Distribution and quantification of corticotropin-releasing hormone (CRH) in the brain of the teleost fish Oreochromis mossambicus (tilapia)

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.     

Neurochemical correlates of male polymorphism and alternative reproductive tactics in the Azorean rock-pool blenny,Parablennius parvicornis

In the common Azorean rock-pool blenny, Parablennius parvicornis, males exhibit alternative reproductive morphologies: (1) larger males defend nest sites, provide parental care, have anal glands (involved in pheromone release), testicular glands, and low gonad:body weight ratio (GSI) and (2) smaller, younger, males do not defend nests, have reduced glands and high GSI. These smaller non-nesting males behave as satellites (associated with nests) or sneakers (moving among nests), attempting to achieve parasitic fertilizations via sperm competition. In non-mammals, arginine vasotocin (AVT) is a key hypothalamic peptide involved in the control of reproductive behavior and physiology, and several fish species that exhibit alternative male reproductive morphs show polymorphism in AVT brain chemistry. We conducted an immunocytochemical study to generate comparative data on this intertidal blenny. Our analysis showed no difference in AVT-immunoreactive cell number or size between the male morphs, which is consistent with studies on other fish, including blennies. The number of AVT cells was positively correlated to fish body mass, while cell size showed no such relation. If corrected for body mass, the smaller non-nesting males have significantly more cells than the large nesting males. Our data suggest that the size and number of forebrain AVT cells develops initially to allow for reproduction in the young non-nesting males and this pattern does not appear to change when males take on the nesting morphotype later in life. This result appears to be consistent in many fishes with alternative male morphotypes.     

Synthesis and biological activities of a photoaffinity probe for vasotocin and oxytocin receptors

The present study describes the synthesis and biological activities of the photoreactive vasotocin analog 1–deamino[8–lysine(N^e–4–azidobenzoyl)] vasotocin ([Mpa¹, Lys(N^e–4–azidobenzoyl)⁸]vasotocin). The analog was obtained by introducing the photoreactive aryl azido group at the e–amino group of Lys⁸ in [Mpa¹, Lys⁸]-vasotocin, which was synthesized by the solid phase method. In the isolated toad urinary bladder the photoaffinity analog of vasotocin retained hydroosmotic activity in the absence of u.v.-light. After irradiation the osmotic water flow across the bladder wall increased. Moreover, the water permeability remained high during repeated periods of washout, suggesting that the analog formed covalent complexes with vasotocin receptors in the toad bladder. In the rat uterotonic assay the photoreactive vasotocin analog was without photoactivation a mild agonist. These studies suggest that the photoaffinity analog of vasotocin might be useful for the isolation of vasotocin receptors in low vertebrates and oxytocin receptors in mammals.     

Ostrich MSEL-neurophysin belongs to the class of two-domain “big” neurophysin as indicated by complete amino acid sequence of the neurophysin/copeptin

Mammalian neurohypophyseal hormones, oxytocin and vasopressin, are known to be synthesized as part of two larger precursors containing, respectively, a VLDV-neurophysin and a MSEL-neurophysin together with its associated glycopeptide. Starting from ostrich neurohypophyses, a “big” neurophysin was isolated and chemically characterized. Following sequence determination of the CNBr-derived fragments and of peptides obtained from trypsin and V8-protease digestion of the oxidized protein, this “big” neurophysin was found to contain an MSEL-neurophysin moiety (94 residues) still covalently associated with the COOH-terminal glycopeptide (38 residues, copeptin). This study demonstrates that the ostrich MSEL-neurophysin sequence closely resembles all known MSEL-neurophysin sequences and that, furthermore, it does not contain the single amino acid insertion shown previously in the ostrich VLDV-neurophysin. It is also shown that the stretch of amino acids, linking the MSEL-neurophysin and the copeptin, is clearly different from its mammalian homologues and lacks the Arg residue normally recognized by the cleaving enzyme. This study also demonstrates that the ostrich copeptin is more closely related to the amphibian copeptin sequence than to its mammalian homologue, leading to the hypothesis that two families of copeptin molecules might exist. Thus, the ostrich MSEL-neurophysin-copeptin molecule is the first “big” neurophysin reported in birds and, together with the guinea pig and amphibian homologues, represents the third example of partial or no neurophysin-copeptin cleavage.     

Influence of Age on August Levels of Pineal Immunoreactive Arginine Vasotocin in Rats

The present study was undertaken to evaluate carefully the influence of age on physiological levels of arginine vasotocin-like peptide in rat pineal glands. Glands were collected from male and female rats aged 13, 33, 53, and 73 days on August 4, 12, and 19, 1984. Individual extracts were assayed for arginine vasotocin (AVT) by radioimmunoassay. The results confirm our previous observation that rat pineal AVT immunoactivity (iAVT) increases significantly during August each year; and in this study, each group of rats reached the same peak level of iAVT (700-750 pg/gland) regardless of age or gender. Thus we do not confirm a previously reported decrease in AVT activity with age. In our studies thus far, season of the year is the physiological variable with the most significant influence on pineal AVT activity levels.     

