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Neuropeptide AF is associated with short-term reduced food intake in rats
Behavioural Brain Research 219 (2011) 351–353
Contents lists available at ScienceDirect
Behavioural Brain Research journal homepage: www.elsevier.com/locate/bbr
Short communication
Neuropeptide AF is associated with short-term reduced food intake in rats Brandon A. Newmyer, Mark A. Cline ∗ Department of Biology, Radford University, Radford, VA, USA
a r t i c l e
i n f o
Article history: Received 13 September 2010 Received in revised form 26 November 2010 Accepted 5 December 2010 Available online 13 December 2010
a b s t r a c t Neuropeptide AF (NPAF), a member of the RFamide family, was centrally administered to rats to determine its effect on food intake. Rats responded with a linear dose-dependent reduction in cumulative food intake up to 3 h post injection, but when analyzed on a noncumulative basis, food intake was only significantly reduced at 0.5 h. To our knowledge, this is the first report of NPAF-induced reduction in food intake in a mammalian model. © 2010 Elsevier B.V. All rights reserved.
Keywords: Rat Food intake Neuropeptide AF NPAF
One group of neuropeptides receiving increased attention for roles in appetite regulation are the RFamides, which share structural similarity by containing an arginine (R) and amidated phenylalanine (F) at their carboxy-terminal ends. Members of this group affect food intake in species ranging from invertebrates such as locusts [1], jellyfish [2], and nematodes [3], to higher vertebrates, including chickens [4,5] and rats [6,7]. That the RFamides arose early in evolution and affect appetite in a broad range of species may imply that they have primary roles in appetite regulation. One member of this group is neuropeptide AF (NPAF), which we demonstrated is associated with satiety in chicks [8]. First isolated from bovine brain extracts by Yang et al. [9], NPAF has been studied primarily for its antiopioid action [10]. Kavaliers [11] showed the antinociceptive effects associated with centrally injected of NPAF in mice; NPAF-treated mice had increased magnitudes and durations of morphine- and stress-induced analgesia. NPAF’s effects on other signaling systems have also been investigated. Recently, Jászberényi et al. [12] demonstrated that centrally administered NPAF stimulates the release of ACTH, corticosterone, and dopamine. Because these signals are associated with appetite [13–15], and that we demonstrated that NPAF reduces food intake in chicks [8], we hypothesized that NPAF would also be associated with reduced food intake in a mammalian model. Thus, to evaluate this hypothesis we centrally administered NPAF to rats and determined its effects on food and water intake for up to 84 h post injection.
∗ Corresponding author. Tel.: +1 540 831 6431; fax: +1 540 831 5129. E-mail address: [email protected] (M.A. Cline). 0166-4328/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2010.12.001
Male Sprague-Dawley rats (35–49 g) were purchased from Harlan Laboratories (Indianapolis, IN) and housed individually in 24 cm × 18 cm × 18 cm hanging stainless steel cages under controlled conditions (24 ± 2 ◦ C, 50 ± 5% relative humidity and lights on 06:00–18:00) with free access to Teklad 2018C (18% crude protein, 5% crude fat, Harlan Laboratories) meal diet and tap water. Feeders were equipped with devices to reduce spillage and water bottles with anti-drip sippers. All experimental procedures were performed according to the National Research Council Publication, Guide for Care and Use of Laboratory Animals, and were approved by the Radford University Institutional Animal Care and Use Committee. Upon reaching 230–245 g, rats were anesthetized with isofluorane and a stainless-steel guide cannula (11 mm, 22 gauge) was stereotaxically implanted into the left lateral cerebroventricle (coordinates relative to bregma: X: −1.5, Y: −0.8, Z: −3.6, based on anatomy described in [16]) and immobilized using dental cement and stainless steel surgical screws. Buprenorphine (0.05 mg/kg body wt, subcutaneously) was administered post-anesthesia for analgesia. Fluid was replaced by subcutaneous injection of 0.9% NaCl to balance fluid loss at 3:1. A dummy injector remained in the guide cannula when it was not in use. Rats, after returning to pre-surgery weight, were administered 50 pmol angiotensin II and monitored for a dipsogenic response to confirm correct placement of cannula. Rats that did not drink at least 5 mL of water within 30 min of angiotensin II injection were removed from the study [17]. A preliminary trial was conducted to ascertain the duration and magnitude of the potential reduction in food intake following central NPAF. Nine 24 h-fasted rats on a cross-over design were randomly assigned to receive either 0 (0.9% NaCl, vehicle only)
352
B.A. Newmyer, M.A. Cline / Behavioural Brain Research 219 (2011) 351–353
Fig. 1. Cumulative (top) and noncumulative (bottom) food intake after central injection of NPAF to rats measured for 84 h post injection. Values are the mean ± SE; value above each SE is that group’s food intake expressed as a percentage of the vehicle group within that time; * denotes difference within a time point (P < 0.05).
