Material and methods periods. Feeding behavior was assessed by counting the

126 H naturally occurring fragment isolated from ovine hypo- pellets Moore Clark, St. Andrews, NB, Canada. Fish thalamus [40] appears to be the most potent fragment. The were acclimated under these standard conditions for ap- numbers in the fragments derive from the predicted signal proximately 2 weeks before the start of an experiment. peptide cleavage site in the long form of CART [14]. Other Forty-eight hours prior to experimentation, two fish were CART fragments including CART 54–105, CART 60– moved into an observation tank and starved. 105, CART 61–105 [43], CART 55–76 and CART 62–76 [32] have also been shown to cause a dose- 2.2. Intracerebroventricular i.c.v. injections dependent feeding inhibition in rats. Immunoneutralization of CART by administration of rabbit anti-CART 55–102 Brain i.c.v. injections were administered following results in higher food intake in rats, suggesting that CART . procedures described by Peter and Gill [37] Briefly, is an endogenous satiety factor [32,42]. following deep anesthesia, a three-sided flap was cut in the NPY-containing terminals are present around CART roof of the skull using a dentist drill equipped with a peptide-positive cell bodies in the paraventricular nucleus circular saw. The flap was then folded to the side, exposing of the hypothalamus [3,9,32] suggesting a regulation of the brain. Fish were then placed in a specially designed NPY on CART output. Decreased levels of the adipocyte stereotaxic apparatus. The needle of a 5-ml microsyringe hormone leptin, as seen during fasting or in obese rats with was stereotaxically placed in the preoptic region of the impaired leptin action, cause a fall in the expression of brain third ventricle according to coordinates 11.0, M, D CART mRNA [2,48] which can be restored by peripheral 1.2 taken from the stereotaxic atlas of the goldfish brain administration of leptin [29]. Leptin treatment activates [37]. Following injection of 2 ml of test solution, the CART hypothalamic neurons [16]. In turn, intracereb- needle was withdrawn and the space in the cranial cavity roventricular injection of recombinant CART inhibits filled with teleost physiological saline [4]. The skull flap NPY-induced feeding [32]. was put back in place, and secured by surgical thread. Fish In fish, there is limited information on the neural control were then returned to their tanks, and normally recovered of feeding behavior. Recent studies show that orexigenic from anesthesia within 2–5 min. All experiments were peptides such as NPY [33,35], galanin [10], and orexins carried out in accordance with the principles published in [46], and anorexigenic peptides such as corticotropin-re- the Canadian Council on Animal Care’s ‘‘guide to the care leasing factor Bernier, Andrusky, Volkoff and Peter, and use of experimental animals’’. unpublished; [11], cholecystokinin [20], serotonin [12] and bombesin [19] are involved in the regulation of feeding in goldfish. 2.3. Observational experiments To determine if CART peptides are involved in feeding in goldfish, we tested the effects of different doses of two Fish were tested in random order in terms of treatment recombinant CART fragments, CART 62–76 and CART and days. For each experiment, two fish were placed in a 55–102, on the feeding behavior of food-restricted fish single observation tank to avoid stress due to isolation and and in fish subjected to NPY- or orexin A OXA-induced to allow for an accurate observation of feeding behavior feeding. As CART peptides might have various physiolog- and food consumption. Observations were made for each ical functions and influence other behaviors besides feed- individual fish. An approximate 4 bw ration of pellets ing, we also examined the effects of CART fragments on per fish was administered at 15 min post-injection. Be- behavioral activity. havioral observations and measurement of food consump- tion commenced upon entry of pellets into the tank, and were made for 1 h. Observations were divided in 15-min

