H . Volkoff, R.E. Peter Brain Research 887 2000 125 –133
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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
other doses tested, during all time intervals, compared to 3.1. Effects of human CART 62 –76 and CART 55 –
saline-injected controls. At 30 and 60 min after the 102 i.c.v. injections on food intake of food-restricted
presentation of food, there was a significant difference goldfish
between the number of pellets consumed by 1 and 50 ng g treated fish.
At 0.5 and 1 ng g, CART 62–76 had no significant At both 30 and 60 min, 1 and 5 ng g CART 55–102
effects on the cumulative number of pellets consumed at caused a greater reduction in food intake than CART
30 or 60 min following presentation of food compared to 62–76 at the same dosages. There was no difference in
the saline group Fig. 1. Doses of 5 ng g caused a food intake between the two fragments at 0.5, 10 and 50
significant decrease in food intake after 60 min, but not ng g, at either time interval.
128 H
3.2. Effects of human CART 62 –76 and CART 55 – ng g CART 55–102 caused a greater reduction in food
102 i.c.v. injections on food intake of NPY-treated intake than co-treatment with NPY and CART 62–76 at
goldfish the same dosages. At 60 min, co-treatment with 5 ng g
NPY and 2.5 ng g CART 55–102 caused a greater Fish treated with 5 ng g NPY exhibited a higher food
reduction in food intake than co-treatment with NPY and intake compared to the saline group at both 30 and 60 min
CART 62–76 at the same dosages. There was no Fig. 2.
difference in food intake between the two fragments at any At 30 min, NPY-induced feeding was significantly
other dose at either time interval. reduced by co-treatment with CART 62–76 at doses of
2.5, 5 and 10 ng g. At 60 min, NPY-induced feeding was reduced by co-treatment with CART 62–76 at all doses
3.3. Effects of human CART 55 –102 i.c.v. injections tested. Fish co-treated with 5 ng g NPY and CART 62–
on food intake of orexin A-treated goldfish 76 at doses of 5 and 10 ng g had levels of feeding similar
to the saline group at 30 and 60 min. Fish treated with 10 ng g OXA alone exhibited a higher
NPY-induced feeding was reduced by CART 55–102 food intake compared to the saline group at both 30 and 60
at all doses tested, at both 30 and 60 min. All groups of min Fig. 3.
fish co-treated with NPY and CART 55–102 had levels At 30 min, orexin A-induced feeding was reduced by
of feeding similar to the saline group. There were no co-treatment with CART 55–102 at doses of 5 and 10
significant differences between the five co-treatment ng g. At 60 min, OXA-induced feeding was reduced by
groups at either 30 or 60 min. co-treatments with of CART 55–102 at doses of 2, 5
At 30 and 60 min, co-treatment with 5 ng g NPY and and10 ng g. At 30 and 60 min, only fish co-treated with 10
0.5 ng g CART 55–102, and with 5 ng g NPY and 1 ng g OXA and 10 ng g CART 55–102 exhibited a food
intake significantly lower than the saline controls.
Fig. 2. Effects of i.c.v. injection of recombinant CART peptides on food intake of NPY-treated goldfish 30 A and 60 min B following
presentation of food. Fish were i.c.v. injected with either saline n520, Fig. 3. Effects of i.c.v. injection of recombinant CART 55–102 on food
NPY 5 ng g n512, a combination of NPY CART 62–76 5 0.5 intake of orexin A-treated goldfish 30 A and 60 min B following
ng g, n58, 5 1 ng g, n58; 5 2.5 ng g, n58; 5 5 ng g, n58; 5 10 presentation of food. Fish were i.c.v. injected with either saline n520,
ng g, n59, or a combination of NPY CART 55–102 5 0.5 ng g, orexin A OXA 10 ng g n58, or a combination of OXA CART
n59; 5 1 ng g, n510; 5 2.5 ng g, n57; 5 5 ng g, n58; 5 10 ng g, 55–102 10 0.5 ng g, n58; 10 1 ng g, n510; 10 2 ng g, n57; 10 5
n59. Fish received food 15 min post-i.c.v. injection, and were observed ng g, n57; 10 10 ng g, n56. Fish received food 15 min post-i.c.v.
for 1 h. Data are mean6S.E.M. Bars with dissimilar superscripts indicate injection, and were observed for 1 h. Data are mean6S.E.M. Bars with
groups that differ significantly. dissimilar superscripts indicate groups that differ significantly.
