- Email: [email protected]
Feeding response to central orexins
Brain Research 821 Ž1999. 535–538
Short communication
Feeding response to central orexins Donald C. Sweet a
a,b
, Allen S. Levine
a,b
, Charles J. Billington
b,c
, Catherine M. Kotz
a,b,)
UniÕersity of Minnesota, Department of Food Science and Nutrition, St. Paul, MN 55108, USA b Veterans Affairs Medical Center, Research SerÕice, Minneapolis, MN 55417, USA c UniÕersity of Minnesota, School of Medicine, Minneapolis, MN 55455, USA Accepted 5 January 1999
Abstract Orexin A and orexin B were microinjected into the perifornical hypothalamus ŽPFH., lateral hypothalamus ŽLH., hypothalamic paraventricular nucleus ŽPVN., and ventral tegmental area ŽVTA. of male Sprague–Dawley rats. Orexin B Ž15 nmol. was also injected into the lateral cerebral ventricle Ži.c.v... Orexin A ŽG 500 pmol. stimulated feeding in the PFH and LH, but not in the VTA or PVN. Orexin B stimulated feeding only when injected i.c.v. q 1999 Published by Elsevier Science B.V. All rights reserved. Keywords: Orexin; Hypocretin; Hypothalamic area, lateral; Hypothalamic area, perifornical; Feeding behavior; Hypothalamus
The orexins are a recently identified class of neuropeptides that were first described by de Lecea et al., who referred to them as ‘hypocretins’ Žfor ‘hypothalamic incretins’. w6x. Orexin A and orexin B are 33- and 28-amino acid peptides, respectively, sharing 46% identity. Both peptides are coded by the same gene. The presence of orexin mRNA, prepro-orexin, and orexin A peptide has been demonstrated in the lateral hypothalamus ŽLH. and perifornical hypothalamus ŽPFH. w13,14x. Because the LH plays a predominant role in feeding behavior w3x, Sakurai et al. w14x tested whether these same compounds influence feeding. In that study, i.c.v. orexin A and orexin B elicited a dose-dependent feeding response, and fasted rats showed increased expression of orexin mRNA in the thalamicrhypothalamic portion of the brain w14x. However, no information regarding a site-specific response to the orexins was reported. Through a series of experiments, we sought to identify orexin-sensitive siteŽs. by injecting orexin A and orexin B into five different brain locations and measuring feeding response. Because orexin mRNA, prepro-orexin, and orexin A peptide are abundant in the LH and PFH w13,14x, both of those sites were tested. The ventral tegmental area ŽVTA. was tested because the LH has axonal projections to the
) Corresponding author. Veterans Affairs Medical Center, Research 151, One Veterans Drive, Minneapolis, MN 55417. Fax: q1-612-7252093; E-mail: [email protected]
VTA w8x, and the VTA has been implicated in food intake regulation w2,11x. The hypothalamic paraventricular nucleus ŽPVN. was tested because of its key role in food intake regulation and energy homeostasis w4x. Different rats were used to study each site. In all experiments, male Sprague–Dawley rats ŽHarlan, Madison, WI; Charles River, Portage, MI. were housed in individual hanging wire cages and allowed ad lib tap water and chow pellets ŽTeklad 8604. from a wire hopper. Room temperature was maintained at 21–228C with a 24 h photoperiod Ž12 h light cycle began at 0700.. Rats were anesthetized with either sodium pentobarbital Ž60 mgrkg i.p.. or a combination of ketamine and xylazine Ž100 mgrkg i.p. and 10 mgrkg i.p., respectively. and mounted onto a stereotaxic apparatus ŽKopf, Tujunga, CA.. Stainless steel guide cannulae ŽPlastics One, Roanoke, VA. were implanted at the stereotaxic coordinates indicated in Table 1, which were determined using the brain atlas of Paxinos and Watson w12x. Rats were injected with flunixin meglumide Ž2.5 mgrkg, s.c.. for post-surgical analgesia, fitted with dummy cannulae, and allowed to recover for at least one week prior to any experimentation. Correct cannula placement was determined in experiments 1–4a by cryosectioning brains and staining for histological verification. Placement was considered acceptable if within 0.2 mm of the target site. In experiment 4b, PVN-cannulated rats were injected with 0.5 mgr0.5 ml NPY, and correct placement was assumed if rats consumed more than 2.5 g chow within 2 h. In experiment 5,
0006-8993r99r$ - see front matter q 1999 Published by Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 1 1 3 6 - 1
D.C. Sweet et al.r Brain Research 821 (1999) 535–538
536 Table 1 Guide cannula stereotaxic coordinates Site
Lateral Žmm from midline.
