A confocal microscopic study of synaptic inputs to gonadotropin-releasing hormone cells in mouse brain: regional differences and enhancement by estrogen.

Even though the cells producing gonadotropin-releasing hormone (GnRH) are scattered in the basal forebrain, a large proportion of them is present in the organum vasculosum of the lamina terminalis (OVLT) and in the preoptic area. The present studies were undertaken to investigate whether there is any difference in the number of synaptic inputs between GnRH cells located in the OVLT and those located at more anterior levels of the brain. Immunohistochemical staining for the synaptic marker synaptophysin coupled with confocal microscopy was employed to analyze synaptic inputs to GnRH cells located at the two levels examined. The results indicate that GnRH cells in the OVLT region receive a greater number of synaptophysin-immunoreactive appositions as compared with those located in the anterior septum. This supports the existence of subsets among the GnRH cells located in the basal forebrain. The effect of estradiol on the number of synaptophysin-immunoreactive appositions onto GnRH cells was also studied. Treatment of ovariectomized mice with estradiol significantly enhanced the number of synaptophysin-immunoreactive appositions to GnRH cells located at both levels examined. Thus the effect of estrogen on GnRH cells may be mediated in part by changes in the number of synaptic contacts.

Galanin immunoreactivity in mouse basal forebrain: sex differences and discrete projections of galanin-containing cells beyond the blood-brain barrier.

The distribution of galanin-immunoreactive (GAL-IR) cell bodies in the basal forebrain of mice was investigated. The overall pattern of staining for GAL in the area of brain analyzed was similar to that reported in other species with noticeable variations. Distinctive groups of GAL-IR cells were present in the bed nucleus of stria terminalis (BNST), supraoptic nucleus, retrochiasmatic supraoptic nucleus (SOR), magnocellular paraventricular nucleus, arcuate nucleus (ARC) and the nucleus circularis which is one of the cell groups belonging to the accessory magnocellular system. Comparison of the number of GAL-IR cells between the sexes indicated sexual dimorphism in the BNST, SOR and the ARC. As compared with female mice, the mean number of GAL-IR cells/section in the BNST and the SOR was higher and that in the ARC was lower in the males. Unlike in rats, the preoptic area contained mostly scattered GAL-IR cell bodies. Intraperitoneal injection of the retrograde tracer fluoro-gold in male mice resulted in uptake of fluoro-gold by selective GAL-IR cell groups in the basal forebrain suggesting that only some of these cell groups may project outside the blood-brain barrier whereas others may be involved in intracerebral neural transmission.

Intrahypothalamic injection of a cell line secreting gonadotropin-releasing hormone results in cellular differentiation and reversal of hypogonadism in mutant mice.

GT1 is an immortalized cell line that synthesizes and secretes the neurohormone gonadotropin-releasing hormone (GnRH). We have placed these cells into the brains of adult mutant hypogonadal (hpg) mice, which lack a functional GnRH gene, to determine whether such cells could differentiate in situ and support gonadal development. Immunocytochemical detection of GnRH revealed that these cells migrated widely in the central nervous system and elaborated axonal processes which on rare occasion projected to the normal target, the median eminence. Using a battery of antibodies, we demonstrated that these cells could cleave the GnRH precursor and that the amidated decapeptide as well as other cleavage products were present. The presence of biologically active material and its appropriate secretion were further documented by gonadal growth in both males and females. The morphological differentiation of the GT1 cells correlated with the density of cells injected. Those remaining within the injection site and/or forming a tumor retained a simple, rounded or fibroblastic appearance. Those cells that migrated into the host away from such tumors assumed the simple fusiform shape of normal GnRH neurons with dendrites extending from one or both poles. When cell density was drastically reduced a much more complex dendritic arbor was elaborated. These data suggest that such cell lines can be useful in reversing genetic defects and in studying such processes as GnRH neuronal migration, axonal targeting, and cytological differentiation.

Application of a fluorescent dye to study connectivity between third ventricular preoptic area grafts and host hypothalamus.

The mutant hypogonadal (hpg) mouse lacks a functioning gene for the neurohormone gonadotropin releasing hormone (GnRH). Previous studies from our laboratory had indicated that the initiation and maintenance of reproductive function in these mice could be brought about by the implantation of normal fetal grafts into adult hosts. Testicular or ovarian growth and other indicators of normal neurosecretory output were always accompanied by survival of GnRH neurons and growth of GnRH axons into the host median eminence where such axons terminate on the hypophysial portal capillaries. To determine if other connections exist between graft and the host hypothalamus, small crystals of the carbocyanine dye, 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (DiI) were applied to either graft or host after fixation of the brain. Tissue sections were analyzed for retrograde and and anterograde movement of the dye. When crystals were placed on the graft, labeled axons were found in the host median eminence or in the host hypothalamus taking an arching trajectory toward the median eminence. Retrogradely labeled neurons in the host were few in number and largely confined to the host arcuate nucleus. With DiI crystals applied to the basal hypothalamus, labeled axons were distributed widely in the host but much sparser in the graft. Axons appeared to enter primarily at sites where the graft and host interface lacked an ependymal lining. Small numbers of retrogradely labeled neurons were also seen in the graft. Most were cells of very simple morphology and were distributed randomly in the graft. When double label experiments were carried out most DiI positive cells in the graft contained GnRH. These results indicate the connectivity between host hypothalamus and the third ventricular preoptic area grafts exists but is limited in nature.

