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Notes: Abstract: An isocratic high–performance liquid chromatographic technique was developed to measure levels of γ–aminobutyric acid (GABA), glutamate, and taurine in the brain and pituitary of goldfish. Accuracy of this procedure for quantification of these compounds was established by evaluating anesthetic and postmortem effects and by selectively manipulating GABA concentrations by intraperitoneal administration of the glutamic acid decarboxylase (GAD) inhibitor 3–mercaptopropionic acid or the GABA transaminase inhibitor γ–vinyl GABA. The technique provided a simple, rapid, and reliable method for evaluating the concentrations of these amino acids without the use of complex gradient chromatographic systems. To investigate the relationship between neurotransmitter amino acids and the control of pituitary secretion of gonadotropin, the effects of injection of taurine, GABA, or monosodium glutamate on GABA, glutamate, taurine, and, in some instances, monoamine concentrations in the brain and pituitary were evaluated and related to serum gonadotropin levels. Injection of taurine caused an elevation in serum gonadotropin concentrations. In addition, injection of the taurine precursor hypotaurine but not the taurine catabolite isetheonic acid elevated serum gonadotropin levels. Intracerebroventricular injection of either GABA or taurine also elevated serum gonadotropin concentrations. Pretreatment of recrudescent fish with α–methyl–p–tyrosine reduced pituitary dopamine concentrations and also potentiated the serum gonadotropin response to taurine. Injection of monosodium glutamate caused an increase of glutamate content in the pituitary at 24 h; this was followed by a decrease at 72 h after administration. Pituitary GABA, taurine, and dopamine concentrations underwent a transient depletion after monosodium glutamate administration, and this was associated with an elevation of serum gonadotropin content. The increase in serum gonadotropin concentrations in response to a gonadotropin–releasing hormone analogue was potentiated by pretreatment with monosodium glutamate. This article demonstrates that procedures causing elevation in GABA and taurine concentrations stimulate gonadotropin release in a teleost fish.

Notes: Testosterone and oestradiol can modulate GABA synthesis in sexually regressed goldfish. Here we investigated their effects on the mRNA expression of two isoforms of the GABA synthesizing enzyme glutamate decarboxylase (GAD65 and GAD67, EC 4.1.1.15). Full-length GAD clones were isolated from a goldfish cDNA library and sequenced. Goldfish GAD65 encodes a polypeptide of 583 amino acid residues, which is 77% identical to human GAD65. Goldfish GAD67 encodes a polypeptide of 587 amino acid residues and is 82% identical to human GAD67. Goldfish GAD65 and GAD67 are 63% identical. Sexually regressed male and female goldfish were implanted with solid silastic pellets containing testosterone, oestradiol or no steroid. Semiquantitative PCR analysis showed that oestradiol significantly increased GAD65 mRNA expression in female hypothalamus and telencephalon, while testosterone resulted in a significant increase only in telencephalon. GAD67 mRNA levels were not affected by steroids in females. In contrast, both steroids induced significant decreases of GAD65 and GAD67 mRNA levels in male hypothalamus, but had no effect on GAD mRNA expression in male telencephalon. Our results indicate that modulation of GAD mRNA expression is a possible mechanism for steroid action on GABA synthesis, which may have opposite effects in males and females.

Notes: The effects of neuropeptide Y (NPY) on growth hormone (GH) and gonadotropin-II (GtH-II) release in different reproductive stages were studied using perfused pituitary fragments of female goldfish. The GH and GtH-II release responses to 5-min pulses of NPY were relatively small in sexually regressed fish (July), intermediate in recrudescent fish (December), and maximal in sexually mature (= prespawning) fish (May). To test if sex steroids can modulate NPY action, the effects of in vivo implantation of 17β-estradiol (E2) and testosterone (T) (both at 100 μg/g dosage) on NPY-induced GH and GtH-II secretion were examined. In sexually regressed goldfish, implantation of T significantly enhanced NPY-induced GH and GtH-ll release from perfused pituitary fragments; implantation of E2 potentiated the NPY-induced GtH-II, but not GH release. However, steroid implantation did not affect responses to NPY when this experiment was repeated using pituitaries from sexually mature fish. To test the hypothesis that steroids may act directly at the level of the pituitary to potentiate NPY action, pituitary fragments taken from sexually regressed goldfish were incubated with 100 nM T for 24 h, and the GH and GtH-ll responses to 5-min challenges of NPY assessed in the presence of T. Both GH and GtH-ll responses to NPY were not affected by treatment with T in vitro, suggesting that T does not act directly at the level of the pituitary. Since we have found that gonadotropin-releasing hormone (GnRH) in part mediates the effects of NPY on GH and GtH-ll release, the possibility that steroids may potentiate the actions of NPY on GnRH release were also examined. In sexually regressed fish, NPY did not alter GnRH release either from pituitary fragments or preoptic anterior hypothalamic slices. When fish were pretreated with E2 and T by in vivo implantation, NPY significantly stimulated the release of GnRH. Taken together, these results demonstrate that: 1) there is a seasonal variation of NPY action on GH and GtH-ll release in the female goldfish; 2) sex steroids, especially T, potentiate the effects of NPY on GH and GtH-ll release in sexually regressed fish, when the endogenous steroid levels are low; and 3) the seasonality of NPY actions and the potentiation by steroids may be mediated, at least in part, by enhanced stimulation of GnRH release.