Posterior Žmm from bregma.
Ventral Žmm from skull surface.
PFH LH VTA PVN i.c.v.
1.2 2.0 0.8 0.5 1.5
2.8 2.1 5.0 1.9 1.0
7.6 a 7.2 a 7.2 a 7.3 a 3.5 b
a b
Injector tip protruded an additional 1.0 mm. Injector tip protruded an additional 0.5 mm.
i.c.v.-cannulated rats were injected with 5 mgr5 ml angiotensin II, and correct placement was assumed if rats consumed more than 5 g water within 0.5 h. Rats with misplaced cannulae or failed bioassays were excluded from all experiments. All procedures involving animals were approved by the Subcommittee on Animal Studies, Veterans Affairs Medical Center, Minneapolis, MN. Orexin A and orexin B ŽPhoenix Pharmaceuticals, Mountain View, CA. were dissolved in 0.9% saline immediately prior to first use, and, when not in use, solutions were stored at 48C for a maximum of three weeks. For experiments 1–4, 300 nl orexin A, orexin B, and saline were injected at an approximate rate of 20 nlrs. For experiment 5, 5 ml orexin B and saline were injected i.c.v. Peptide and saline delivery were verified by movement of an airspace in the injection tubing. Injectors were left in place for 15 s after each injection to allow diffusion of peptide from the injector tip. Food and hoppers were weighed at baseline. Food, hoppers, and spillage were then weighed at 2 and 4 h following all injections. Food intake was measured as weight difference from baseline less spillage. Rats were allowed a 48 h washout period between injections. All injections were given between 1400 and 1500 h. Food intake in experiment 1 was analyzed separately at 0–2 h and 2–4 h, using a separate single factor ANOVA for each peptide. Post hoc analysis for experiment 1 was performed using Fisher’s PLSD. Food intake in experiments 2–5 was analyzed using a separate repeated measures ANOVA for each peptide, with time and treatment serving as within-subjects factors. Experiment 1: orexins in the PFH. Four separate feeding studies were conducted in the PFH. For experiment 1a, 17 PFH-cannulated rats were divided into five groups and injected with either 5 pmol orexin A Ž n s 2., 50 pmol orexin A Ž n s 4., 5 pmol orexin B Ž n s 3., 50 pmol orexin B Ž n s 4., or saline Ž n s 4.. For experiment 1b, nine PFH-cannulated rats were injected with 250 pmol orexin A Ž n s 3., 250 pmol orexin B Ž n s 3., or saline Ž n s 3.. In experiment 1c, eight PFH-cannulated rats were injected with either 500 pmol orexin A Ž n s 3. or saline Ž n s 5.. In experiment 1d, nine PFH-cannulated rats were injected with either 500 pmol orexin B Ž n s 3. or saline Ž n s 6.. A
single-factor ANOVA of food intake by saline-injected rats was performed to test for a day effect Ž F4,13 s 1.936, p s 0.164., and data from all experiments were combined and analyzed as described above. During the 0–2 h period, a main effect of orexin A in the PFH was observed Ž F4,26 s 5.152, p s 0.003; Fig. 1.. Injection of 500 pmol orexin A in the PFH resulted in a significant increase in food intake Ž p - 0.001; Fig. 1.. During the 2–4 h period, there was a main effect of orexin A Ž F4,26 s 3.173, p s 0.030.. A dose of 500 pmol orexin A significantly increased feeding between 2–4 h Ž p s 0.004, data not shown.. No effect was observed after injection of 5, 50, or 250 pmol orexin A. Injection of orexin B in the PFH did not increase feeding at any dose. Experiment 2: orexins in the LH. Twelve LH-cannulated rats were injected with 50, 250, 500, and 1,000 pmol orexin A and orexin B in a repeated measures design. For control, all rats were injected with saline on three separate days and food intake was measured. The average of the three measurements served as a baseline for each