Maternal regulation of the hypothalamic-pituitary-adrenal axis in the 20-day-old rat: consequences of laboratory weaning.

There is a large body of evidence that the development of the hypothalamic-pituitary-adrenal (HPA) system in the rat is under maternal regulation. One method used to study the influence of the dam-pup interaction in neonates and weanlings is the separation of mother and litter for 24 h. Previous studies showed that, even at the time of weaning, maternal deprivation results in a dysregulation of the HPA axis at multiple levels. However, the maternal deprivation paradigm usually includes deprivation of food and water, and it was not clear to which extent the observed effects are due to either maternal cues or dehydration and fasting. The primary purpose of the present study was to determine the role of fasting and/or maternal separation on the HPA axis at the time of weaning. Pups at 20 days after parturition are capable of self-feeding and no longer require tactile stimulation to induce eliminative functions. The results indicated that 24 h of fasting led to increased basal levels and further increases in stress induced corticosterone secretion. Fasting also appeared to contribute to the down regulation of basal glucocorticoid receptor mRNA in the hippocampus. In contrast, abrupt weaning irrespective of fasting or dehydration resulted in a suppressed adrenocorticotropin hormone response to an injection of isotonic saline. Although there was an effect of maternal separation on corticotropin-releasing factor mRNA in the paraventricular nucleus, this effect was further exacerbated by the absence of food. Finally, all rats that were separated from their dams showed more efficient negative-feedback. Thus, different aspects of the HPA system appear to respond differentially to either the absence of food or the absence of the mother or both.

Stress-induced alterations in corticotropin-releasing hormone and vasopressin gene expression in the paraventricular nucleus during ontogeny.

From postnatal day (PND) 4 to 14, neonates display a minimal pituitary-adrenal response to mild stress, the so-called 'stress hyporesponsive period' (SHRP). During the SHRP, maternal deprivation (MD) alters the pituitary-adrenal system, enabling neonates to become endocrine responsive to specific stimuli. We have previously reported that during the SHRP, mild stress enhances corticotropin-releasing hormone (CRH) messenger RNA (mRNA) expression in the paraventricular nucleus (PVN). Insofar as elevated CRH mRNA was observed both in the presence and absence of adrenocorticotropin (ACTH) release, we hypothesized that other ACTH secretagogues may participate in the pituitary stress response. During the SHRP, does arginine vasopressin (AVP) complement the actions of CRH which might be reflected centrally by the enhanced biosynthesis of both neuropeptides? To test this hypothesis we examined the time course of stress-induced CRH and AVP mRNA in the PVN at PND 6, 12, and 18. As an index of neural activity, c-fos mRNA in the PVN was also examined. Restraint was used as the stressor and MD was employed to enable an endocrine response during the SHRP. Despite the absence of stress-induced ACTH, in nondeprived pups during the SHRP, CRH mRNA was rapidly enhanced. In their maternally deprived (DEP) counterparts, ACTH levels were increased, and a significant induction of CRH mRNA was only observed at day 12. AVP mRNA levels were elevated in DEP 12-day-old pups at 15, 30 and 60 min. In rats beyond the SHRP, plasma ACTH levels, CRH and AVP mRNA were all enhanced following restraint. At PND 18, elevated CRH mRNA was not observed until 4 h after stimulus. Following restraint, c-fos mRNA was increased at all three ages, although the magnitude of c-fos response was less during the SHRP. These results demonstrate that when restraint elicits prototypical ACTH release, the neonatal central response is to enhance the biosynthesis of both AVP and CRH. If nucleic acid changes correlate with release, the increased synthesis of both neuropeptides may indicate the potential for AVP to synergize with CRH during the neonatal stress response.

Properties of the melatonin-generating system of the sailfin molly, Poecilia velifera.

The properties of the melatonin-generating system of a tropical teleost, the sailfin molly (Poecilia velifera), were investigated in vitro in a series of experiments using static or perifusion culture techniques. The properties examined included photic entrainment, circadian rhythmicity under continuous light (LL) and continuous darkness (DD), functionality of the melatonin-generating system at birth, and presence of multiple circadian oscillators in the molly pineal. Pineal glands or skull caps with the pineal gland firmly attached were dissected from adult and new-born fishes, respectively, and placed into static or perifusion culture at constant temperature (27 degrees C) depending upon the experiment. Melatonin release in samples was quantified by RIA. Rhythmic melatonin release was observed from isolated adult pineals under 12L:12D and 14L:10D, with low amounts of melatonin released during the light and high amounts during the dark. Melatonin release was inhibited by LL. However, under DD, melatonin release was robust and rhythmic with a circadian period (Tau) that ranged between 21.3 and 27.0 h (n = 21). Pineals from new-born (1-day old) mollies released melatonin rhythmically under a light:dark cycle and DD in both static and perifusion culture. Melatonin release from half and quarter pineals of adult mollies under DD was robust and rhythmic with circadian periods that ranged between 22.5 and 29.0 h (n = 19). Taken together, these data show that the molly pineal is photosensitive, fully functional from birth, and contains multiple circadian oscillators (at least four) regulating melatonin production.