Fig. 2. Cumulative (top) and noncumulative (bottom) water intake after central injection of NPAF to rats measured for 84 h post injection. Values are the mean ± SE; value above each SE is that group’s food intake expressed as a percentage of the vehicle group within that time; * denotes difference within a time point (P < 0.05).
or 16 nmol NPAF for a total injection volume of 4 L. Rats were injected 7 d post surgery. The two replicates were performed 5 d apart. Food and water intake were measured to 84 h post injection. Data were analyzed using analysis of variance (ANOVA) at each time point, with the model food intake as a function of treatment, replicate, and treatment by replicate interaction. Replicate was not significant and was thus removed from the secondary ANOVA analysis. Rats that received NPAF reduced their food intake at 3 h post injection when data were analyzed on a both a cumulative (Fig. 1(top)) and non-cumulative (Fig. 1(bottom)) basis, but this effect was not significant at any other time point. Water intake was also concurrently measured during this preliminary trial and was not significantly affected at any observation time when analyzed on a cumulative basis (Fig. 2(top)). However, when analyzed on a noncumulative basis, water intake was reduced during the 24–36 h interval (Fig. 2(bottom)). Based on these data, we concluded that NPAF was associated with short-term reduced food intake in rats. We then designed a more comprehensive food and water intake experiment to better understand the dose-response and duration of this effect. Eight rats were assigned to receive 0, 4, 8, or 16 nmol NPAF as part of a Latin Square design, such that each rat received each treatment. Each replicate of the Latin Square was conducted 5 d apart, a time span based on our preliminary trial. At the end of the fourth replicate, a secondary angiotensin II test was performed to reconfirm proper cannula placement and function. Data were analyzed similarly as in the preliminary trial and Duncan’s was used as a post hoc test to separate means. To determine the dose response relationship at each time, treatment effects were also partitioned into linear and quadratic contrasts. As was demonstrated in the pre-
liminary trial, NPAF was associated with reduced cumulative food intake 3 h post injection (Fig. 3(top)). Groups of rats that received 8 nmol NPAF had reduced food intake up to 2 h post injection, while those treated with 16 nmol had food intake reduction to 3 h post injection. By 4 h post injection NPAF-induced food intake reduction had subsided. However, when data were analyzed on a non-cumulative basis, food intake was reduced only at 0.5 h post injection (Fig. 1(bottom)) in rats that received 8 and 16 nmol NPAF. Because noncumulative food intake was not affected after 0.5 h, we interpret this as the potential perception of satiety associated with NPAF sustained only 0.5 h. Thus, we hypothesize that the cumulative 3 h response in Fig. 3 (top) was an artifact of what had occurred prior to 0.5 h post injection. That food intake was not increased in any group of rats treated with NPAF at any observation time may be interpreted as there was no compensatory food intake following subsidence of NPAF-induced food intake reduction. This thesis is further supported by the preliminary study, where food intake was not increased in NPAF-treated rats for at least 84 h following NPAFinduced food intake reduction cumulatively or noncumulatively (Fig. 1). Water intake was not affected (Fig. 4). NPAF is closely related to neuropeptide FF (NPFF), both of which originate from the same gene through alternative splicing [9,18], and bind central NPFF1 and NPFF2 receptors in the hypothalamus [19]. Although the two peptides share some sequence homology, their effects on food intake differ: NPFF was associated with an anorexic effect much longer in duration than NPAF (8 h post injection) at a lower dose (10 nmol), as well as a dipsogenic effect from 0.5 to 8 h post injection [7]. The differential responses of NPAF and NPFF in this species may be partly explained by their receptor affini-