rat. A significant and dose-dependent effect of treatment was observed when orexin A was injected into the LH Ž F4,9 s 4.525, p s 0.005, Fig. 2.. Rats injected with 500 and 1000 pmol orexin A ate significantly more at 0–2 h than their baseline levels Ž p s 0.009, p - 0.001, respectively; Fig. 2.. In the 2–4 h time period, rats injected with orexin A ate less Žnon-significant. than their baseline levels, resulting in a similar cumulative food intake by 4 h Ždata not shown.. Orexin B had no effect on feeding. Experiment 3: orexins in the VTA. Experiment 3a was a repeated measures design in which 12 VTA-cannulated
Fig. 1. Orexin A in the PFH. Bars represent 2 h food intake of rats injected with orexin A in the PFH. Asterisk Ž). indicates significant difference from saline controls Ž p- 0.001.. Rats injected with 5 and 250 pmol did not eat, therefore no bar is depicted in the graph.
D.C. Sweet et al.r Brain Research 821 (1999) 535–538
Fig. 2. Orexin A in the LH. Bars represent 2 h food intake of rats injected with orexin A in the LH. Asterisk Ž). indicates significant difference from saline controls Ž p- 0.01..
rats were injected with 500 pmol orexin A and saline. For experiment 3b, a separate set of 11 VTA-cannulated rats were injected with 50, 250, and 500 pmol of orexin B and saline in a repeated measures design. Neither orexin A nor orexin B significantly affected feeding when injected into the VTA. Experiment 4: orexins in the PVN. Experiment 4a was a repeated measures design in which eight PVN-cannulated rats were injected with 500 pmol orexin A and saline. For experiment 4b, a separate set of eight PVN-cannulated rats were injected with 50, 250, and 500 pmol orexin B and saline in a repeated measures design. Neither orexin A nor orexin B significantly affected feeding when injected into the PVN. Experiment 5: orexin B i.c.Õ. Experiment 5 was a repeated measures design in which eight i.c.v.-cannulated rats were injected with 15 nmol orexin B and saline into the right lateral ventricle. No main effect of treatment or time was observed; however, there was a significant interaction of treatment and time Ž F1,7 s 5.970, p s 0.045; Fig. 3.. Food intake during the 0–2 h period was significantly increased when rats were injected with orexin B Ž p s 0.024; Fig. 3.. There was no significant effect on feeding during the 2–4 h period. This feeding response and the lack of such a response following orexin B injection into the PFH, LH, VTA, and PVN suggest the presence of other unidentified siteŽs. of action for orexin B in the brain. More work will need to be done to determine these siteŽs.. To compare directly the feeding effects of orexin A and orexin B in all brain nuclei tested, we plotted feeding
537
Fig. 3. Orexin B i.c.v. Bars represent 2 h food intake of rats injected with orexin B i.c.v. Asterisk Ž). indicates significant difference from saline controls Ž ps 0.024..
above baseline for both orexin A and orexin B Ž500 pmol. at 2 h in each site ŽFig. 4.. Depicted in this manner, the data clearly show that the PFH and LH are feeding-sensitive sites for orexin A. During the first 2 h of the 4 h measurement period, orexin A significantly stimulated feeding in the PFH and the LH ŽFigs. 1, 2 and 4.. During the 2–4 h period, rats injected with 500 pmol orexin A in the PFH ate significantly more than those injected with
Fig. 4. Response to orexins in all tested brain nuclei. Bars represent food intake Ž0–2 h. of rats injected with either 500 pmol orexin A Žnarrow hatches. or 500 pmol orexin B Žwide hatches. as difference from controls. Asterisk Ž). indicates significant difference from saline controls Ž p0.01..
