The recent characterization of the corticotropin-releasing hormone (CRH) prehormone of the fish tilapia (Oreochromis mossambicus) showed that more variation exists between vertebrate CRH amino acid sequences than recognized before. The present study investigates whether the deviating composition of tilapia CRH coincides with an atypical distribution of CRH in the brain. For this purpose we applied immunohistochemistry, as well as radioimmunoassay (RIA) quantification in brain slices. The results are plotted in a new atlas and reconstruction of the tilapia brain. The largest population of CRH-immunoreactive (ir) neurons is present in the lateral part of the ventral telencephalon (Vl). Approximately tenfold less CRH-ir neurons are observed in the preoptic and tuberal region. The CRH-ir neurons observed in the preoptic region are parvocellular and do not, or hardly, display arginine-vasotocin (AVT) immunoreactivity. CRH-ir neurons are also present in the glomerular layer of the olfactory bulb, in the periventricular layer of the optic tectum, and caudal to the glomerular nucleus. A very dense plexus of CRH-ir terminals is located in the most rostral part of the dorsal telencephalon. This region has not been described in other teleosts and is in the present study subdivided into the anterior part of the dorsal telencephalon (Da) and the anterior part of the laterodorsal telencephalon (Dla). High densities of CRH-ir terminals were observed in and around Vl, in the tuberal region, around the rostral part of the lateral recess, and in the caudal part of the vagal lobe. In the pituitary, CRH-ir terminals are concentrated in the neuro-intermediate lobe. Overall, the immunohistochemical and quantitative data correlated well, as the RIA CRH profile in serial 160-microm slices revealed four peaks, which corresponded with major ir-cell groups and terminal fields. Our results strongly suggest that the CRH-ir cells of Vl project to the rostro-dorsal telencephalon. Consequently, they may not be primarily involved in regulation of pituitary cell types but may subserve other functions. The presence of a CRH-containing Vl-Da/Dla projection seems to be restricted to the most modern group of teleosts, i.e., the Acanthopterygians. Further anatomic indications for non-pituitary-related functions of CRH are found in the vagal lobe and the optic tectum of tilapia. Although the low CRH content of the preoptic region reported here for tilapia may be typical for unstressed fish, the fact remains that remarkably few CRH-ir neurons are involved in regulating the pituitary. Overall, the CRH distribution in the brain of tilapia is more widespread than previously reported for other teleosts.

A d r e n o c o r tic o tr o p ic H o r m o n e in R e la tio n to In te rre n a l F u n c tio n d u r in g S t r e s s in T ila p ia ( O r e o c h r o m is m o s s a m b i c u s )

High concentrations (up to 600 pg/ml) of corticotropinreleasing hormone (CRH) were detected in plasma of the teleost fish Oreochromis mossambicus (tilapia) when screening peripheral tissues of tilapia exposed to stress. Notably, the plasma CRH response to stressors in tilapia is much more pronounced than that in higher vertebrates, such as rats. After characterisation by RIA, by spiking plasma with synthetic tilapia CRH and by methanol-acid extraction, it is concluded that the immunoreactive (ir) material in plasma represents tilapia CRH . Results indicate that a CRH-binding protein is absent in tilapia plasma.Unstressed fish had plasma CRH levels under the limit of detection (,2 pg/ml), but following capture stress plasma CRH levels (170-300 pg/ml) as well as plasma cortisol levels (120 ng/ml) increased rapidly to plateau levels, which were reached after approximately 5 min.Tilapia CRH 1-41 tested at concentrations between 10 11 and 10 7 M in vitro did not stimulate the cortisol release from interrenal tissue. Also pretreatment of interrenal tissue with 10 9 M CRH did not sensitise the cortisolproducing cells to a subsequent ACTH challenge. Fortyeight hours of net confinement or 48 h of cortisol treatment abolished the plasma CRH response and cortisol response to capture stress. The rapidity of the plasma CRH response and its inhibition after 48 h of stress or cortisol treatment point to release by central nervous tissue. Therefore the distribution of glucocorticoid receptors (GRs) in the brain and pituitary of tilapia was investigated. Main GR-ir cell clusters were found in the medial part (Dm) and posterior part of the dorsal telencephalon, in the preoptic region, in the inferior lobe of the hypothalamus and in the cerebellum.We conclude from comparison of CRH brain contents of unstressed and stressed fish that plasma CRH was released by CRH-ir cells located in the lateral part of the ventral telencephalon (Vl), and suggest that the cortisol feedback on CRH release by Vl is mainly exerted via the forebrain Dm region. We propose that CRH is mobilised during stress to fulfil peripheral functions, such as the regulation of circulating leukocytes or of cardiac output, as CRH receptors have been reported in these organs for fish species.

No abstract

Although hypothalamic corticotropin-releasing hormone (CRH) is involved in the stress response in all vertebrate groups, only a limited number of studies on this neuroendocrine peptide deals with non-mammalian neuroendocrine systems. We determined the cDNA sequence of the CRH precursor of the teleost Oreochromis mossambicus (tilapia) and studied the biological potency of the CRH peptide in a homologous teleost bioassay. Polymerase chain reaction (PCR) with degenerate and specific primers yielded fragments of tilapia CRH cDNA. Full-length CRH cDNA (988 nucleotides) was obtained by screening a tilapia hypothalamus cDNA library with the tilapia CRH PCR products. The precursor sequence (167 amino acids) contains a signal peptide, the CRH peptide and a motif conserved among all vertebrate CRH precursors. Tilapia CRH (41 aa) displays between 63% and 80% amino acid sequence identity to CRH from other vertebrates, whereas the degree of identity to members of the urotensin I/urocortin lineage is considerably lower. In a phylogenetic tree, based on alignment of all full CRH peptide precursors presently known, the three teleost CRH precursors (tilapia; sockeye salmon, Oncorhynchus nerka; white sucker, Catostomus commersoni) form a monophyletic group distinct from amphibian and mammalian precursors. Despite the differences between the primary structures of tilapia and rat CRH, maximally effective concentrations of tilapia and rat CRH were equally potent in stimulating adrenocorticotropic hormone (ACTH) and alpha-MSH release by tilapia pituitaries in vitro. The tilapia and salmon CRH sequences show that more variation exists between orthologous vertebrate CRH structures, and teleost CRHs in particular than previously recognized. Whether the structural differences reflect different mechanisms of action of this peptide in the stress response remains to be investigated.