B.A. Newmyer, M.A. Cline / Behavioural Brain Research 219 (2011) 351–353
353
ties; NPFF specifically binds both NPPF1 and NPFF2 receptors while NPAF is associated most with NPFF2 [19]. Further investigation on the specific cellular pathways of these two peptides may clarify this. NPAF appears to cause similar appetite-related effects across different species; NPAF appears to be a short-acting regulator of satiety in both rats and chicks, and the threshold for NPAF-induced reduction in food intake in both models falls somewhere between 4 and 8 nmol [8]. The duration of this effect is also similar between these models; in both cases reduced food intake was evident at 0.5 h post injection and sustained through 3 h post injection when analyzed cumulatively. Thus, the central mechanisms associated with NPAF-induced reduction in food intake may have undergone little modification during divergent evolution of birds and mammals. That an anorexic model hyper-responds while an obese model shows latency to response to NPAF [20], and that the appetiterelated effects of NPAF are conserved between rats and chicks, supports that the NPAF system may be a logical target for the pharmacological reversal of eating disorders. Acknowledgement The authors would like to thank Bryan Pittman for his contributions to this project, which made it possible. References
Fig. 3. Cumulative (top) and noncumulative (bottom) food intake after central injection of NPAF to rats measured for 24 h post injection. Values are the mean ± SE; value above each SE is that group’s food intake expressed as a percentage of the vehicle group within that time; bars with different superscripts are different from each other within a time point (P < 0.05).
Fig. 4. Cumulative (top) and noncumulative (bottom) water intake after central injection of NPAF to rats measured for 24 h post injection. Values are the mean ± SE; value above each SE is that group’s food intake expressed as a percentage of the vehicle group within that time; bars with different superscripts are different from each other within a time point (P < 0.05).
[1] Hill SR, Orchard I. In vitro analysis of the digestive enzymes amylase and alpha-glucosidase in the midguts of Locusta migratoria L. in response to the myosuppressin, SchistoFLRFamide. J Insect Physiol 2005;51:1–9. [2] Mackie GO, Marx RM, Meech RW. Central circuitry in the jellyfish Aglantha digitale. IV. Pathways coordinating feeding behaviour. J Exp Biol 2003;206:2487–505. [3] de Bono M, Bargmann C. Natural variation in a neuropeptide Y receptor homolog modifies social behavior and food response in C. elegans. Cell 1998;94:679–89. [4] Tachibana T, Saito S, Tomonaga S, Takagi T, Saito E, Nakanishi T, et al. Effect of central administration of prolactin-releasing peptide on feeding in chicks. Physiol Behav 2004;80:713–9. [5] Newmyer BA, Cline MA. Neuropeptide SF is associated with reduced food intake in chicks. Behav Brain Res 2009;205:311–4. [6] Murase T, Arima H, Kondo K, Oiso Y. Neuropeptide FF reduces food intake in rats. Peptides 1996;17:353–4. [7] Sunter D, Hewson AK, Lynam S, Dickson SL. Intracerebroventricular injection of neuropeptide FF, an opioid modulating neuropeptide, acutely reduces food intake and stimulates water intake in rats. Neurosci Lett 2001;313:145–8. [8] Cline MA, Newmyer BA, Smith ML. The anorectic effect of neuropeptide AF is associated with satiety-related hypothalamic nuclei. J Neuroendocrinol 2009;21:595–601. [9] Yang HYT, Fratta W, Majane EA, Costa E. Isolation, sequencing, synthesis, and pharmacological characterization of two brain neuropeptides that modulate the action of morphine. Proc Natl Acad Sci USA 1985;82:7757–61. [10] Panula P, Aarnisalo AA, Wasowicz K. Neuropeptide FF, a mammalian neuropeptide with multiple functions. Prog Neurobiol 1996;48:461–87. [11] Kavaliers M. Inhibitory influences of mammalian FMRFamide (Phe-MetArg-Phe-amide)-related peptides on nociception and morphine- and stressinduced analgesia in