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The brain monoamines serotonin (5-hydroxytryptamine; 5-HT) and dopamine (DA) both play an integrative role in behavioural and neuroendocrine responses to challenges, and comparative models suggest common mechanisms for dietary modulation of transmission by these signal substances in vertebrates. Previous studies in teleosts demonstrate that 7 d of dietary administration with L-tryptophan (Trp), the direct precursor of 5-HT, suppresses the endocrine stress response. The present study investigated how long the suppressive effects of a Trp-enriched feed regimen, at doses corresponding to two, three or four times the Trp levels in commercial feed, last in juvenile Atlantic cod (Gadus morhua) when the fish are reintroduced to a diet with standard amino acid composition. We also wanted to determine whether Trp supplementation induced changes in brain monoaminergic neurochemistry in those forebrain structures innervated by DAand 5-HTergic neurons, by measuring regional activity of DA and 5-HT in the lateral pallial regions (Dl) of the telencephalon and nucleus lateralis tuberis (NLT) of the hypothalamus. Dietary Trp resulted in a dose-dependent suppression in plasma cortisol among fish exposed to confinement stress on the first day following experimental diet; however, such an effect was not observed at 2 or 6 d after Trp treatment. Feeding the fish with moderate Trp doses also evoked a general increase in DA and 5-HT-ergic activity, suggesting that these neural circuits within the NLT and Dl may be indirectly involved in regulating the acute stress response.Key words: L-Tryptophan: Atlantic cod: Stress response: Nucleus lateralis tuberis: Lateral pallial telencephalonThe monoamines serotonin (5-hydroxytryptamine; 5-HT) and dopamine (DA) are important neurotransmitters/modulators involved in integration of the behavioural and neuroendocrine responses to stress (1 -4) . Comparative models suggest common mechanisms for dietary modulation of release and transmission by these signal substances in vertebrates. For instance, it has been demonstrated that dietary supplementation with the essential amino acid L-tryptophan (Trp), the immediate precursor of 5-HT, enhances brain serotonergic activity in teleosts (5 -8) and mammals, including man (9) . In general, the synthesis of central 5-HT depends largely on the plasma concentration ratio between Trp and other large neutral amino acids (LNAA), since they all enter the brain through the same carrier transporter located on the blood -brain barrier (10,11) . Once inside the neuron, Trp is transformed by tryptophan-5-hydroxylase (EC 1.14.16.4) into 5-hydroxytryptophan, which * Corresponding author: Dean Basic, email dean.basic@nvh.no Abbreviations: 1xTrp, control feed containing 0·47 % L-tryptophan; 2xTrp, experimental feed containing 0·82 % L-tryptophan; 3xTrp, experimental feed containing 1·14 % L-tryptophan; 4xTrp, experimental feed containing 1·65 % L-tryptophan; 5-HIAA, 5-hydroxyindoleacetic acid; 5-HT, 5-hydroxytryptamine (serotonin); DA, dopamine; Dl, lateral pall...

Primary cell culture is a powerful tool commonly used by scientists to study cellular properties and mechanisms of isolated cells in a controlled environment. Despite vast differences in the physiology between mammals and fish, primary cell culture protocols from fish are often based on mammalian culture conditions, often with only minor modifications. The environmental differences affect not only body temperature, but also blood serum parameters such as osmolality, pH, and pH buffer capacity. As cell culture media and similar working solutions are meant to mimic characteristics of the extracellular fluid and/or blood serum to which a cell is adapted, it is crucial that these parameters are adjusted specifically to the animal in question. The current protocol describes optimized primary culture conditions for medaka (Oryzias latipes). The protocol provides detailed steps on how to isolate and maintain healthy dissociated pituitary cells for more than one week and includes the following steps: 1. the adjustment of the osmolality to the values found in medaka blood plasma, 2. the adjustment of the incubation temperature to normal medaka temperature (here in the aquarium facility), and 3. the adjustment of the pH and bicarbonate buffer to values comparable to other fish species living at similar temperatures. The results presented using the described protocol promote physiologically meaningful results for medaka and can be used as a reference guide by scientists making primary cell cultures from other non-mammalian species.