538
D.C. Sweet et al.r Brain Research 821 (1999) 535–538
saline ŽFig. 1.. However, the low numbers of subjects and non-counterbalanced design of that experiment warrant further studies to verify this sustained effect. Administration of orexin A in the LH stimulates meal initiation but does not affect total consumption over 4 h. This raises the possibility that orexin A functions via different pathways than neuropeptide Y ŽNPY., which stimulates persistent hyperphagia over 4 h w5,10x. Neither peptide elicited a feeding response when injected into the PVN. This is in contrast to recent work by Dube et al. w7x, who observed increased feeding 2 h after injecting 1 nmol orexin A into the PVN of adult male rats. A possible explanation for this observed difference is that the dose necessary to elicit a feeding response in the PVN lies somewhere between 500 pmol and 1 nmol. The function of orexin A in the PFH and LH are consistent with the roles of these areas in feeding behavior. NPY acts in the PFH to stimulate feeding w9,15,16x, and lesions of the LH induce anorexia w1x. Prior to the discovery of the orexins, no satisfactory mechanism for LH feeding stimulatory effects had been advanced. Clearly, the means by which orexin A in the LH and PFH stimulates feeding and the potential for interaction with other components of the appetiterenergy balance regulatory network must still be defined.
w4x
w5x
w6x
w7x
w8x
w9x
w10x w11x
w12x w13x
Acknowledgements w14x
This work was supported by funds from the Minnesota Obesity Center ŽP30 DK-50456..
References w15x w1x B.K. Anand, J.R. Brobeck, Hypothalamic control of food intake in rats and cats, Yale J. Biol. Med. 24 Ž1951. 123–146. w2x A. Badiani, P. Leone, M.B. Noel, J. Stewart, Ventral tegmental area opioid mechanisms and modulation of ingestive behavior, Brain Res. 670 Ž1995. 264–276. w3x L.L. Bernardis, L.L. Bellinger, The lateral hypothalamic area revis-
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ited: ingestive behavior, Neurosci. Biobehav. Rev. 20 Ž1996. 189– 287. C.J. Billington, A.S. Levine, Hypothalamic neuropeptide Y regulation of feeding and energy metabolism, Curr. Opin. Neurobiol. 2 Ž1992. 847–851. J.T. Clark, P.S. Kalra, W.R. Crowley, S.P. Kalra, Neuropeptide Y and human pancreatic polypeptide stimulates feeding behavior in rats, Endocrinology 115 Ž1984. 427–429. L. de Lecea, T.S. Kilduff, C. Peyron, X. Gao, P.E. Foye, P.E. Danielson, C. Fukuhara, E.L. Battenberg, V.T. Gautvik, F.S.N. Bartlett, W.N. Frankel, A.N. van den Pol, F.E. Bloom, K.M. Gautvik, J.G. Sutcliffe, The hypocretins: hypothalamus-specific peptides with neuroexcitatory activity, Proc. Natl. Acad. Sci. USA 95 Ž1998. 322–327. M.G. Dube, S.P. Kalra, P.S. Kalra, Food intake elicited by central administration of orexins: identification of hypothalamic sites of action, Soc. Neurosci. 24 Ž1998. 448, Žabstract.. H.J. Groenewegen, H.W. Berendse, Connections of the subthalamic nucleus with ventral striatopallidal parts of the basal ganglia in the rat, J. Comp. Neurol. 294 Ž1990. 