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Complementary deoxyribonucleic acid (cDNA) encoding goldfish preproghrelin was identified using rapid amplification of the cDNA ends (RACE) and reverse transcription (RT)-polymerase chain reaction (PCR). The 490 bp cDNA encodes a 103 amino acid preproghrelin which has a 26 amino acid signal region, 19 amino acid mature peptide and a 55 amino acid C-terminal peptide region. The mature peptide region of goldfish ghrelin has two putative cleavage sites and amidation signals (GRR); one after 12 amino acids and the other after 19 amino acids. The serine (S) in the second amino acid position in the "active core" of ghrelin is substituted with threonine (T). The goldfish ghrelin gene has four exons and three short introns and resembles the human ghrelin gene. Ghrelin messenger RNA (mRNA) expression was detected in the brain, pituitary, intestine, liver, spleen and gill by RT-PCR followed by Southern blot analysis, and in the intestine by Northern blot. Intracerebroventricular (ICV) injection of n-octanoylated goldfish ghrelin (1-19) stimulates food intake in goldfish.

A stereotaxic apparatus and technique for electrode placement in forebrain nuclei of the goldfish is described. An atlas of forebrain nuclei is given. Some new nuclear regions are described and the nomenclature of several nuclei is discussed.A stereotaxic technique for electrode placement in hypothalamic nuclei of the goldfish was described by Peter ('70). Peter's technique was based on the use of a specially designed holder for fish. Other authors have implanted elytrodes for stimulation of forebrain regions in teleost fish using a modified small mammal stereotaxic apparatus (e.g., Demski and Knigge, '71; Demski and Gerald, '72; Demski, '73). However, the technique used in these studies does not facilitate duplication of electrode implantation sites.The sterentaxic technique for goldfish described by Peter ('70) was accompanied by a parasagittal outline drawing showing some hypothalamic nuclei. This restricted the use of the technique to the few stereotaxic coordinates given with the description. The present paper describes a stereotaxic atlas of the forebrain nuclei of goldfish, and describes in detail a stereotaxic apparatus and technique modified after Peter ('70). Some new forebrain nuclei have 31so been described. orbital. The orbital clamps were mounted on the upright extension of slides that could be moved laterally in guides machined into the base plate of the fish holder (figs. l a , lb). The grooves of the orbital clamps were 49 mm above the surface of the base plate. MATERIALS AND METHODS Stereotaxic apparatusThe mouth bar of the fish holder (figs. l a , lb) was a screwbolt three-sixteenths inch diameter by one inch (4.76 mm by 25.40 mm). The mouth bar was mounted on the upright extension of a slide that could be moved anterior-posterior in a groove machined into the base plate. The mouth bar was clamped to the upright so that its upper surface was 2 mm below the plane of the grooves in the orbital clamps. The slide on which the mouth bar was mounted was bolted to the base plate when the instrument was in use. It was positioned so that the posterior tip of the mouth bar was 1.5 mm anterior of the leading edges of the tips of the orbital clamps when they were positioned in the mid-sagittal plane of the fish holder.A Plexiglas extension attached to the micromanipulator served as the holder for the electrode vice ( fig. la). The planes of operation of the micromanipulator were squared to the planes of operation of the fish holder by using a spirit level and a plumb line.Surgical procedures and use of stereotaxic apparatus The surgical procedures of Peter ('70) were used with slight modification. The fish were anesthetized, to the point at which all visible muscular movement ceased, with 0.1 % tricaine methanesulphonate. They were weighed, wrapped in moistened tissue paper, and positioned in a trough-shaped support a s described previously. Using a Foredom Model CC cable engine with a contra angle handpiece (Foredom Electric Company, Bethel, Connecticut), and a Meisinger 232-35 circular saw blad...

Fingerling rainbow trout, acclimated to hatchery raceways or laboratory aquaria, had low plasma cortisol levels (≤ 2 ng/mL), with no apparent daily cycle in levels. Netting of cohorts out of an aquarium, without agitation, did not cause a rise in plasma cortisol concentrations in remaining fish. However, following 90 s of handling and confinement by netting, fingerlings had a sharp rise in plasma cortisol to a peak at 15 min, and then a gradual decline to the basal level over 2 h. Gentle agitation and intermittent restraint with a dip net in the aquarium caused a gradual increase in plasma cortisol concentrations. Intense handling and severe confinement caused a rapid increase in plasma cortisol to a plateau, two to four times greater than the peak levels found in fingerlings subjected to the less vigorous stressors; high levels of plasma cortisol were maintained to the median tolerance limit. During a stocking operation, there was a rapid rise in plasma cortisol concentrations during the initial capture from the rearing ponds, and high levels were maintained through to stocking into the lake 6 h and 10 min later, although a small decrease occurred during transit while the fish were in the truck tanks. After stocking, plasma cortisol concentrations in caged fingerlings did not decrease to the basal level until 8 d poststocking.Key words: rainbow trout, fingerlings, plasma cortisol, cycles, handling stress, stocking stress