Edythe L. P. Anthony

LHRH neurons and their projections in humans and other mammals: Species comparisons

Using light microscopic immunocytochemistry, we have identified LHRH neurons and their projections in humans, monkeys, ferrets, bats and rats. In all these species, LHRH neurons project to the vascular contact zone of the ME, but positions of perikarya vary. This cell population, confined largely to rostral forebrain regions in rats, expands into the medial basal hypothalamus in humans, rhesus monkeys, ferrets and bats. Accompanying this expansion is an augmentation of extrahypothalamic LHRH projections. In rats, LHRH projections are primarily confined to the ME and OVLT. In humans, monkeys, ferrets and bats, however, there are also substantial projections to the posterior pituitary, habenular complex and amygdala. Although the significance of these extrahypothalamic projections is unknown, LHRH may function at some of these sites as a neuromodulator. Humans, monkeys, ferrets and bats further differ from rats in the apparent presence of mature decapeptide within perikarya. Whether variations in the dynamics of maturation of LHRH are related to differences in location of these neurons is currently under investigation.

Neuronal systems immunoreactive with antiserum to lamprey gonadotropin-releasing hormone in the brain of Petromyzon marinus.

SummaryThe role of gonadotropin-releasing hormone (GnRH) in mammalian reproduction has been studied extensively; however, the role of a structurally different, but related, decapeptide is not well characterized in the most primitive class of vertebrates, Agnatha. Utilizing an antiserum directed to the recently characterized lamprey GnRH, we examined immunoreactive neuronal perikarya and nerve fibers in sections from the brain of the sea lamprey, Petromyzon marinus, using the unlabeled peroxidase-antiperoxidase method. Neuronal perikarya and fibers were immunopositive with antisera generated to lamprey GnRH and also to certain antisera generated to mammalian GnRH. Immunopositive neuronal perikarya were detected in an arcshaped population extending from ventral to dorsal preoptic areas. Fibers from these cells projected to the neurohypophysis via the preoptico-hypophyseal tract, but in addition also protruded into the third ventricle. Additionally, some fibers coursed along the external surface of the brain, and may also release GnRH into meningeal compartments. The presence of fully processed, mature decapeptide is indicated within neuronal perikarya, as well as in projecting nerve fibers and terminals. No reaction product was detected in sections incubated with an antiserum to the interior amino acid sequences of mammalian LHRH. This finding supports the structure reported for lamprey GnRH by Sherwood et al. (1986).

Biosynthesis of LHRH: Inferences from immunocytochemical studies

Inferences regarding biosynthesis of LHRH in rats are made from immunocytochemical studies using LHRH antisera with varied and specific binding requirements. Immunoreactive perikarya were observed with antisera that could bind putative large molecular weight precursors of LHRH. No cells were detected with an antiserum that requires free decapeptide terminals and could not bind extended precursors. No such differential immunoreactivity was apparent in neuronal processes and neurovascular terminals. Features of intracellular processing of LHRH which can be inferred from these immunocytochemical data are: (1) the decapeptide is initially synthesized within neuronal cell bodies as a larger molecular weight peptide, extended at both the N- and C-terminals; (2) processing occurs as the newly synthesized material is transported along neuronal processes; and (3) intermediate molecular forms are converted to the active decapeptide primarily in distal portions of neuronal fibers, including the neurovascular terminal. Immunocytochemical observations in other mammalian species (humans, monkeys, ferrets and bats) allow us to further suggest that the dynamics of maturation of this hormone may differ among mammals.

Corticotropin‐releasing Factor in the Adrenal Medulla

Immunoreactive and bioactive corticotropin-releasing factor has been identified in the adrenal gland of dogs, rats and humans. Radioimmunoassay and immunohistochemical experiments have clearly demonstrated that localization of the peptide is confined to the adrenal medulla. CRF-containing cells have a characteristic appearance and are often found in close association with blood vessels. Electron microscopic studies suggest that CRF is secreted at blood vessels within the adrenal medullary vasculature. CRF has also been identified in pheochromocytomas. The amount of the peptide made by such tumors is highly variable as the CRF content of pheochromocytomas may be 20 to 100 times higher or lower than that of normal adrenal tissue. The pathophysiological importance of CRF in pheochromocytomas is unknown. Excessive secretion of the peptide into the peripheral circulation may cause prolonged activation of the pituitary adrenal axis. The peptide may also act within the tumor, although its role remains obscure. Studies on chronically cannulated, awake dogs have shown that CRF is secreted into adrenal venous blood. A gradient exists between adrenal venous and peripheral arterial blood, as CRF is undetectable peripherally under resting conditions. Hemorrhage, a hemodynamic stimulus known to activate a sympathetic adrenal response, increases the CRF secretory rate. The time course of CRF secretion in response to this stimulus parallels that of epinephrine secretion. The physiological significance of adrenal medullary CRF remains to be determined. Although CRF has been shown to affect catecholamine secretion, the peptide appears to be only a weak secretagogue for catecholamines. We suggest that CRF may affect local blood flow within the adrenal medulla and may modify catecholamine secretory rates via this mechanism. The localization of CRF cells in close apposition to blood vessels supports this hypothesis.