A GABAergic habenulo-raphe pathway mediation of the hypnogenic effects of vasotocin in cat

Electrolytic lesions, performed in the lateral habenula of cats, specifically altered the sleep-wakefulness cycle and completely prevented the usual actions of intraventricularly administered vasotocin, which are to induce non-rapid eye movement sleep and to suppress rapid eye movement sleep. These alterations are (i) selectively related to lateral habenula, since similar lesions performed in thalamus 2 mm lateral to lateral habenula, were unable to prevent the actions of vasotocin or to reproduce the sleep alterations observed after habenular lesions, and (ii) reversible, since at eight days after habenular lesions there is a total return to normal of the sleep-wakefulness parameters, and vasotocin is able again to induce its hypnogenic effects. Opposite effects, characterized by an increase in non-rapid eye movement sleep and a decrease in rapid eye movement sleep, could be induced by a short (10 min) electrical stimulation of the lateral habenula, but not if the stimulating electrodes are placed 2 mm more laterally. Picrotoxin, a gamma-aminobutyrate antagonist, injected intraventricularly in normal cats was without any apparent effect on the sleep-wakefulness cycle if administered in a dose of 1 ng, but had sleep-increasing effects when administered in a dose of 100 ng. However, the smaller dose of picrotoxin (1 ng), when administered 15 min before vasotocin, completely blocked the hypnogenic effect of vasotocin. It is suggested that vasotocin acts within the brain by activating a descending gamma-aminobutyrate-containing habenulo-raphe pathway, and that this pathway plays an important role in the induction and/or organization of the sleep-wakefulness cycle.     

Failure to detect radioimmunoassayable arginine vasotocin in mammalian pineals

The presence of arginine vasopressin (AVP) and arginine vasotocin (AVT) in pineal and neural lobes of a variety of species was evaluated by radioimmunoassay using two antisera. One antiserum recognizes only AVP, while the other cross-reacts 100% with both AVP and AVT. Only traces of AVP were detected in toad and chicken pituitaries and chicken pineals, tissues found to contain abundant AVT. On the other hand, pineals from rats and rabbits, while containing measurable quantities of AVP, did not contain detectable levels (less than 6 pg) of AVT. These results fail to demonstrate the presence of AVT in rat and rabbit pineals and strongly question the role of AVT as a physiological pineal hormone in mammals.     

A potential cardiovascular mechanism for the behavioral effects of central and peripheral arginine vasotocin

Few studies have attempted to separate the behavior modulating actions of central and peripheral arginine vasotocin/arginine vasopressin (AVT/AVP) from their roles in the endocrine and cardiovascular stress response. This series of experiments explored the relative contributions of AVT's central and peripheral mechanisms on altering behavior while simultaneously investigating AVT's effects on the behavioral, cardiovascular, and corticosteroid responses to acute stressors in European starlings (Sturnus vulgaris). Results illustrated peripheral AVT's ability to decrease feeding, drinking, preening, and overall activity. Effects on feeding, drinking, and preening were similar after AVT injection into the lateral ventricles, but the central effects were not blocked by dPTyr(Me)AVP, a selective AVT receptor blocker. This suggests that AVT's behavioral effects may not depend solely on a receptor mediated central mechanism. However, both central and peripheral AVT attenuated heart rate independent of increased plasma corticosterone. These data suggest that AVT's behavioral effects may be modulated by a corticosterone-independent cardiovascular mechanism.     

Identification and localization of neurohypophysial peptides in the brain of a caecilian amphibian,Typhlonectes natans (amphibia: Gymnophiona)

The amphibian order Gymnophiona contains more than 150 different species of caecilians. The characterization and distribution of neurohypophysial peptides, however, has not been described for any member of this order. By using high-performance liquid chromatography, radioimmunoassay, and mass spectrometry, we identified the peptide arginine vasotocin (AVT) in brain and pituitary extracts from the caecilian Typhlonectes natans. By using immunocytochemistry, we found five populations of AVT-immunoreactive (AVT-ir) cells in the brain of T. natans. AVT-ir cell bodies were located in the preoptic area, amygdala pars medialis, ventral thalamus, dorsal hypothalamic nucleus, and nucleus of the solitary tract. AVT-ir fibers and terminal fields were widespread. We also identified a mesotocin-like peptide. The distribution of this peptide in the brain of T. natans was more restricted than the distribution of AVT. Mesotocin-like-immunoreactive cell bodies were located almost exclusively in the preoptic area, with only a few other cells located in the amygdala pars medialis. This caecilian species, therefore, possesses neurohypophysial peptides that are similar in their structure and distribution to the peptides found in anuran and urodele amphibian orders. J. Comp. Neurol. 394:139–151, 1998. © 1998 Wiley-Liss, Inc.