mice. Neurosci Lett 1990;115:307–12. [12] Jászberényi M, Bagosi Z, Thurzó B, Földesi I, Szabó G, Telegdy G. Endocrine, behavioral and autonomic effects of neuropeptide AF. Horm Behav 2009;56:24–34. [13] Kamara AK, Kamara KS, Castonguay TW. Effect of ACTH on weight gain, food intake, and plasma glucose concentrations in Sprague-Dawley rats. Appetite 1992;19:191. [14] Bligh ME, Douglass LW, Castonguay TW. Corticosterone modulation of dietary selection patterns. Physiol Behav 1993;53:975–82. [15] Schwartz MW, Woods SC, Porte Jr D, Seeley RJ, Baskin DG. Central nervous system control of food intake. Nature 2000;404:661–71. [16] Paxinos G, Watson C. The Rat Brain in Stereotaxic Coordinates. Elsevier; 2007. [17] Samson WK, Bagley SL, Ferguson AV, White MM. Orexin receptor subtype activation and locomotor behavior in the rat. Acta Physiol 2010;198:313–24. [18] Perry SJ, Hang EY, Cronk D, Bagust J, Sharma R, Walker RJ, et al. A human gene encoding morphine-modulating peptides related to NPFF and FMRFamide. FEBS 1997;409:426–30. [19] Bonini JA, Jones KA, Adham N, Forray C, Artymyshyn R, Durkin MM, et al. Identification and characterization of two G protein-coupled receptors for Neuropeptide FF. J Biol Chem 2000;275:39324–31. [20] Newmyer BA, Siegel PB, Cline MA. Neuropeptide AF differentially affects reduced food intake in lines of chickens selected for high or low body weight. J Neuroendocrinol 2010;22:593–8.
Contents lists available at ScienceDirect
Behavioural Brain Research journal homepage: www.elsevier.com/locate/bbr
Short communication
Neuropeptide AF is associated with short-term reduced food intake in rats Brandon A. Newmyer, Mark A. Cline ∗ Department of Biology, Radford University, Radford, VA, USA
a r t i c l e
i n f o
Article history: Received 13 September 2010 Received in revised form 26 November 2010 Accepted 5 December 2010 Available online 13 December 2010
a b s t r a c t Neuropeptide AF (NPAF), a member of the RFamide family, was centrally administered to rats to determine its effect on food intake. Rats responded with a linear dose-dependent reduction in cumulative food intake up to 3 h post injection, but when analyzed on a noncumulative basis, food intake was only significantly reduced at 0.5 h. To our knowledge, this is the first report of NPAF-induced reduction in food intake in a mammalian model. © 2010 Elsevier B.V. All rights reserved.
Keywords: Rat Food intake Neuropeptide AF NPAF
One group of neuropeptides receiving increased attention for roles in appetite regulation are the RFamides, which share structural similarity by containing an arginine (R) and amidated phenylalanine (F) at their carboxy-terminal ends. Members of this group affect food intake in species ranging from invertebrates such as locusts [1], jellyfish [2], and nematodes [3], to higher vertebrates, including chickens [4,5] and rats [6,7]. That the RFamides arose early in evolution and affect appetite in a broad range of species may imply that they have primary roles in appetite regulation. One member of this group is neuropeptide AF (NPAF), which we demonstrated is associated with satiety in chicks [8]. First isolated from bovine brain extracts by Yang et al. [9], NPAF has been studied primarily for its antiopioid action [10]. Kavaliers [11] showed the antinociceptive effects associated with centrally injected of NPAF in mice; NPAF-treated mice had increased magnitudes and durations of morphine- and stress-induced analgesia. NPAF’s effects on other signaling systems have also been investigated. Recently, Jászberényi et al. [12] demonstrated that centrally administered NPAF stimulates the release of ACTH, corticosterone, and dopamine. Because these signals are associated with appetite [13–15], and that we demonstrated that NPAF reduces food intake in chicks [8], we hypothesized that NPAF would also be associated with reduced food intake in a mammalian model. Thus, to evaluate this hypothesis we centrally administered NPAF to rats and determined its effects on food and water intake for up to 84 h post injection.