Immunocytochemical localization of LHRH in the median eminence, infundibular stalk, and neurohypophysis

SummaryThe distribution of luteinizing hormone-releasing hormone (LHRH) was studied by light-microscopic immunocytochemistry in the hypothalamo-pituitary complex of humans, monkeys, ferrets, bats, and rats. LHRH-immunoreactive fibers were identified in the median eminence of all these species, but the precise location of these fibers varied. In rats, the vast majority of LHRH fibers in the median eminence was confined to the external zone. In contrast, in bats, most of the LHRH fibers were located in the internal zone. While these two species represent opposite extremes in distribution of LHRH fibers within the median eminence, intermediate conditions were found in humans, monkeys, and ferrets, as considerable numbers of fibers occurred in both internal and external zones. In addition to fibers in the median eminence, large numbers of LHRH-immunoreactive fibers were identified traversing the infundibular stalk and entering the neural lobe of the pituitary in all species examined except the rat. In rats, only occasional fibers were observed in the infundibular stalk, and they did not project into the neural lobe. However, in humans, monkeys, ferrets, and bats, groups of LHRH-immunoreactive fibers extended well into the substance of the posterior pituitary. Most of these fibers appeared to terminate near the adenohypophysis, but others coursed away from the anterior lobe and penetrated deeper portions of the neural lobe. These observations, made in several mammalian species, indicate that multiple routes may exist in the median eminence/stalk/pituitary complex for the delivery of LHRH to the anterior pituitary.

1 – Endocrinology of Reproduction in Bats: Central Control

Publisher Summary This chapter deals with the physiology of bats, focusing on the endocrinology of reproduction. Wild-caught bats, sampled at different phases of their reproductive cycles, have provided the basis for most of the current knowledge. As a result, most studies have been descriptive in nature, documenting apparent changes in hypothalamic and pituitary activity that occur over the course of male and female reproductive cycles. However, efforts to maintain healthy captive bat colonies, in which individuals not only survive over the long term, but also reproduce, are becoming increasingly successful. These advances are creating new research environments in which investigators can conduct studies that are more experimental in design. This chapter reviews information derived from all these sources. The chapter places primary emphasis upon form and function of the hypothalamic-pituitary complex in bats within the broader context of their reproductive physiology. Immunocytochemical studies of GnRH neurons have revealed a characteristic distribution of perikarya that differs in potentially significant ways from that seen in other mammalian groups. Differences in GnRH transport routes are also suggested by abundant neuronal projections that extend into the pituitary neural lobe as well as by the architecture of the portal vasculature, which at least in M. lucifugus appears to be composed primarily of short portal vessels. Distributions of pituitary gonadotropes and lactotropes have been determined by immunocytochemistry. In combination with information derived from plasma gonadotropin assays, these studies have provided insight into the seasonal dynamics of LH-, FSH- and prolactin-secreting cells in species exhibiting varied reproductive patterns. Research has brought to light that delayed ovulation may result from long-loop negative feedback of steroids on the hypothalamic-pituitary complex. However, it is yet to be determined whether seasonal modifications in hypothalamic and pituitary sensitivity to steroids contribute to this mechanism.