607–622. F.B. Jolicoeur, S.M. Bouali, A. Fournier, S. St-Pierre, Mapping of hypothalamic sites involved in the effects of NPY on body temperature and food intake, Brain Res. Bull. 36 Ž1995. 125–129. A.S. Levine, J.E. Morley, Neuropeptide Y: a potent inducer of consummatory behavior in rats, Peptides 5 Ž1984. 1025–1029. M.B. Noel, R.A. Wise, Ventral tegmental injections of morphine but not U-50,488H enhance feeding in food-deprived rats, Brain Res. 632 Ž1993. 68–73. G. Paxinos, C. Watson, The Rat Brain in Stereotaxic Coordinates, San Diego, 1986. C. Peyron, D.K. Tighe, A.N. van den Pol, L. de Lecea, H.C. Heller, J.G. Sutcliffe, T.S. Kilduff, Neurons containing hypocretin Žorexin. project to multiple neuronal systems, J. Neurosci. 18 Ž1998. 9996– 10015. T. Sakurai, A. Amemiya, M. Ishii, I. Matsuzaki, R.M. Chemelli, H. Tanaka, S.C. Williams, J.A. Richardson, G.P. Kozlowski, S. Wilson, J.R. Arch, R.E. Buckingham, A.C. Haynes, S.A. Carr, R.S. Annan, D.E. McNulty, W.S. Liu, J.A. Terrett, N.A. Elshourbagy, D.J. Bergsma, M. Yanagisawa, Orexins and orexin receptors: a family of hypothalamic neuropeptides and G protein-coupled receptors that regulate feeding behavior, Cell 92 Ž1998. 573–585. B.G. Stanley, W. Magdalin, A. Seirafi, W.J. Thomas, S.F. Leibowitz, The perifornical area: the major focus of Ža. patchily distributed hypothalamic neuropeptide Y-sensitive feeding systemŽs., Brain Res. 604 Ž1993. 304–317. B.G. Stanley, W.J. Thomas, Feeding responses to perifornical hypothalamic injection of neuropeptide Y in relation to circadian rhythms of eating behavior, Peptides 14 Ž1993. 475–481.
Short communication
Feeding response to central orexins Donald C. Sweet a
a,b
, Allen S. Levine
a,b
, Charles J. Billington
b,c
, Catherine M. Kotz
a,b,)
UniÕersity of Minnesota, Department of Food Science and Nutrition, St. Paul, MN 55108, USA b Veterans Affairs Medical Center, Research SerÕice, Minneapolis, MN 55417, USA c UniÕersity of Minnesota, School of Medicine, Minneapolis, MN 55455, USA Accepted 5 January 1999
Abstract Orexin A and orexin B were microinjected into the perifornical hypothalamus ŽPFH., lateral hypothalamus ŽLH., hypothalamic paraventricular nucleus ŽPVN., and ventral tegmental area ŽVTA. of male Sprague–Dawley rats. Orexin B Ž15 nmol. was also injected into the lateral cerebral ventricle Ži.c.v... Orexin A ŽG 500 pmol. stimulated feeding in the PFH and LH, but not in the VTA or PVN. Orexin B stimulated feeding only when injected i.c.v. q 1999 Published by Elsevier Science B.V. All rights reserved. Keywords: Orexin; Hypocretin; Hypothalamic area, lateral; Hypothalamic area, perifornical; Feeding behavior; Hypothalamus
