for KARs in feeding selective stimulation. In the present study, we used Feeding the KAR agonist, (RS)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl) propanoic acid Kainate (ATPA), which selectively binds the GluR5 subunit of KARs, to stimulate feeding, Glutamate presumably via KAR activation. Using ATPA, we tested whether: (1) LH injection of ATPA ATPA elicits feeding, (2) prior treatment with the non-selective AMPA/KAR antagonist, CNQX, iGluR suppresses ATPA-elicited feeding, and (3) LH injection of ATPA elicits behavioral patterns specific for feeding. We found that injection of ATPA (0.1 and 1 nmol) elicited an intense feeding response (e.g., 4.8±1.6 g) that was blocked by LH pretreatment with CNQX, but was unaffected by pretreatment with the AMPAR selective antagonist, GYKI 52466. Furthermore, minute-by-minute behavioral analysis revealed that LH injection of ATPA increased time spent feeding to 55% of the initial test period with little or no effects on other behaviors at any time. In contrast, LH injection of KA similarly increased feeding but also produced intense locomotor activity. These data suggest that selective activation of LH KARs containing GluR5 subunit(s) is sufficient to elicit feeding. © 2007 Elsevier B.V. All rights reserved.

1. Introduction these ionotropic glutamate receptor (iGluR) subtypes may play a role in feeding stimulation (Duva et al., 2002; Hettes et al., One of our major goals has been to determine the role of LH 2003; Khan et al., 1999; Stanley et al., 1993a,b, 1996). Addi- glutamate in the stimulation of feeding behavior. Toward tionally, we have demonstrated that LH pretreatment with this end, we have employed agonists of N-methyl-D-aspartate the NMDA receptor (NMDAR) antagonist, D(−)-2-amino-5- (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxale propionate phosphonopentanoic acid (DAP-5), blocked food intake eli- (AMPA), and kainate (KA) receptors to provide evidence that cited by subsequent LH injection of NMDA without affecting

⁎ Corresponding author. Department of Psychology, University of California-Riverside, Riverside, CA 92521, USA. Fax: +1 951 827 3985. E-mail address: [email protected] (B.G. Stanley).

2. Results

2.1. Locus of injection sites

Histological analysis of the cannulation sites confirmed that the injections were typically centered in the middle region of the LH along its longitudinal axis, at least 0.3 mm lateral to the fornix. A representative example is provided in Fig. 1. The behavioral data from animals with placements outside of the LH were excluded from analysis.

2.1.1. Experiment 1: LH injection of the KAR agonist, ATPA, elicits feeding in a dose-dependent manner As shown in Fig. 2, ATPA at 0.1 and 1.0 nmol elicited eating at intervals from 30 min to 4 h post injection. One-way ANOVAs revealed significant effects of dose at the 30 min, 1 h, and 4 h post