Presence of an intrapineal circadian oscillator in the teleostean family Poeciliidae.

In most fish, rhythmic melatonin production is controlled by circadian oscillators located within the pineal (=pineal clocks) that are reset daily by the ambient light:dark (LD) cycle. However, one question that has yet to be addressed concerns the phylogenetic distribution of the pineal clock within fish families. We tested whether a pineal clock identified in the sailfin molly (Poecilia velifera) in an earlier study is also present in some other representatives of the teleostean family Poeciliidae. Isolated pineals from adults belonging to the genus Poecilia (P. velifera albino, P. reticulata, and P. sphenops), genus Xiphophorus (X. helleri and X. maculatus), and genus Limia (L. vittata) were obtained and cultured under LD and/or continuous darkness (DD) at constant temperature (27 degrees C). With one exception, free-running rhythms in melatonin release with circadian periodicities ranging from 19.5 to 27.4 h (n = 26) were detected in isolated pineals from all poeciliid representatives tested under DD exposure. In addition, rhythmic melatonin production was also observed in isolated pineals of some representatives tested from all three genera under LD exposure, suggesting the property of direct photosensitivity. Taken together, these data suggest that a circadian oscillator residing in the pineal of the sailfin molly also appears to be present in all of the poeciliid representatives tested in our system, supporting the notion that the presence of a pineal clock occurs at the family level of taxonomic organization.

On plasma volume measurement and the effect of experimental stress in the themale tilapia, Oreochromis mossambicus, maintained in fresh water.

Plasma volumes in male tilapia (Oreochromis mossambicus) of different size were estimated following intracardial injection of radioiodinated human serum albumin ((125)I-HSA), coupled with short-term, early sampling transient response analysis of 1251-HSA disappearance from the plasma pool. This approach circumvents vascular marker leakage problems associated with constant steady state indicator dilution methods, minimizes some sampling and mixing problems, and simplifies analysis of the data. Changes in hematological parameters due to experimental stress were also studied, because the fish were not chronically cannulated. Results were used in a novel way to correct estimates of plasma volume upward by 15%, thereby providing a potentially useful alternative approach to vascular volume measurement in species where stress-eliminating or reducing techniques, e.g., cannulation, are impractical or infeasible. Hematrocrits increased 38% at the onset, from 24.9% to 34.4%, and remained essentially constant during the 60 minute kinetic study, and plasma osmolalities increased 7%. Corrected plasma volumes Vp (ml) were a linear function of body weight (BW). The group mean Vp was 2.93% of BW and corresponding blood volumes were 3.9% of BW.

Patterns of thyroxine and triiodothyronine in serum and follicle-bound oocytes of the tilapia, Oreochromis mossambicus, during oogenesis.

This study describes simultaneous measurements of thyroid hormones, thyroxine (T4) and triiodothyronine (T3), in the oocytes and serum of a female teleost fish over a complete reproductive cycle. We have identified patterns in circulating T4 and T3 levels as well as their accumulation into oocytes during the reproductive cycle of the tilapia (Oreochromis mossambicus). This is the first description of the patterns with which thyroid hormones accumulate in teleost oocytes. The sampling strategy used in the study eliminated the possible influences of covarying environmental factors that may affect thyroid hormone levels independently of reproductive events. Hormones in serum and oocytes were measured by radioimmunoassay utilizing miniature Sephadex columns. The total content of both thyroid hormones in the oocytes increased throughout most of the ovarian cycle as the oocytes increased in size from less than 2 mg to approximately 6.5 mg by ovulation. By contrast, concentrations of thyroid hormones in the oocytes rose only during the first third of post-spawning oocyte growth (up to approximately 2 mg) before attaining plateaus at approximately 6 ng/g for T4 and 13 ng/g for T3. Serum concentrations of T4 and T3 varied in cyclical patterns during oogenesis, dropping to lows of 3.4 ng/ml (T4) and 2.7 ng/ml (T3) when the oocytes were 1.5 and 2 mg, respectively, and then increasing to 6.5 ng/ml (T4) and 4.8 ng/ml (T3) when the oocytes reach approximately 6 mg. The concentrations of both hormones decreased shortly before spawning. Maximum concentrations of thyroid hormones in the oocytes were reached approximately 10 days prior to those in the serum. Although the serum levels of T4 were greater than those of T3, the reverse was found in the oocytes. Triiodothyronine appears to be accumulated selectively over T4 and the patterns with which both thyroid hormones accumulate in the oocytes of the tilapia do not appear to be tied to serum levels.