The orexins are a recently identified class of neuropeptides that were first described by de Lecea et al., who referred to them as ‘hypocretins’ Žfor ‘hypothalamic incretins’. w6x. Orexin A and orexin B are 33- and 28-amino acid peptides, respectively, sharing 46% identity. Both peptides are coded by the same gene. The presence of orexin mRNA, prepro-orexin, and orexin A peptide has been demonstrated in the lateral hypothalamus ŽLH. and perifornical hypothalamus ŽPFH. w13,14x. Because the LH plays a predominant role in feeding behavior w3x, Sakurai et al. w14x tested whether these same compounds influence feeding. In that study, i.c.v. orexin A and orexin B elicited a dose-dependent feeding response, and fasted rats showed increased expression of orexin mRNA in the thalamicrhypothalamic portion of the brain w14x. However, no information regarding a site-specific response to the orexins was reported. Through a series of experiments, we sought to identify orexin-sensitive siteŽs. by injecting orexin A and orexin B into five different brain locations and measuring feeding response. Because orexin mRNA, prepro-orexin, and orexin A peptide are abundant in the LH and PFH w13,14x, both of those sites were tested. The ventral tegmental area ŽVTA. was tested because the LH has axonal projections to the
) Corresponding author. Veterans Affairs Medical Center, Research 151, One Veterans Drive, Minneapolis, MN 55417. Fax: q1-612-7252093; E-mail: [email protected]
VTA w8x, and the VTA has been implicated in food intake regulation w2,11x. The hypothalamic paraventricular nucleus ŽPVN. was tested because of its key role in food intake regulation and energy homeostasis w4x. Different rats were used to study each site. In all experiments, male Sprague–Dawley rats ŽHarlan, Madison, WI; Charles River, Portage, MI. were housed in individual hanging wire cages and allowed ad lib tap water and chow pellets ŽTeklad 8604. from a wire hopper. Room temperature was maintained at 21–228C with a 24 h photoperiod Ž12 h light cycle began at 0700.. Rats were anesthetized with either sodium pentobarbital Ž60 mgrkg i.p.. or a combination of ketamine and xylazine Ž100 mgrkg i.p. and 10 mgrkg i.p., respectively. and mounted onto a stereotaxic apparatus ŽKopf, Tujunga, CA.. Stainless steel guide cannulae ŽPlastics One, Roanoke, VA. were implanted at the stereotaxic coordinates indicated in Table 1, which were determined using the brain atlas of Paxinos and Watson w12x. Rats were injected with flunixin meglumide Ž2.5 mgrkg, s.c.. for post-surgical analgesia, fitted with dummy cannulae, and allowed to recover for at least one week prior to any experimentation. Correct cannula placement was determined in experiments 1–4a by cryosectioning brains and staining for histological verification. Placement was considered acceptable if within 0.2 mm of the target site. In experiment 4b, PVN-cannulated rats were injected with 0.5 mgr0.5 ml NPY, and correct placement was assumed if rats consumed more than 2.5 g chow within 2 h. In experiment 5,
0006-8993r99r$ - see front matter q 1999 Published by Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 1 1 3 6 - 1
D.C. Sweet et al.r Brain Research 821 (1999) 535–538
536 Table 1 Guide cannula stereotaxic coordinates Site
Lateral Žmm from midline.
Posterior Žmm from bregma.
Ventral Žmm from skull surface.
PFH LH VTA PVN i.c.v.
1.2 2.0 0.8 0.5 1.5
2.8 2.1 5.0 1.9 1.0
7.6 a 7.2 a 7.2 a 7.3 a 3.5 b
a b
Injector tip protruded an additional 1.0 mm. Injector tip protruded an additional 0.5 mm.