injection times [F3,44 =6.76, 8.39, and 4.26 respectively (p<0.01)]. Fig. 1 – Cannula localized to the LH. Digital photomicrograph Compared to the dilute aCSF intake score of 0.1±0.4 g at 30 min of coronal thionin stained section showing representative post injection, the 0.1 and 1.0 nmol doses of ATPA increased cannula placement in the LH. Scale bar equals 1 mm. consumption to 2.8±0.7 g and 4.8±1.6 g, respectively, with no statistically significant further increases at later intervals. These data suggest that the presumed stimulation of LH KARs by ATPA feeding elicited by LH injection of AMPA or KA (Stanley et al., rapidly elicits feeding and that the effect is short-lived. 1996). While these data strongly suggest that activation of NMDARs in the LH elicits feeding, they also indicate that sti- 2.1.2. Experiment 2: LH injection of the non-selective AMPA/ mulation of AMPA receptors (AMPARs) and/or KA receptors KAR antagonist, CNQX, but not the AMPAR selective antagonist, (KARs) can elicit feeding in the absence of NMDAR activation. GYKI 52466, blocks subsequent ATPA-elicited feeding Determining the relative contributions of the AMPA and KA To examine whether ATPA- or KA-elicited feeding might result iGluR subtypes to glutamatergic feeding mechanisms has primarily or exclusively from activation of KARs, as opposed to been difficult due to the paucity of agonists and antagonists concurrent activation of AMPARs, we tested whether LH pre- selective for each of these receptors (Lauridsen et al., 1985). treatment with CNQX or GYKI 52466 would suppress ATPA- or However, (RS)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl) KA-elicited feeding. As shown in Fig. 3A and consistent with the propanoic acid (ATPA) has been demonstrated to be a selec- results of Experiment 1, LH injection of ATPA elicited eating tive agonist for KARs containing the GluR5 subunit (Lauridsen (10.9±2.6 g at 30 min) and pretreatment with CNQX blocked that et al., 1985; Pinheiro and Mulle, 2006), with over 100× greater potency for KARs than AMPARs (Clarke et al., 1997; Hoo et al., 1999). Here, we attempt to determine whether selective stimulation of KARs in the LH of satiated rats is sufficient to elicit feeding. Several problems were associated with the presumed stimulation of KARs in our previous research (Stanley et al., 1993a,b). First, KA, the agonist we employed, is not specific for KARs versus AMPARs (Krogsgaard-Larsen et al., 1980). Second, KA is a potent neurotoxin and has been used to lesion brain tissue, including the LH (Lenard et al., 1988). Lastly, LH injection of KA produces intense and prolonged behavioral hy- perctivity (Stanley et al., 1993a). This is troublesome as feeding behavior can occur as a component of general arousal (Antel- man and Szechtman, 1975; Antelman et al., 1976; Morley et al., 1983) in the absence of direct activation of neural mechanisms specific to feeding stimulation. We suspected that using ATPA might resolve some of the complications associated with KA injection. Therefore, we tested the selective GluR5 agonist, ATPA, to determine whether: (1) LH injection of ATPA would Fig. 2 – ATPA injected into the LH elicits feeding. Cumulative elicit feeding, (2) this feeding could be suppressed by prior food intake (mean grams±SEM) 0.5, 1, and 4 h following injection of an AMPA/KAR antagonist, and (3) behavioral injection of aCSF vehicle, or ATPA at 0.01, 0.1, and changes elicited by its injection were specifically related to 1 nmol (N=12). *p<0.05, by one-way ANOVA and food intake. For comparison, we conducted similar tests using Student–Newman–Keuls test compared to vehicle (aCSF) KA. control injection at matched times. 180 BRAIN RESEARCH 1184 (2007) 178– 185

Fig. 3 – ATPA- and KA-elicited feeding are suppressed by pretreatment with CNQX but not by pretreatment with GYKI 52466. Cumulative food intake (mean g±SEM) 30 min, 1, and 4 h post injection as a function of treatment. Prior treatment with CNQX (3 nmol) (A) but not GYKI 52466 (10 nmol) (C), blocked feeding elicited by ATPA (1 nmol, N=15) for the duration of the test. The veh+ATPA treatment elicited significantly greater feeding than any other treatment at matched times. Prior treatment with CNQX (3 nmol) (B) but not GYKI 52466 (10 nmol) (D), transiently suppressed feeding elicited by KA (0.3 nmol, N=15) by 41% for the first hour post injection. By 4 h post injection, there was no difference between scores for veh+KA and CNQX+KA treatments (*p<0.05; by two-way ANOVA and Student–Newman–Keuls test compared to veh+veh control treatment. ^p<0.05 by two-way ANOVA and Student–Newman–Keuls test compared to CNQX+KA treatment). effect, reducing the elicited intake to 0.9±0.3 g (p<0. 05, by SNK). 10 nmol of GYKI 52466 did not affect either the feeding responses This blockade of ATPA-elicited feeding was maintained for the induced by subsequent injection of ATPA or KA. Collectively, duration of the 4 hr test period. Two-way ANOVA revealed a these data may indicate that ATPA- and KA-elicited feeding significant effect of treatment (F3,96 =15.9, p<0.0001) and time occurs by selective stimulation of KARs.