∗ Corresponding author. Tel.: +1 540 831 6431; fax: +1 540 831 5129. E-mail address: [email protected] (M.A. Cline). 0166-4328/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2010.12.001
Male Sprague-Dawley rats (35–49 g) were purchased from Harlan Laboratories (Indianapolis, IN) and housed individually in 24 cm × 18 cm × 18 cm hanging stainless steel cages under controlled conditions (24 ± 2 ◦ C, 50 ± 5% relative humidity and lights on 06:00–18:00) with free access to Teklad 2018C (18% crude protein, 5% crude fat, Harlan Laboratories) meal diet and tap water. Feeders were equipped with devices to reduce spillage and water bottles with anti-drip sippers. All experimental procedures were performed according to the National Research Council Publication, Guide for Care and Use of Laboratory Animals, and were approved by the Radford University Institutional Animal Care and Use Committee. Upon reaching 230–245 g, rats were anesthetized with isofluorane and a stainless-steel guide cannula (11 mm, 22 gauge) was stereotaxically implanted into the left lateral cerebroventricle (coordinates relative to bregma: X: −1.5, Y: −0.8, Z: −3.6, based on anatomy described in [16]) and immobilized using dental cement and stainless steel surgical screws. Buprenorphine (0.05 mg/kg body wt, subcutaneously) was administered post-anesthesia for analgesia. Fluid was replaced by subcutaneous injection of 0.9% NaCl to balance fluid loss at 3:1. A dummy injector remained in the guide cannula when it was not in use. Rats, after returning to pre-surgery weight, were administered 50 pmol angiotensin II and monitored for a dipsogenic response to confirm correct placement of cannula. Rats that did not drink at least 5 mL of water within 30 min of angiotensin II injection were removed from the study [17]. A preliminary trial was conducted to ascertain the duration and magnitude of the potential reduction in food intake following central NPAF. Nine 24 h-fasted rats on a cross-over design were randomly assigned to receive either 0 (0.9% NaCl, vehicle only)
352
B.A. Newmyer, M.A. Cline / Behavioural Brain Research 219 (2011) 351–353
Fig. 1. Cumulative (top) and noncumulative (bottom) food intake after central injection of NPAF to rats measured for 84 h post injection. Values are the mean ± SE; value above each SE is that group’s food intake expressed as a percentage of the vehicle group within that time; * denotes difference within a time point (P < 0.05).
Fig. 2. Cumulative (top) and noncumulative (bottom) water intake after central injection of NPAF to rats measured for 84 h post injection. Values are the mean ± SE; value above each SE is that group’s food intake expressed as a percentage of the vehicle group within that time; * denotes difference within a time point (P < 0.05).