i.c.v.-cannulated rats were injected with 5 mgr5 ml angiotensin II, and correct placement was assumed if rats consumed more than 5 g water within 0.5 h. Rats with misplaced cannulae or failed bioassays were excluded from all experiments. All procedures involving animals were approved by the Subcommittee on Animal Studies, Veterans Affairs Medical Center, Minneapolis, MN. Orexin A and orexin B ŽPhoenix Pharmaceuticals, Mountain View, CA. were dissolved in 0.9% saline immediately prior to first use, and, when not in use, solutions were stored at 48C for a maximum of three weeks. For experiments 1–4, 300 nl orexin A, orexin B, and saline were injected at an approximate rate of 20 nlrs. For experiment 5, 5 ml orexin B and saline were injected i.c.v. Peptide and saline delivery were verified by movement of an airspace in the injection tubing. Injectors were left in place for 15 s after each injection to allow diffusion of peptide from the injector tip. Food and hoppers were weighed at baseline. Food, hoppers, and spillage were then weighed at 2 and 4 h following all injections. Food intake was measured as weight difference from baseline less spillage. Rats were allowed a 48 h washout period between injections. All injections were given between 1400 and 1500 h. Food intake in experiment 1 was analyzed separately at 0–2 h and 2–4 h, using a separate single factor ANOVA for each peptide. Post hoc analysis for experiment 1 was performed using Fisher’s PLSD. Food intake in experiments 2–5 was analyzed using a separate repeated measures ANOVA for each peptide, with time and treatment serving as within-subjects factors. Experiment 1: orexins in the PFH. Four separate feeding studies were conducted in the PFH. For experiment 1a, 17 PFH-cannulated rats were divided into five groups and injected with either 5 pmol orexin A Ž n s 2., 50 pmol orexin A Ž n s 4., 5 pmol orexin B Ž n s 3., 50 pmol orexin B Ž n s 4., or saline Ž n s 4.. For experiment 1b, nine PFH-cannulated rats were injected with 250 pmol orexin A Ž n s 3., 250 pmol orexin B Ž n s 3., or saline Ž n s 3.. In experiment 1c, eight PFH-cannulated rats were injected with either 500 pmol orexin A Ž n s 3. or saline Ž n s 5.. In experiment 1d, nine PFH-cannulated rats were injected with either 500 pmol orexin B Ž n s 3. or saline Ž n s 6.. A
single-factor ANOVA of food intake by saline-injected rats was performed to test for a day effect Ž F4,13 s 1.936, p s 0.164., and data from all experiments were combined and analyzed as described above. During the 0–2 h period, a main effect of orexin A in the PFH was observed Ž F4,26 s 5.152, p s 0.003; Fig. 1.. Injection of 500 pmol orexin A in the PFH resulted in a significant increase in food intake Ž p - 0.001; Fig. 1.. During the 2–4 h period, there was a main effect of orexin A Ž F4,26 s 3.173, p s 0.030.. A dose of 500 pmol orexin A significantly increased feeding between 2–4 h Ž p s 0.004, data not shown.. No effect was observed after injection of 5, 50, or 250 pmol orexin A. Injection of orexin B in the PFH did not increase feeding at any dose. Experiment 2: orexins in the LH. Twelve LH-cannulated rats were injected with 50, 250, 500, and 1,000 pmol orexin A and orexin B in a repeated measures design. For control, all rats were injected with saline on three separate days and food intake was measured. The average of the three measurements served as a baseline for each rat. A significant and dose-dependent effect of treatment was observed when orexin A was injected into the LH Ž F4,9 s 4.525, p s 0.005, Fig. 2.. Rats injected with 500 and 1000 pmol orexin A ate significantly more at 0–2 h than their baseline levels Ž p s 0.009, p - 0.001, respectively; Fig. 2.. In the 2–4 h time period, rats injected with orexin A ate less Žnon-significant. than their baseline levels, resulting in a similar cumulative food intake by 4 h Ždata not shown.. Orexin B had no effect on feeding. Experiment 3: orexins in the VTA. Experiment 3a was a repeated measures design in which 12 VTA-cannulated
Fig. 1. Orexin A in the PFH. Bars represent 2 h food intake of rats injected with orexin A in the PFH. Asterisk Ž). indicates significant difference from saline controls Ž p- 0.001.. Rats injected with 5 and 250 pmol did not eat, therefore no bar is depicted in the graph.