(F3,96 =17.44, p<0.0001), but no interaction between the two

(F9,91 =1.163). 2.1.3. Experiment 3: LH injection of ATPA initiates behavioral As shown in Fig. 3B, LH pretreatment with CNQX transiently activity distinct to feeding suppressed feeding elicited by subsequent injection of KA. Two- Casual observations during Experiment 2 suggested that while way ANOVA revealed a significant effect of treatment (F3,114 = KA produced intense behavioral hyperactivity (Stanley et al.,

18.98, p<0.001), time post injection (F3,114 =57.81,p<0.001), as well 1993a), LH injection of ATPA had only a small effect. To quan- as an interaction between the two (F9,108 =2.84,p<0.01).At30min tify this, we systematically observed the behavioral effects of post injection, KA elicited a robust feeding response [(12.2±1.5 g LH injection of ATPA or KA. As shown in Fig. 4A, the immediate (⁎p<0.05 by SNK)] and CNQX pretreatment reduced this by 41% to and most salient effect of LH injection of ATPA (1.0 nmol, 7.1±1.16 g (^p<0.05 by SNK). However, by 4 h post injection, N=13) was to increase the time spent engaged in feeding be- pretreatment with CNQX was no longer effective in suppressing havior. This response peaked 10 min post injection, when the KA-elicited feeding. In contrast, as shown in Figs. 3C and 2D, rats spent an average of 55% of their time eating, and remained BRAIN RESEARCH 1184 (2007) 178– 185 181

Fig. 4 – Behavioral responses elicited by LH injection of ATPA are selective for feeding while LH injection of KA elicits feeding accompanied and followed by general arousal. Time spent engaged in typical behaviors was averaged over 5 min increments. Injection of ATPA (1 nmol or its dilute aCSF control, N=13) or KA (1 nmol or its aCSF control, N=10) occurred at T=6 min. Changes in behavioral activity are given as percent of total time. Categories include eating, drinking, grooming, locomotion, alert, or resting/sleeping (for definition see text). (A) ATPA markedly increased time spent engaged in eating while (B) KA increased time spent engaged in feeding followed by a marked increased time spent engaged in locomotion (*p<0.05; by two-way ANOVA and Student–Newman–Keuls test compared to vehicle control treatment for matched activity and time). (C) Behavioral results of LH injection of ATPA and KA are presented here for direct comparison (*p<0.05; by two-way ANOVA and Student–Newman–Keuls test comparing ATPA and KA treatments for matched activity and time). 182 BRAIN RESEARCH 1184 (2007) 178– 185

behaviors declined to near zero. These data suggest that the feeding produced by LH injection of KA is accompanied and followed by an intense generalized behavioral hyperactivity. In Fig. 4C, scores for the ATPA and KA injections are presented together for direct comparison. Early in the test period, the latency, duration, and time course of feeding stimulation were similar upon LH injection of ATPA or KA. However, the magnitude of feeding produced by LH injection of ATPA was

greater than that induced by KA injection [F1,252 =11.2 (treat-

ment), F11,252 =1.97 (time post injection), F11,252 =3.84(interaction) p<0.05, by two-way ANOVA and p<0.05 by SNK]. However, starting at 20 min into the test, the behavior of the ATPA injected rats began to return to control values. In contrast, the KA injected rats exhibited locomotion, to the virtual exclusion of other behaviors. When compared directly, it is clear that LH injection Fig. 5 – Both ATPA and KA elicit feeding during of ATPA and KA produced markedly different effects on general minute-by-minute analysis of behavior. Cumulative food behavioral activity. This difference might be due to KA's intake (mean grams±SEM) was measured at the completion presumed activation of a broader population of LH KARs, thereby eliciting a more general effect on behavior. of behavioral analysis in the experiment following injection of ATPA (1 nmol or its dilute aCSF control, N=13) or KA (1 nmol or its aCSF control, N=10). *p<0.05, by one-way ANOVA and Student–Newman–Keuls test compared to 3. Discussion vehicle (aCSF) control injection. Although we had previously demonstrated that feeding is elicited by LH injection of glutamate or ionotropic glutamate elevated for nearly 30 min [F1,132 =30.8 (treatment), F11,132 =8.6 receptor (iGluR) agonists and that NMDA receptor stimulation