or 16 nmol NPAF for a total injection volume of 4 L. Rats were injected 7 d post surgery. The two replicates were performed 5 d apart. Food and water intake were measured to 84 h post injection. Data were analyzed using analysis of variance (ANOVA) at each time point, with the model food intake as a function of treatment, replicate, and treatment by replicate interaction. Replicate was not significant and was thus removed from the secondary ANOVA analysis. Rats that received NPAF reduced their food intake at 3 h post injection when data were analyzed on a both a cumulative (Fig. 1(top)) and non-cumulative (Fig. 1(bottom)) basis, but this effect was not significant at any other time point. Water intake was also concurrently measured during this preliminary trial and was not significantly affected at any observation time when analyzed on a cumulative basis (Fig. 2(top)). However, when analyzed on a noncumulative basis, water intake was reduced during the 24–36 h interval (Fig. 2(bottom)). Based on these data, we concluded that NPAF was associated with short-term reduced food intake in rats. We then designed a more comprehensive food and water intake experiment to better understand the dose-response and duration of this effect. Eight rats were assigned to receive 0, 4, 8, or 16 nmol NPAF as part of a Latin Square design, such that each rat received each treatment. Each replicate of the Latin Square was conducted 5 d apart, a time span based on our preliminary trial. At the end of the fourth replicate, a secondary angiotensin II test was performed to reconfirm proper cannula placement and function. Data were analyzed similarly as in the preliminary trial and Duncan’s was used as a post hoc test to separate means. To determine the dose response relationship at each time, treatment effects were also partitioned into linear and quadratic contrasts. As was demonstrated in the pre-
liminary trial, NPAF was associated with reduced cumulative food intake 3 h post injection (Fig. 3(top)). Groups of rats that received 8 nmol NPAF had reduced food intake up to 2 h post injection, while those treated with 16 nmol had food intake reduction to 3 h post injection. By 4 h post injection NPAF-induced food intake reduction had subsided. However, when data were analyzed on a non-cumulative basis, food intake was reduced only at 0.5 h post injection (Fig. 1(bottom)) in rats that received 8 and 16 nmol NPAF. Because noncumulative food intake was not affected after 0.5 h, we interpret this as the potential perception of satiety associated with NPAF sustained only 0.5 h. Thus, we hypothesize that the cumulative 3 h response in Fig. 3 (top) was an artifact of what had occurred prior to 0.5 h post injection. That food intake was not increased in any group of rats treated with NPAF at any observation time may be interpreted as there was no compensatory food intake following subsidence of NPAF-induced food intake reduction. This thesis is further supported by the preliminary study, where food intake was not increased in NPAF-treated rats for at least 84 h following NPAFinduced food intake reduction cumulatively or noncumulatively (Fig. 1). Water intake was not affected (Fig. 4). NPAF is closely related to neuropeptide FF (NPFF), both of which originate from the same gene through alternative splicing [9,18], and bind central NPFF1 and NPFF2 receptors in the hypothalamus [19]. Although the two peptides share some sequence homology, their effects on food intake differ: NPFF was associated with an anorexic effect much longer in duration than NPAF (8 h post injection) at a lower dose (10 nmol), as well as a dipsogenic effect from 0.5 to 8 h post injection [7]. The differential responses of NPAF and NPFF in this species may be partly explained by their receptor affini-
B.A. Newmyer, M.A. Cline / Behavioural Brain Research 219 (2011) 351–353
353
ties; NPFF specifically binds both NPPF1 and NPFF2 receptors while NPAF is associated most with NPFF2 [19]. Further investigation on the specific cellular pathways of these two peptides may clarify this. NPAF appears to cause similar appetite-related effects across different species; NPAF appears to be a short-acting regulator of satiety in both rats and chicks, and the threshold for NPAF-induced reduction in food intake in both models falls somewhere between 4 and 8 nmol [8]. The duration of this effect is also similar between these models; in both cases reduced food intake was evident at 0.5 h post injection and sustained through 3 h post injection when analyzed cumulatively. Thus, the central mechanisms associated with NPAF-induced reduction in food intake may have undergone little modification during divergent evolution of birds and mammals. That an anorexic model hyper-responds while an obese model shows latency to response to NPAF [20], and that the appetiterelated effects of NPAF are conserved between rats and chicks, supports that the NPAF system may be a logical target for the pharmacological reversal of eating disorders. Acknowledgement The authors would like to thank Bryan Pittman for his contributions to this project, which made it possible. References
Fig. 3. Cumulative (top) and noncumulative (bottom) food intake after central injection of NPAF to rats measured for 24 h post injection. Values are the mean ± SE; value above each SE is that group’s food intake expressed as a percentage of the vehicle group within that time; bars with different superscripts are different from each other within a time point (P < 0.05).
Fig. 4. Cumulative (top) and noncumulative (bottom) water intake after central injection of NPAF to rats measured for 24 h post injection. Values are the mean ± SE; value above each SE is that group’s food intake expressed as a percentage of the vehicle group within that time; bars with different superscripts are different from each other within a time point (P < 0.05).