D.C. Sweet et al.r Brain Research 821 (1999) 535–538
Fig. 2. Orexin A in the LH. Bars represent 2 h food intake of rats injected with orexin A in the LH. Asterisk Ž). indicates significant difference from saline controls Ž p- 0.01..
rats were injected with 500 pmol orexin A and saline. For experiment 3b, a separate set of 11 VTA-cannulated rats were injected with 50, 250, and 500 pmol of orexin B and saline in a repeated measures design. Neither orexin A nor orexin B significantly affected feeding when injected into the VTA. Experiment 4: orexins in the PVN. Experiment 4a was a repeated measures design in which eight PVN-cannulated rats were injected with 500 pmol orexin A and saline. For experiment 4b, a separate set of eight PVN-cannulated rats were injected with 50, 250, and 500 pmol orexin B and saline in a repeated measures design. Neither orexin A nor orexin B significantly affected feeding when injected into the PVN. Experiment 5: orexin B i.c.Õ. Experiment 5 was a repeated measures design in which eight i.c.v.-cannulated rats were injected with 15 nmol orexin B and saline into the right lateral ventricle. No main effect of treatment or time was observed; however, there was a significant interaction of treatment and time Ž F1,7 s 5.970, p s 0.045; Fig. 3.. Food intake during the 0–2 h period was significantly increased when rats were injected with orexin B Ž p s 0.024; Fig. 3.. There was no significant effect on feeding during the 2–4 h period. This feeding response and the lack of such a response following orexin B injection into the PFH, LH, VTA, and PVN suggest the presence of other unidentified siteŽs. of action for orexin B in the brain. More work will need to be done to determine these siteŽs.. To compare directly the feeding effects of orexin A and orexin B in all brain nuclei tested, we plotted feeding
537
Fig. 3. Orexin B i.c.v. Bars represent 2 h food intake of rats injected with orexin B i.c.v. Asterisk Ž). indicates significant difference from saline controls Ž ps 0.024..
above baseline for both orexin A and orexin B Ž500 pmol. at 2 h in each site ŽFig. 4.. Depicted in this manner, the data clearly show that the PFH and LH are feeding-sensitive sites for orexin A. During the first 2 h of the 4 h measurement period, orexin A significantly stimulated feeding in the PFH and the LH ŽFigs. 1, 2 and 4.. During the 2–4 h period, rats injected with 500 pmol orexin A in the PFH ate significantly more than those injected with
Fig. 4. Response to orexins in all tested brain nuclei. Bars represent food intake Ž0–2 h. of rats injected with either 500 pmol orexin A Žnarrow hatches. or 500 pmol orexin B Žwide hatches. as difference from controls. Asterisk Ž). indicates significant difference from saline controls Ž p0.01..
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D.C. Sweet et al.r Brain Research 821 (1999) 535–538
saline ŽFig. 1.. However, the low numbers of subjects and non-counterbalanced design of that experiment warrant further studies to verify this sustained effect. Administration of orexin A in the LH stimulates meal initiation but does not affect total consumption over 4 h. This raises the possibility that orexin A functions via different pathways than neuropeptide Y ŽNPY., which stimulates persistent hyperphagia over 4 h w5,10x. Neither peptide elicited a feeding response when injected into the PVN. This is in contrast to recent work by Dube et al. w7x, who observed increased feeding 2 h after injecting 1 nmol orexin A into the PVN of adult male rats. A possible explanation for this observed difference is that the dose necessary to elicit a feeding response in the PVN lies somewhere between 500 pmol and 1 nmol. The function of orexin A in the PFH and LH are consistent with the roles of these areas in feeding behavior. NPY acts in the PFH to stimulate feeding w9,15,16x, and lesions of the LH induce anorexia w1x. Prior to the discovery of the orexins, no satisfactory mechanism for LH feeding stimulatory effects had been advanced. Clearly, the means by which orexin A in the LH and PFH stimulates feeding and the potential for interaction with other components of the appetiterenergy balance regulatory network must still be defined.
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Acknowledgements w14x
This work was supported by funds from the Minnesota Obesity Center ŽP30 DK-50456..
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