Identification of gonadotropin releasing hormone (GnRH) neurons projecting to the median eminence from third ventricular preoptic area grafts in hypogonadal mice.

Functional gonadotropin releasing hormone (GnRH) neurosecretory activity can be restored in genetically hypogonadal (hpg) adult mice with grafts of GnRH-containing fetal or neonatal septal-preoptic area (S/POA) tissue. Neurons implanted into the third ventricle of the host brain survive and send out axons which innervate one of the normal targets of these neurons, the median eminence (ME). Fibers terminate near primary portal vessels where GnRH is available for release into the vasculature, and this axonal outgrowth is essential for the stimulation of gonadotropin secretion, gonadal and accessory sex structure growth, gametogenesis, and fertility. Although it is known that GnRH axons reach their target, it is not known if all such neurons in a graft contribute to the projection. Taking advantage of the fact that axons in the ME, the sole host target, are outside the blood-brain barrier (BBB), long-term grafted animals were injected intraperitoneally with a retrograde tracer, Fluorogold (FG). Normal male mice were injected for comparison. Animals were sacrificed 5 days after injection and brain sections in the area of the graft were stained immunocytochemically for GnRH. In the normal male mice, two-thirds of the GnRH neurons were double-labeled with FG. In grafted individuals which showed increased gonadal growth, the percentage of labeled cells ranged from 17 to 75%. The results indicate that despite tissue injury, ectopic location, and a vastly reduced population, transplanted fetal GnRH neurons recapitulate a pattern seen in normal intact mice where some but not all neurons were capable of capturing a peripherally delivered tracer.

Effect of neonatal androgenization on positive feedback in female mice.

Exposure of female mice to androgens within 5 days of birth impairs fertility. Such treatment in rats results in a post-pubertal acyclic state of persistent vaginal cornification and in an inability, when ovariectomized, to show normal positive feedback on luteinizing hormone (LH) release in response to steroid challenge. In the present study, we explored whether neonatally androgenized mice demonstrate positive feedback. Female mice were administered 100 micrograms of testosterone propionate (TP) on either Day 1 (TP1) or Day 5 (TP5) after birth, or vehicle on Day 1 (SO1). Androgen-treated mice had a statistically significant advance in onset of vaginal opening as compared with vehicle-treated mice. All mice that received TP entered constant vaginal estrus, whereas those given vehicle showed variable cytology. All mice were ovariectomized at 7 wk of age and received Silastic capsules containing a priming dose of 17 beta-estradiol. When all mice were challenged 1 wk later with sequential administration of estradiol benzoate and progesterone, a significant increase in plasma LH level was present only in the vehicle-treated mice. We conclude that neonatal androgenization defeminizes the neuroendocrine mechanisms controlling gonadotropin release.

Diabetes in Western Australian children: descriptive epidemiology.

The prevalence and incidence of diabetes mellitus in the age group zero to 14 years in Western Australia were determined from a survey by means of Schools Health Services. Additional information from the State's computer-linked hospital records system, the State's only children's hospital, diabetic clinics and physicians enabled virtually complete ascertainment of cases of childhood diabetes. Only 60% of school-age diabetic children were known to school nurses before the survey, but the nurses were able to identify two-thirds of the remainder during the survey. Among non-Aboriginal children, the prevalence of diabetes in the age group zero to 14 years was 0.59 per 1000 children and the incidence was 12.3 per 100,000 children per year. These rates are somewhat lower than those that have been reported from the United Kingdom and North America, and substantially lower than the rates that were reported from Scandinavia. All but one of the diabetic children who were identified required insulin and were assumed to be insulin-dependent. An excess of boys was found. None of 8715 Aboriginal or part-Aboriginal children had insulin-dependent diabetes mellitus, which indicates that this racial group has a low prevalence of this condition. In case--control studies, which used questionnaires for parents, no significant trends were found in relation to the history of immunizations or of specific viral illnesses except for a past history of varicella which was less frequent in diabetic children. A past history of established breast-feeding (of more than one week) was less frequent in diabetic children, as was the ingestion of vitamin C supplements before the onset of diabetes. Some evidence for a seasonality of onset was obtained. The diabetic children were absent from school for more days and had more admissions to hospital than did non-diabetic children. The majority of diabetic children were prescribed insulin twice a day or more often (84%); performed home blood-glucose monitoring (74%); and attended hospital diabetic clinics (91%).

