c-fos expression in the putative avian suprachiasmatic nucleus.

c-fos induction was investigated as a potential component in the avian photic entrainment pathway and as a possible means of locating the central pacemaker in birds. In both quail (Coturnix coturnix japonica) and starlings (Sturnus vulgaris) exposure to 1 h of light induced Fos-lir in the visual suprachiasmatic nucleus but not in the medial suprachiasmatic nucleus. However, the degree of c-fos induction in the visual suprachiasmatic nucleus was similar at different circadian times despite the fact that the light pulses caused differential phase shifts in the locomotor rhythm. For golden hamsters the same experiment resulted in significantly different levels of Fos-lir in the suprachiasmatic nucleus, as well as different phase shifts. Starlings and hamsters were also entrained to T-cycles that caused a large daily phase shift (T = 21.5 h in starlings, T = 22.67 hours in hamsters), or no daily phase shift (T = free running period). No difference in the induced levels of Fos-lir in the visual suprachiasmatic nucleus region was observed between the two groups of starlings, but in hamsters there were significantly different levels of Fos-lir in the suprachiasmatic nucleus between the two groups.

Photoperiodic activation of fos-like immunoreactive protein in neurones within the tuberal hypothalamus of Japanese quail.

Photoperiodic stimulation of quail (Coturnix coturnix japonica) resulted in the appearance of a nuclear fos-like protein within neurones of the basal tuberal hypothalamus. On transfer to long days the number of neurones containing this fos-like immunoreactivity increased from about 150 to 700, the neurones being scattered throughout the length of the tubero-infundibular complex. This activation had occurred by early in the second long day and was maintained for at least three long days. Over this period circulating levels of LH increased seven-fold, indicating that photoperiodic induction had taken place in the birds. A similar time-course of fos-like induction occurred in castrated quail exposed to a single long day and then returned to short days. Activation mirrored the long-term changes in LH secretion found in this paradigm and fos-like immunoreactivity showed the same "carry-over" characteristics of photoperiodic induction, being maximal two days after the quail had been exposed to the single long day (and were again on short days) and when LH secretion was at its maximum. Activation of fos-like immunoreactive cells did not take place when long-day quail were transferred to short photoperiods. The evidence supports the view that the neurones being activated are involved in a specific fashion in the avian photoperiodic response.

Photoperiodic induction in vitro: the dynamics of gonadotropin-releasing hormone release from hypothalamic explants of the Japanese quail.

The Japanese quail is a photoperiodic animal that under certain experimental conditions can respond to a single long day with a wave of LH secretion. Such a system offers an opportunity to analyze the photoneuroendocrine changes as they occur in real time, especially as all of the neural machinery (photoreceptor, clock, and GnRH system) is believed to lie within the hypothalamus. The first detectable rise in LH occurs at about hour 23 of the long day, and this single inductive event leads to prolonged LH secretion lasting for up to 2 weeks and peaking 2-4 days after the dawn of the long day. The size of the quail's hypothalamus is such that the entire structure, including both the GnRH cell bodies and the median eminence, can be cultured for some hours, and the rates of GnRH release measured therefrom. The present experiments used hypothalamic explants from quail at different times throughout the photoperiodic response, superfused them for up to 7.5 h in vitro, and measured the dynamics of GnRH release. A significant step increase of 80% in GnRH release occurred between hours 22.5 and 23 in quail that had been exposed to a long day: an equivalent change was not found in hypothalami taken from quail maintained only under short day lengths. In explants taken from quail at the peak of LH secretion (53 h after dawn of the long day), the rates of GnRH release were double those found in control quail not exposed to the long day. Explants taken 14 days after the long day, when LH secretion had subsided fully, showed no difference in GnRH release between photo-stimulated and control quail. These results suggest that photoperiodic induction involves a timed increase in GnRH release, and the rise at hour 23 is believed to represent photoperiodic induction actually taking place within the brain in vitro. They also suggest that the wave of LH secretion triggered by the single long day is, at least in part, a neuroendocrine or neural phenomenon; this confirms earlier indirect evidence to this effect.

Variation in hypothalamic gonadotrophin-releasing hormone content, plasma and pituitary LH, and in-vitro testosterone release in a long-distance migratory bird, the garden warbler (Sylvia borin), under constant photoperiods.