(time post injection), F11,132 =8.5 (interaction), p<0.01, by two- appears to mediate some aspects of natural feeding via action way ANOVA and p<0.05, by SNK compared to vehicle control in the tuberal LH (Duva et al., 2002; Khan et al., 1999; Stanley injection for matched time]. As shown in Fig. 5, this yielded a et al., 1993a,b, 1996), we had not, before now, presented evi- total food intake of 11.3±2.1 g versus 0.7±0.3 g vehicle injection dence of a role for specific KAR activation in feeding stimu- (p<0.01 by paired t-test). A concurrent decrease in percent time lation. The data presented here extend our previous findings spent either resting/sleeping [F1,132 =24.5 (treatment), F11,132 = (Stanley et al., 1993a,b), as they suggest that LH injection of

14.3 (time post injection), F11,132 =9.2 (interaction), p<0.01, by ATPA or KA elicits feeding, at least in large part, by activating two-way ANOVA and p<0.05, by SNK] or alert [F1,132 =1.5 KARs within this region. Additionally, they are among the first

(treatment, ns), F11,132 =3.2 (time post injection), F11,132 =6.66 to describe the initiation of a complex goal oriented behavior (interaction), p<0.01, by two-way ANOVA and p<0.05, by SNK] by administration of an iGluR agonist targeted specifically to was observed after ATPA injection when compared to vehicle GluR5 KARs (Benvenga et al., 1995; Pinheiro and Mulle, 2006). control injection. Small isolated instances of increased time That LH injection of KA elicits feeding reliably and dose spent drinking or in locomotion were also exhibited by ATPA- dependently had already been established (Stanley et al., injected rats up to 30 min post injection. These data demon- 1993a,b). Here, we demonstrate that LH injection of ATPA also strate that LH injection of ATPA increases the time spent en- induced feeding in a dose-dependent manner and that this gaged in feeding behavior with relatively minor and brief ATPA-elicited response was blocked by LH pretreatment with effects on other behaviors. the non-selective AMPAR/KAR antagonist, CNQX. Further- Comparable tests with KA (1 nmol, N=10) produced similar more, LH pretreatment with CNQX suppressed KA-elicited patterns of feeding, but very different effects on locomotion. feeding by ∼40%. These data may suggest that CNQX an- As shown in Fig. 4B, at 10 min post injection, the time spent tagonizes GluR5 KARs in the LH sufficiently to block ATPA- eating increased to 28%, but this value failed to achieve induced stimulation of feeding, but may be less effective in significance over aCSF control injection [F1,99 =3.4 (treatment, antagonizing the broader population of LH KARs upon which ns), F11,99 =3.0 (time post injection, p<0.05), F11,99 =0.9 (inter- KA may act to elicit feeding. Perhaps equally important, pre- action, ns) by two-way ANOVA]. However, as shown in Fig. 5, treatment with the AMPAR selective antagonist, GYKI 52466, because KA-injected rats ate rapidly, this time spent feeding did not suppress ATPA- or KA-induced feeding, suggesting yielded an intake of 8.5±3.0 g, which was significantly greater that ATPA- and KA-elicited feeding can occur in the absence of than the 3.0±0.9 g eaten after the aCSF control injection AMPAR stimulation. These data imply that specific stimula- (p<0.01, by paired t-test). As for locomotion, this had increased tion of KARs, which have been localized to hypothalamic significantly by 10 min post injection and continued to in- regions (Eyigor et al., 2005; van den Pol et al., 1994) including crease subsequently, reaching 100% 35 min into the test and the LH (Eyigor et al., 2001; Herman et al., 2000) and which remained at or near this level for the duration of the test received apparent glutamatergic synaptic inputs (Henny and period [F1,99 =139.0 (treatment), F11,99 =21.7 (time post injec- Jones, 2006b), is sufficient to elicit feeding behavior. tion), F11,99 =29.8 (interaction), p<0.001, by two-way ANOVA In experiments aimed at determining the behavioral ef- and p<0.05, by SNK]. Concurrently, incidence of all other fects of LH KAR stimulation, we observed that the initial effect BRAIN RESEARCH 1184 (2007) 178– 185 183 of LH injected ATPA or KA was to increase feeding behavior, tides which have been shown to increases glutamate release with both agonists eliciting similar food intakes. While ATPA's from cultured hypothalamic cells (Sweet et al., 1999) and other effect was relatively selective