2. Material and methods periods. Feeding behavior was assessed by counting the

number of feeding acts. A ‘feeding act’ was defined as 2.1. Experimental animals occasions when fish approached a pellet. Feeding acts were considered complete CFA when the fish consumed the Male goldfish ranging from 30 to 55 g in weight were pellet and incomplete when the fish approached a pellet purchased from Ozark Fisheries, Stoutland, MO. As gen- and either engulfed it and then ‘spat’ it out, or ‘bumped’ it der basal differences in CART mRNA [21], orexin A with a closed mouth. The total number of feeding acts mRNA [41] and NPY mRNA [38] expression have recent- TFA was calculated by adding the ‘complete’ to the ly been reported in rats, we chose to use only males in our ‘incomplete’ feeding acts. Pellets were either floating or study. Fish were kept under a simulated photoperiod of sinking. Food consumption was converted to milligrams of 16L:8D in 65-l tanks which received a constant flow of food consumed wet body weight time feeding based on aerated water at 188C. The sides of the tanks were opaque the mean pellet weight fed to fish. The total number of acts to minimize external disturbances. Fish were fed a 2 wet TA was defined as the sum of the number of feeding acts body weight bw ration once a day 10:00 h, with and non-feeding acts. The number of non-feeding acts commercially prepared 2.5 mm31 cm cylindrical trout reflected locomotor searching behavior and was assessed H . Volkoff, R.E. Peter Brain Research 887 2000 125 –133 127 by counting the times fish mouthed, picked up and spat gravel, or ‘bumped’ any object in the tank air stone, wall or their tank mate. A tremor was defined as any rapid change in direction or rapid shaking of the head or the body. 2.4. Reagents Human CART 62–76 Tyr–Gly–Val–Pro–Met–Cys– Asp–Ala–Gly–Glu–Gln–Cys–Ala–Val, human CART 55–102 Val–Pro–Ile–Tyr–Glu–Lys–Lys–Tyr–Gly– Gln –Val – Pro – Met – Cys – Asp – Ala–Gly–Glu–Gly–Cys– Ala – Val – Arg – Lys – Gly–Ala–Arg–Ile–Gly – Lys–Leu – Cys–Asp – Cys – Pro – Arg – Gly–Thr–Ser–Cys–Asn–Ser – Phe–Leu–Leu–Lys–Cys–Leu and human orexin A Glp–Pro–Leu–Pro – Asp–Cys–Cys–Arg–Gln–Lys–Thr – Cys – Ser – Cys – Arg–Leu – Tyr–Glu–Leu–Leu–His–Gly – Ala – Gly – Asn – His – Ala – Ala – Gly – Ile–Leu–Thr–Leu – NH2 were purchased from American Peptide Company Sunnyvale, CA. The numbers in CART correspond to the number in the long form of the pro-CART protein with 102 amino acids. Throughout this manuscript, unless specified otherwise, the term ‘CART’ refers to peptides. Goldfish NPY was a gift from Dr. J.E. Rivier Clayton Foundation Laboratories for Peptide Biology, The Salk Institute for Biological Studies, La Jolla, CA, USA. Stock solutions of peptides were made and subsequently diluted Fig. 1. Effects of i.c.v. injection of recombinant CART peptides on food in fish physiological saline [4]. intake of food-restricted goldfish 30 A and 60 min B following presentation of food. Fish were i.c.v. injected with either saline n520, CART 62–76 0.5 ng g, n58; 1 ng g, n59; 5 ng g, n59; 10 ng g, n512; 50 ng g, n58, or CART 55–102 0.5 ng g, n510; 1 ng g, 2.5. Statistics n59; 5 ng g, n510; 10 ng g, n59; 50 ng g, n511. Fish received food 15 min post-i.c.v. injection, and were observed for 1 h. Data are Statistical analyses for food intake, number of acts and mean6S.E.M. Bars with dissimilar superscripts indicate groups that differ significantly. number of tremors were conducted using ANOVA fol- lowed by Student–Newman–Keuls range test. Significance was considered at P,0.05. All error bars indicate standard 2 error of means S.E.M.. Two tailed x and Fisher’s exact test were used for comparison of the frequencies of after 30 min. Doses of 10 and 50 ng g induced a tremors between saline controls and treated fish. Data significant decrease in food intake at 30 and 60 min. At 60 analyses were performed with Instat 2.0 software for min, the feeding level of fish treated with 10 and 50 ng g MacIntosh. CART 62–76 was significantly lower than in fish treated with a dose of 5 ng g. At 0.5 ng g, CART 55–102 had no significant effects on food consumption compared to the saline group. CART

3. Results 55–102 significantly decreased food consumption at all