Accessory olfactory bulb transplants correct hypogonadism in mutant mice.

Transplantation of normal fetal gonadotropin-releasing hormone (GnRH) neurons from the accessory olfactory bulb (AOB) to the third ventricle of GnRH-deficient adult mutant mice reverses the genetically determined reduction in pituitary hormones and poorly developed gonads. The transplanted heterotopic AOB neurons adapt their morphology and secretory functions to what is observed with preoptic GnRH neurons when transplanted into deficient mice and in the normal intact mature animal. This suggests the presence of median eminence trophic factors affecting the growth, terminal sprouting, and functional behavior of the transplanted neurons.

Relationships between behavioral rhythms, plasma corticosterone and hypothalamic circadian rhythms.

Circadian rhythms in physiological processes and behaviors were compared with hypothalamic circadian rhythms in norepinephrine (NE) metabolites, adrenergic transmitter receptors, cAMP, cGMP and suprachiasmatic nucleus (SCN) arginine vasopressin (AVP) in a single population of rats under D:D conditions. Eating, drinking and locomotor activity were high during the subjective night (the time when lights were out in L:D) and low during the subjective day (the time when lights were on in L:D). Plasma corticosterone concentration rose at subjective dusk and remained high until subjective dawn. Binding to hypothalamic alpha 1- and beta-adrenergic receptors also peaked during the subjective night. Cyclic cGMP concentration was elevated throughout the 24-hr period except for a trough at dusk, whereas DHPG concentration peaked at dawn. Arginine vasopressin levels in the suprachiasmatic nucleus peaked in the middle of the day. No rhythm was found either in binding to the alpha 2-adrenergic receptor, or in MHPG or cAMP concentration. Behavioral and corticosterone rhythms, therefore, are parallel to rhythms in hypothalamic alpha 1- and beta-receptor binding and NE-release. Cyclic GMP falls only at dusk, suggesting the possibility that cGMP inhibits activity much of the day and that at dusk the inhibition of nocturnal activity is removed. SCN AVP, on the other hand, peaking at 1400 hr, may play a role in the pacemaking function of the SCN that drives these other rhythms.

The effects of the GnRH agonist, [(imBzl)-D-His6, Pro9-NEt]-GnRH, on the response to stress in rats.

The antigonadal activity of potent gonadotropin-releasing hormone (GnRH) analogs may be associated with undesirable secondary effects on other aspects of the endocrine system. In preliminary studies, rats treated with GnRH agonists were noted to have altered adrenal weights. To determine whether this was associated with alterations in the pituitary-adrenal axis we have studied male and female rats treated with the GnRH agonist [(imBzl)-D-His6, Pro9-NEt]-GnRH (GnRH-A). Animals receiving daily doses of this peptide showed normal adrenal corticosteroid responsivity to both ether and immobilization stress. Brain, pituitary, and plasma concentrations of immunoreactive (IR) beta-endorphin and ACTH were unaffected in treated animals after four weeks of daily injections, although after one week plasma levels of the two hormones were transiently elevated in female rats. Adrenal, thyroid, and pituitary lobe weights were unchanged with treatment, except for an increase in anterior pituitary weight in males receiving the lowest dose of GnRH-A. In conclusion, long-term treatment with GnRH-A, while significantly affecting gonadal and secondary sexual tissue, had little impact on the hypothalamo-pituitary-adrenal system or on stress responsivity in rats.

Brain grafts reverse hypogonadism of gonadotropin releasing hormone deficiency.

Hypogonadism in the mutant hpg mouse is characterized by a deficiency of hypothalamic gonadotropin releasing hormone (GnRH). Affected male mice exhibit immature reproductive organs, small abdominal testes and low pituitary and plasma gonadotropin concentrations. Recent studies have demonstrated the potential of fetal brain transplants to establish functional connections with host tissues. We therefore sought to use this approach to correct the hpg deficit. Fetal preoptic area (POA) (a site of GnRH production) from unaffected animals of the hpg strain was transplanted into the anterior third ventricle of adult hpg mice. We report that in such implanted animals, killed 2 months post-implantation, the POA grafts contained GnRH neurones, from which GnRH-positive fibres could be traced to capillaries of the median eminence. Hypothalamic GnRH and pituitary and plasma gonadotropin concentrations were increased compared with levels in untreated (hpg) animals. The testes were enlarged and had descended into the scrotum. Evidence of full spermatogenesis and interstitial cell development was present in testicular sections. No such effects were seen with transplants of cortical tissue.