to feeding behavior, KA-injected brain regions in vivo (John et al., 2003). Orexins, thought to be rats exhibited an intense and sustained general hyperactivity. generated primarily by LH neurons (De Lecea et al., 1998), many This difference in behavioral effects might be explained if of which receive apparent glutamatergic synaptic inputs ATPA selectively activates KARs containing a GluR5 subunit, (Henny and Jones, 2006a), have been implicated in feeding, while KA activates a broader population of LH KARs and sleep regulation, and circadian rhythm (Willie et al., 2001). thereby has a more general effect on behavior. Alternatively, Orexin A (also called hypocretin1) elicits feeding when injected KA may act as an agonist at both the AMPAR and KAR subtypes into the LH, the paraventricular nucleus (PVN), and the within the LH (Keinanen et al., 1990), with the combined perifornical hypothalamus (PFH) (Dube et al., 1999; Sweet activation resulting in behavioral hyperactivity. However, this et al., 1999) suggesting that it might play multiple roles in seems unlikely, since casual observations during our experi- feeding regulation. Furthermore, PVN injection of an NPY an- ments testing GYKI 52466 on KA-elicited feeding did not tagonist prior to ICV injection of orexin (Jain et al., 2000; Woods suggest a decrease in hyperactivity (nor feeding) under these et al., 1998) blocked orexin-elicited food intake, suggesting a conditions. possible NPY/orexin interaction to control feeding. We hope to Collectively these data may suggest that selective activa- determine if KARs might also interact with orexin in the LH or tion of KARs is sufficient to elicit feeding behavior in the other regions to mediate central mechanisms of feeding. absence of AMPAR stimulation. However, before attempting to clarify the aspects of feeding control mechanisms that might be impacted by LH KARs, we sought to first determine whether 4. Experimental procedures the behavior induced by the presumed KAR activation with ATPA was specific to feeding or was instead associated with 4.1. Subjects and surgery the pattern of general hyperactivity produced by LH injections of KA (Stanley et al., 1993a). The underlying goal was to de- Male Sprague–Dawley rats (350–500 g), bred in our viviarium, termine whether KAR stimulation initiates feeding directly or were used in this study. They were individually housed at as an indirect consequence of general arousal. Our results constant temperature (21 °C) on a 12:12 h light/dark cycle, and revealed that ATPA elicited feeding with minimal behavioral were maintained and tested on a milk-mash diet consisting of hyperactivity, suggesting that this effect can be produced Purina rat chow (500 g), sucrose (400 g), and evaporated milk without excessive general arousal, supporting a role for sti- (354 ml). Rats had ad libitum access to food and water at all mulation of KARs, particularly those containing GluR5 subunit times, and on test days they were given fresh mash 1.5 h prior (s), in LH mechanisms of feeding stimulations. More generally, to testing to ensure satiety. they provide evidence that ATPA is a useful pharmacological Stereotaxic procedures were used to implant a unilateral tool for in vivo studies of KAR function, which is important 18.0 mm long, 26 gauge (o.d.=0.46 mm), stainless steel guide given KA's reported neurotoxicity (Hampson and Manalo, cannula into the LH of rats anesthetized with inhaled metofane 1998; Kiskin et al., 1986; Patneau and Mayer, 1991) and broad or injected intraperitoneally with sodium pentobarbital (50 mg/ spectrum of receptor effects. kg of body weight). Stereotaxic coordinates were determined Collectively, these data may suggest that selective activa- empirically following the atlas of Paxinos and Watson (1986) tion of KARs is sufficient to elicit feeding behavior in the with placements 6.1 mm anterior to the interaural line, 1.8 mm absence of AMPAR stimulation. Supporting this suggestion, lateral to the midsagittal sinus, and 8.2 mm ventral to the anatomical and electrophysiological data from other systems surface of the skull. The incisor bar was positioned 3.3 mm indicate that KAR and AMPAR mediated events are separable below the interaural line. Cannulas were secured to the skull (Christoffersen and Meltzer, 1995; Kidd and Isaac, 1999; Li with dental acrylic and 6 stainless steel screws just penetrating et al., 1999; van den Pol et