Changes in concentrations of hypothalamic gonadotrophin-releasing hormone (GnRH), pituitary and plasma LH, testicular mass, in-vitro release of testosterone, body mass and migratory activity were measured in male garden warblers (Sylvia borin) kept from November to June under a constant photo-period of 12.8 h. Under such conditions garden warblers gradually change from the photorefractory to the photosensitive state and gonadal recrudescence then occurs. Hypothalamic GnRH content was low from December to March, but increased in April to reach the highest levels in June. The spontaneous increase in GnRH was paralleled by increases in pituitary LH content, testicular mass and in-vitro testosterone release. Body mass decreased 1 month and nocturnal activity 2 months before the spontaneous increase in GnRH. Ovine LH increased in-vitro testosterone release over basal release at all times. The results suggest that in garden warblers (1) changes in hypothalamic GnRH content can occur under constant photoperiodic condition, (2) the gradual change from the photorefractory to the photosensitive state is not characterized by a gradual increase in hypothalamic content of GnRH (cf. starlings), and (3) Leydig cells are capable of testosterone release even during the photorefractory state.

Photoperiodic control of the development of the LHRH neurosecretory system of European starlings (Sturnus vulgaris) during puberty and the onset of photorefractoriness.

The development of the reproductive system was studied in juvenile starlings during the acquisition of photosensitivity, the attainment of sexual maturation after photostimulation and the subsequent onset of photorefractoriness, using immunohistochemistry for LHRH and radioimmunoassay measurements of hypothalamic, pituitary and plasma hormone concentrations. The first stage of sexual development induced by exposure of photorefractory immature starlings to short days (8 h light:16 h darkness; 8L:16D) was characterized by a decrease in pituitary prolactin content within 1 week and an increase in hypothalamic LHRH content, in the size of the LHRH perikarya and in the intensity of immunostaining in the median eminence in 4-6 weeks. Sexual maturation occurring after exposure to long days (18L:6D) was associated with further increases in LHRH content and cell size, and increases in LH and prolactin concentrations. During testicular regression, LHRH perikarya were reduced in size and staining intensity but LHRH immunostaining in the median eminence and content in the hypothalamus remained high until gonadal regression was almost complete. Prolactin levels were maximal during testicular regression. These results suggest that gonadal regression is initiated by a reduction in LHRH synthesis and possibly, in addition, an external inhibitory influence on LHRH release. Hypothalamic LHRH content eventually declined and LHRH immunostaining in the median eminence was much reduced in fully photorefractory starlings maintained under long days.

Evidence for a daily rhythmicity in the acute release of luteinizing hormone in response to electrical stimulation in the Japanese quail.

This study was undertaken to examine the effect of electrical stimulation of the hypothalamus at different times of day on luteinizing hormone (LH) secretion in male castrated quail on short days (8L:16D). The posterior hypothalamus was stimulated with square-wave pulses of 80 microA for 2 min through chronically-implanted platinum microelectrodes. Stimulation was carried out on each quail at 4 (treatment A), 10 (B), or 14 h (C) after dawn. Plasma LH levels were increased markedly within 2 min of ending the stimulation but reached basal levels again over the next 20 min or so. The absolute increase was significantly greater in treatment B (10 h after lights on) than at the other times tested. This is consistent with a rhythm in hypothalamic responsivity. The results are discussed in the context of the rhythm of photoinducibility which occurs early in the night and which is used by quail as a photoperiodic clock to regulate seasonal reproduction.

Changes in gonadotrophin-releasing hormone and LH in Japanese quail during the first few days of photostimulation.

Castrated Japanese quail responded to a single long day with a five- to eightfold increase in plasma LH levels. A rise in LH secretion appeared 19-24 h after dawn and LH levels were still increased 3 days later, despite the fact that the birds had been returned to a short daylength. Pituitary LH content decreased, reflecting these changes in secretion, although significant falls in content were only found 36-96 h after dawn, when LH secretion was maximal. Hypothalamic gonadotrophin-releasing hormone content was not altered. One interpretation of this is that increased synthesis of the peptide compensates fully for the increased secretion.

Endocrine changes associated with the termination of photorefractoriness by short daylengths and thyroidectomy in starlings (Sturnus vulgaris).

When starlings (Sturnus vulgaris) are transferred from short to long days, hypothalamic content of gonadotrophin-releasing hormone (GnRH) and pituitary gonadotrophin content eventually decrease, as birds become photorefractory, to values lower than they were on short days. This implies that both should increase as photorefractoriness is terminated some time after transfer from long to short days. Further, since thyroidectomy causes termination of photorefractoriness in birds held on long days, this should also result in an increase in hypothalamic GnRH and pituitary gonadotrophin contents. This study tests these hypotheses. Male starlings were transferred from short to long days for 10 weeks, by which time they should have become photorefractory. One group of birds was then killed; blood was collected, hypothalami and pituitaries were excised, and the stage of moult and testicular weights recorded. The remaining birds were then kept on long days, transferred to short days or thyroidectomized and kept on long days. Groups of birds in each treatment group were killed and sampled 2, 8 and 14 weeks later. Hypothalamic content of GnRH, and pituitary and plasma FSH and prolactin concentrations, were measured by radioimmunoassay. In birds sampled after 10 weeks of long days, hypothalamic content of GnRH was quite low, pituitary and plasma levels of FSH were very low, testes were small and pituitary and plasma prolactin levels were high. In intact birds kept on long days, hypothalamic GnRH content decreased further and remained low. Pituitary and plasma FSH levels remained low, testes remained small and pituitary and plasma prolactin levels decreased slowly.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of age and photoperiod on the responsiveness of the pituitary gland of the vole (Microtus agrestis) to stimulation by GnRH.