The pituitary gland controls many important physiological processes in vertebrates, including growth, homeostasis, and reproduction. As in mammals, the teleost pituitary exhibits a high degree of plasticity. This plasticity permits changes in hormone production and secretion necessary to meet the fluctuating demands over the life of an animal. Pituitary plasticity is achieved at both cellular and population levels. At the cellular level, hormone synthesis and release can be regulated via changes in cell composition to modulate both sensitivity and response to different signals. At the cell population level, the number of cells producing a given hormone can change due to proliferation, differentiation of progenitor cells, or transdifferentiation of specific cell types. Gonadotropes, which play an important role in the control of reproduction, have been intensively investigated during the last decades and found to display plasticity. To ensure appropriate endocrine function, gonadotropes rely on external and internal signals integrated at the brain level or by the gonadotropes themselves. One important group of internal signals is the sex steroids, produced mainly by the gonadal steroidogenic cells. Sex steroids have been shown to exert complex effects on the teleost pituitary, with differential effects depending on the species investigated, physiological status or sex of the animal, and dose or method of administration. This review summarizes current knowledge of the effects of sex steroids (androgens and estrogens) on gonadotrope cell plasticity in teleost anterior pituitary, discriminating direct from indirect effects.

Synthesis and release of FSH and LH are differentially regulated by GnRH, but the mechanisms by which this regulation is achieved are not well understood. Teleost fish are powerful models for studying this differential regulation because they have distinct pituitary cells producing either FSH or LH. By using pituitary cultures from Atlantic cod (Gadus morhua), we were able to investigate and compare the electrophysiological properties of fshb- and lhb-expressing cells, identified by single-cell quantitative PCR after recording. Both cell types fired action potentials spontaneously. The relative number of excitable cells was dependent on reproductive season but varied in opposing directions according to season in the 2 cell types. Excitable and quiescent gonadotropes displayed different ion channel repertoires. The dynamics of outward currents and GnRH-induced membrane responses differed between fshb- and lhb-expressing cells, whereas GnRH-induced cytosolic Ca²⁺ responses were similar. Expression of Ca²⁺-activated K⁺ channels also differed with cell type and showed seasonal variation when measured in whole pituitary. The differential presence of these channels corresponds to the differences observed in membrane response to GnRH. We speculate that differences in ion channel expression levels may be involved in seasonal regulation of hormone secretion as well as the differential response to GnRH in LH- and FSH-producing gonadotropes, through differences in excitability and Ca²⁺ influx.

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Depending on the stage of gonad maturation, as well as other factors, gonadal steroids can exert either a positive or negative feedback at the brain and pituitary level. While this has been demonstrated in many teleost species, little is known about the nature of steroid feedback in Gadiform fish. Using an optimized model system of the Atlantic cod pituitary, the present study investigated the potential effects of two physiologically relevant doses of estradiol, testosterone (TS) or dihydrotestosterone (DHTS) on cell viability and gene expression of gonadotropin subunits (/) and two suggested reproduction-relevant gonadotropin-releasing hormone receptors (/) during three stages of sexual maturity. In general, all steroids stimulated cell viability in terms of metabolic activity and membrane integrity. Furthermore, all steroids affected expression, with the effect depending on both the specific steroid, dose and maturity status. Conversely, only DHTS exposure affected levels, and this occurred only during the spawning season. Using single-cell qPCR, co-transcription of and was confirmed to both and expressing gonadotropes, with being the most prominently expressed isoform. While steroid exposure had no effect on expression, all steroids affected transcript levels in at least one maturity stage. These and previous results from our group point to Gnrhr2a as the main modulator of gonadotropin regulation in cod and that regulation of its gene expression level might function as a direct mechanism for steroid feedback at the pituitary level.