al., 1994). For example, evidence the bone. An obturator was placed in the guide cannula at the suggests that while KARs are localized both pre- and post- completion of surgery and rats were allowed to recover for at synaptically, AMPARs appear to be located primarily within least 7 days. During this time, rats were repeatedly handled and the postsynaptic density of glutamatergic synapses (for re- mock-injected in order to accustom them to the test procedure. view, see Chittajallu et al., 1999; Frerking and Nicoll, 2000; Patneau et al., 1993; Pinheiro and Mulle, 2006). Furthermore, 4.2. Behavioral testing evidence suggests that presynaptic KARs play a role in the regulation of neurotransmitter release at both glutamatergic Tests were conducted during the first half of the light phase of and GABAergic synapses (Chittajallu et al., 1996; Kamiya and the light/dark cycle when rats normally eat very little, and con- Ozawa, 2000; Liu et al., 1999; Patneau et al., 1993; van den Pol, sisted of single or paired injections directly into the LH through a 1991, 1998; Willie et al., 2001; Wong and Mayer, 1993), in- 33-gauge needle projecting 1 mm beyond the guide cannula. KA cluding at presynaptic GABAergic terminals within the hy- [0.33 nmol (0.07 μg) or 1 nmol (0.21 μg)] was delivered into the LH pothalamus (Liu et al., 1999). Given the evidence that feeding in a volume of 0.3 μl using artificial cerebrospinal fluid (aCSF) as can be elicited by GABA receptor blockade in the LH (Kelly the vehicle. ATPA [0.01, 0.1, and 1 nmol (0.0023, 0.023, and et al., 1977), this may suggest that KAR and GABA act in an 0.23 μg, respectively)] and CNQX [3 nmol (0.83 μg)] were delivered μ antagonistic fashion in LH feeding control mechanisms. into the LH in a volume of 0.3 l using a 2:1 dilution of aCSF:dH2O The findings presented here are particularly exciting in (dilute aCSF) because of their enhanced solubility in this vehicle. light of the discovery of the orexin family of neuroactive pep- GYKI 52466 [10 nmol (3.6 μg)] was delivered in a similar manner, 184 BRAIN RESEARCH 1184 (2007) 178– 185 using dimethyl sulfoxide (DMSO) as a vehicle. The aCSF was a 10% formalin. Brains were removed, frozen, and 100 μm thick − sterile mixture of 147 mM Na+,154mMCl,3.0mMK+,1.2mM coronal sections were cut through the extent of the cannula Ca+2, and 0.9 mM Mg+2 (pH 7.4) prepared in our laboratory. track. Sections were stained with thionin and examined Control injections of 0.3 μl aCSF, dilute aCSF, or DMSO were also microscopically. Injection sites were determined by tracing a given. After injection, animals were returned to their home cage projection of the scar made by the injector onto matched atlas and allowed to eat from a pre-weighed food bowl, with sections (Paxinos and Watson, 1986). cumulative food intake measured 30, 60, 120, and 240 min after the injection unless otherwise stated. Tests were con- 4.4. Statistical analysis ducted in counterbalanced order with each subject tested in every condition, and at least 1 day recovery between tests. The food intake data from animals that completed all tests for ATPA, KA, CNQX, and GYKI 52466 were obtained from Tocris their group were analyzed by one-way ANOVA (Experiment 1) Cookson, Inc. All procedures were approved by the University of or by two-way ANOVA across treatments (Experiment 2). California-Riverside, Animal Care Committee. When analysis of variance demonstrated a significant main effect(s), multiple comparison testing using the Student– 4.2.1. Experiment 1: does LH injection of ATPA elicit feeding in Newman–Keuls (SNK) test (Duva et al., 2002; Khan et al., satiated rats? 1999) was then conducted. In Experiment 3, the ATPA and KA To determine if a KAR selective agonist reliably and dose- injection studies were analyzed by two-way ANOVA across dependently elicits food intake, 12 satiated rats were given uni- treatments. The SNK test was used to compare between each lateral LH injections of ATPA (0.01, 0.1, 1 nmol or dilute aCSF) and treatment and its vehicle control score or between ATPA and their subsequent cumulative food intake was measured. KA treatment for matched time and activity. An α of 0.05 or less was deemed significant throughout the study. 4.2.2. Experiment 2: is ATPA- or KA-elicited feeding suppressed by prior LH injection of antagonists of KARs and/or AMPARs? Acknowledgments

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