Male voles were raised from birth to 100 days of age in photoperiods of 16L:8D or 6L:18D. In the long photoperiod testes increased in size between 15 and 80 days of age, and there was an increase in seminal vesicle weight from 60 days of age. Spermatozoa were present in the testes at 60 days of age. In the short photoperiod testicular growth did not begin until 50 days of age with the seminal vesicles beginning to increase at 80 days of age. Spermatozoa were present in testes at 100 days of age. Pituitary secretion in vitro of LH and FSH in response to 1 pmol GnRH, as well as hypothalamic GnRH content, rose to peaks at 50 and 80 days of age respectively in animals exposed to long photoperiods. There was no change in pituitary secretion of FSH in response to GnRH stimulation in animals from the short photoperiod. However, pituitary release of LH in response to 1 pmol GnRH rose to a peak at 80 days of age. Hypothalamic GnRH content rose to a peak at 50 days of age and then declined. The relationship between the hypothalamic GnRH and the sensitivity of the pituitary to GnRH stimulation is compatible with the idea that GnRH can mediate its own receptor numbers.

Hypothalamic gonadotrophin-releasing hormone and pituitary and plasma FSH and prolactin during photostimulation and photorefractoriness in intact and thyroidectomized starlings (Sturnus vulgaris).

Changes in concentrations of hypothalamic gonadotrophin-releasing hormone (GnRH) and pituitary and plasma FSH and prolactin were measured in intact and thyroidectomized female starlings (Sturnus vulgaris) after transfer from short to long photoperiods. In intact birds, hypothalamic GnRH did not increase significantly during the first 6 weeks of photo-stimulation, but by 12 weeks, as birds became photorefractory, it had decreased to levels significantly lower than those before photostimulation. In thyroidectomized birds, which did not become photorefractory, hypothalamic GnRH remained high after 12 weeks of photostimulation. Pituitary FSH increased in both intact and thyroidectomized birds; it then decreased to low levels in intact photorefractory birds, but remained high in thyroidectomized birds. Plasma FSH increased to a peak after 2 weeks, but by 6 weeks it had decreased to low levels in both groups. In intact birds there was a 70-fold increase in pituitary prolactin during the first 6 weeks, and levels were still high after 12 weeks of photostimulation. In thyroidectomized birds, pituitary prolactin remained low. The results suggest that while the initial effect of long daylengths is to cause gonadal maturation, the ultimate effect is to switch off the reproductive system.

Electrical stimulation of the hypothalamus and luteinizing hormone secretion in Japanese quail.

Experiments were undertaken to localize those hypothalamic areas in the male quail (Coturnix coturnix japonica) where electrical stimulation would increase LH secretion. The posterior basal hypothalamus was stimulated with rectangular pulses (height 500 muA) through a bipolar electrode for 6 min, blood samples being taken for LH assay 20 min before, and 2, 10, 20 and 30 min after stimulation. The highest plasma concentration was observed in the 2 min sample. Over the next 30 min the LH level decreased to the resting concentration. The relative increase in LH level was greatest in sexually immature quail and least in photostimulated castrated birds, although the highest absolute levels were seen in the castrated quail. There were no statistical differences between the magnitude of the LH increases in sexually immature, mature and castrated quail. Various hypothalamic regions were then stimulated with a smaller current (200 muA) applied for only 2 min. A highly significant rise in LH followed stimulation of either the tuberal hypothalamus (postero-dorsal part of the infundibular nuclear complex, PD-INC), or the preoptic region (POR) while stimulation 0-5-1-5 mm away from these regions did not change LH secretion. Stimulation of the anterior basal hypothalamus, or of the suprachiasmatic area, caused a significant rise in LH concentration although this was less than that seen after stimulation of the POR. Stimulation in the POR or the PD-INC was ineffective if the tuberal hypothalamus had been deafferentated surgically some days previously. The data complement the studies in which destruction of the PD-INC or the POR by electrolytic lesions has been shown to block photoperiodically induced testicular growth and LH secretion.