[1] Hill SR, Orchard I. In vitro analysis of the digestive enzymes amylase and alpha-glucosidase in the midguts of Locusta migratoria L. in response to the myosuppressin, SchistoFLRFamide. J Insect Physiol 2005;51:1–9. [2] Mackie GO, Marx RM, Meech RW. Central circuitry in the jellyfish Aglantha digitale. IV. Pathways coordinating feeding behaviour. J Exp Biol 2003;206:2487–505. [3] de Bono M, Bargmann C. Natural variation in a neuropeptide Y receptor homolog modifies social behavior and food response in C. elegans. Cell 1998;94:679–89. [4] Tachibana T, Saito S, Tomonaga S, Takagi T, Saito E, Nakanishi T, et al. Effect of central administration of prolactin-releasing peptide on feeding in chicks. Physiol Behav 2004;80:713–9. [5] Newmyer BA, Cline MA. Neuropeptide SF is associated with reduced food intake in chicks. Behav Brain Res 2009;205:311–4. [6] Murase T, Arima H, Kondo K, Oiso Y. Neuropeptide FF reduces food intake in rats. Peptides 1996;17:353–4. [7] Sunter D, Hewson AK, Lynam S, Dickson SL. Intracerebroventricular injection of neuropeptide FF, an opioid modulating neuropeptide, acutely reduces food intake and stimulates water intake in rats. Neurosci Lett 2001;313:145–8. [8] Cline MA, Newmyer BA, Smith ML. The anorectic effect of neuropeptide AF is associated with satiety-related hypothalamic nuclei. J Neuroendocrinol 2009;21:595–601. [9] Yang HYT, Fratta W, Majane EA, Costa E. Isolation, sequencing, synthesis, and pharmacological characterization of two brain neuropeptides that modulate the action of morphine. Proc Natl Acad Sci USA 1985;82:7757–61. [10] Panula P, Aarnisalo AA, Wasowicz K. Neuropeptide FF, a mammalian neuropeptide with multiple functions. Prog Neurobiol 1996;48:461–87. [11] Kavaliers M. Inhibitory influences of mammalian FMRFamide (Phe-MetArg-Phe-amide)-related peptides on nociception and morphine- and stressinduced analgesia in mice. Neurosci Lett 1990;115:307–12. [12] Jászberényi M, Bagosi Z, Thurzó B, Földesi I, Szabó G, Telegdy G. Endocrine, behavioral and autonomic effects of neuropeptide AF. Horm Behav 2009;56:24–34. [13] Kamara AK, Kamara KS, Castonguay TW. Effect of ACTH on weight gain, food intake, and plasma glucose concentrations in Sprague-Dawley rats. Appetite 1992;19:191. [14] Bligh ME, Douglass LW, Castonguay TW. Corticosterone modulation of dietary selection patterns. Physiol Behav 1993;53:975–82. [15] Schwartz MW, Woods SC, Porte Jr D, Seeley RJ, Baskin DG. Central nervous system control of food intake. Nature 2000;404:661–71. [16] Paxinos G, Watson C. The Rat Brain in Stereotaxic Coordinates. Elsevier; 2007. [17] Samson WK, Bagley SL, Ferguson AV, White MM. Orexin receptor subtype activation and locomotor behavior in the rat. Acta Physiol 2010;198:313–24. [18] Perry SJ, Hang EY, Cronk D, Bagust J, Sharma R, Walker RJ, et al. A human gene encoding morphine-modulating peptides related to NPFF and FMRFamide. FEBS 1997;409:426–30. [19] Bonini JA, Jones KA, Adham N, Forray C, Artymyshyn R, Durkin MM, et al. Identification and characterization of two G protein-coupled receptors for Neuropeptide FF. J Biol Chem 2000;275:39324–31. [20] Newmyer BA, Siegel PB, Cline MA. Neuropeptide AF differentially affects reduced food intake in lines of chickens selected for high or low body weight. J Neuroendocrinol 2010;22:593–8.