H . Volkoff, R.E. Peter Brain Research 887 2000 125 –133
129
3.4. Effects of human CART 62 –76 and CART 55 – feeding acts was significantly reduced by treatment with
102 i.c.v. injections on feeding behavior and behavioral CART 62–76 at 10 and 50 ng g and with CART
activity of goldfish 55–102 at 1, 5, 10 and 50 ng g Fig. 4A. Feeding
behavior, as indicated by the total number of feeding acts In food-restricted goldfish, the number of complete
in 60 min, was increased with CART 62–76 at doses of
Fig. 4. Effects of i.c.v. injection of recombinant CART peptides on behavior of food-restricted A, NPY-treated B and orexin A- treated C goldfish. Treatment groups and sample sizes are the same as in Figs. 1–3. Total number of feeding acts TFA includes pellets eaten, pellets engulfed and then spat
out, and pellets bumped with a closed mouth. Complete feeding acts CFA represents the number of pellets or pellet fragments eaten. Total acts TA include feeding acts, as well as non-feeding acts gravel spitting, bumping on walls, any inanimate object in the tank or another fish. Data are
mean6S.E.M. Stars indicate groups that differ from the saline control. Circles and triangles indicate groups that differ from NPY-treated and orexin A-treated fish, respectively.
130 H
Fig. 4. continued
10 and 50 ng g. CART 55–102 had no significant effects significant tremor activity. Co-treatment with 10 ng g
on feeding behavior. The total number of acts was OXA and CART 55–102 at doses of 5 and 10 ng g
significantly increased with both CART fragments at 1, 5, induced a significant increase in the number of fish
10 and 50 ng g as compared to saline controls. In NPY-treated goldfish, the number of complete feed-
ing acts was significantly increased only in fish treated with 5 ng g NPY and in fish co-treated with 5 ng g NPY
and 0.5 or 1 ng g CART 62–76 as compared to saline controls Fig. 4B. The total number of feeding acts was
increased in fish treated with NPY alone and in fish co-treated with 5 ng g NPY and CART 62–76 at doses
of 2.5, 5 and 10 ng g relative to saline controls, but was not significantly different from saline controls in any group
treated with CART 55–102. The total number of acts was increased in fish treated with NPY alone, in fish
co-treated with 5 ng g NPY and CART 62–76 at doses of 2.5, 5 and 10 ng g, and in fish co-treated with NPY 5
ng g and CART 55–102 at doses of 2.5 and 5 ng g, as compared to the saline controls.
In OXA-treated goldfish, the number of complete feed- ing acts was higher in fish treated with OXA alone and in
fish co-treated with 10 ng g OXA and 0.5 or 1 ng g CART 55–102 as compared to the saline controls Fig.
4C. The total number of feeding acts and the total number of acts were both increased in fish treated with OXA alone
relative to saline controls and were significantly lower in fish co-treated with 10 ng g OXA and CART 55–102 at
all doses tested, as compared to OXA alone.
Fig. 5. Effects of i.c.v. injection of CART55–102 on the occurrence of
Tremors were induced by CART 55–102 alone at
tremors in food-restricted, NPY-treated and orexin A-treated goldfish. The data is presented as the frequency of fish displaying tremors in each
doses of 1 ng g and higher, but this induction was only
treatment group A, mean6S.E.M. and the number of occurrences of
significant at doses of 10 and 50 ng g as compared to the
tremors displayed per fish per 60 min B. A tremor was defined as any
saline control Fig. 5. Tremors were not seen in fish
rapid change in direction or shaking of the head or the body. Treatment
treated with saline, NPY alone, or OXA alone. Co-treat-
groups and sample sizes are the same as in Figs. 1–3. Stars indicate
ment with NPY and CART 55–102 did not induce
groups that differ from saline controls.
H . Volkoff, R.E. Peter Brain Research 887 2000 125 –133
131
showing tremors as compared to saline controls. There was of feeding, respectively, as compared to the saline group.
no significant difference in the number of occurrences of The stimulatory effects of i.c.v. injected NPY were sup-
tremors per fish when co-treated with NPY and CART pressed by CART in a dose-dependent manner, with
55–102 or OXA and CART 55–102 compared to CART 55–102 being more potent. In rats, simultaneous
saline controls or to fish treated with 10 ng g CART i.c.v. injection of 4 nmol NPY and 2.4 nmol CART
55–102. Tremors were not observed in fish treated with 55–76 attenuates feeding [32] and co-injection of 5 mg
CART 62–76 alone or in combination with NPY or OXA NPY and CART 55–102 at doses of 0.5, 1 and 2 mg also
data not shown. causes a dose-dependent inhibition of NPY-stimulated
feeding [29,47]. When administered alone at moderate doses 0.5 ng g, neither fragment affected food intake,
4. Discussion suggesting that the inhibition of NPY-induced food intake