Luteinizing hormone-releasing hormone (LH-RH) cells and their projections in the forebrain of the batMyotis lucifugus lucifugus

Abstract Luteinizing hormone-releasing hormone (LH-RH) neurons and their projections were studied by immunocytochemistry in the brains of little brown bats (Myotis lucifugus lucifugus: Chiroptera: Vespertilionidae) as a first step in the study of relationships between these neurons and the seasonal reproductive events characteristic of this species. The majority of immunoreactive neurons in adult male, adult female, and fetal bats were ovoid bipolar cells with one thin and one thicker process, both of which gave rise to fine varicose fibers. LH-RH-immunoreactive perikarya were concentrated in the region of the arcuate nuclei in all bats examined. Perikarya were also consistently found dispersed in the mammillary region, anterior hypothalamus, preoptic areas, septum, diagonal band of Broca, and olfactory tracts; they were occasionally observed in the dorsal hypothalamus, organum vasculosum of the lamina terminalis (OVLT), habenula, amygdala, and cingulate gyrus. LH-RH-immunoreactive fibers projected heavily to the median eminence, infundibular stalk, and posterior pituitary. In extrahypothalamic areas, these fibers were especially abundant in the stria medullaris/habenula and stria terminalis/amygdala, but also contributed to the diagonal band of Broca and the olfactory tracts. Immunoreactive fibers that may be components of many different pathways clustered in the rostral septum and permeated the medial hypothalamus. LH-RH-containing fibers frequently entered the subfornical organ, but were observed less often in the OVLT and only occasionally in the pineal. The organization of the LH-RH system in the little brown bat resembles that of primates, but differs considerably from that in the rat. Anatomical characteristics of the LH-RH system in bats thus suggest that this animal may be a particularly suitable species for further study of neuroendocrine control of reproductive function as it may relate to primates, including humans.

GRF immunoreactive neurons in the paraventricular nucleus of the rat: an immunohistochemical study with monoclonal and polyclonal antibodies

Our study demonstrates a complex GRF neuronal system within the rat hypothalamus. Using both high affinity polyclonal and high specificity monoclonal antibodies to rat (r) GRF, we have substantiated evidence for immunoreactive GRF (GRF-i) perikarya in the parvocellular portion of the paraventricular nucleus. Other hypothalamic areas containing rGRF-positive perikarya include the lateral arcuate nucleus, lateral hypothalamus, perifornical area and dorsomedial nucleus. GRF-i neuronal terminals were seen in the external zone of the median eminence, more rostrally in the periventricular nucleus, and near the suprachiasmatic nucleus and more caudally in the dorsomedial nucleus and ventral premammillary nucleus.

Vaginocervical Stimulation of Ferrets Induces Release of Luteinizing Hormone-Releasing Hormone

Vaginocervical stimulation of ovariectomized estradiol‐primed ferrets (which are reflex ovulators) with a glass rod in the presence of a neck‐gripping male induced an increase in plasma luteinizing hormone (LH) from undetectable levels (≤0.50 ng/ml) before stimulation, to 2.4 ± 0.43 ng/ml 75 min after stimulation (stimulated females). Forty‐eight h after stimulation plasma LH returned to baseline levels (post‐stimulated females). A significant decrease in the number of perikarya containing LH‐releasing hormone (LHRH), detected by immunocytochemistry, was associated with the increase in plasma LH following stimulation. Approximately one half of the number of immunoreactive LHRH neurons (243±27) were detected in the forebrain of stimulated females, compared to those detected in the forebrain of post‐stimulated animals (436 ± 88) using antiserum AR 744. Equivalent results were obtained with a different antiserum (RM 1076) capable of detecting the extended decapeptide, or precursor, as well as partially or fully processed decapeptide.

Morphological differentiation of neuropeptide Y neurons in aggregate cultures of dissociated fetal cortical cells : a model system for glia-neuron paracrine interactions

The temporal changes in the morphological profiles of neuropeptide Y (NPY) neurons and their topographical relationship with glial cells (astrocytes) were characterized in aggregate cultures derived from fetal cortical tissue using immunocytochemical procedures. On day 6 of culture, structures labelled with NPY antibodies were small and uneven in size but many resembled neuronal cell bodies. On day 14, neuronal perikarya were well defined and several morphological types of NPY neurons could be distinguished most of which gave rise to beaded processes: unipolar or multipolar bitufted neurons whose processes branch in close proximity to the cell body; bipolar neurons; and multipolar neurons. On day 23, heavily punctate and asymmetrically labelled cell bodies were dispersed throughout the aggregate; neuronal processes were less conspicuous. At 14 and 23 days, cells expressing glial fibrillary acidic protein (GFAP) and neuronal specific enolase (NSE) were abundantly distributed throughout the aggregate. Using a double immunoreaction on 14-day-old aggregates revealed that GFAP+ cells and their processes were in close apposition to and engulfing the NPY neurons. Thus, dissociated fetal NPY neurons undergo morphological differentiation in culture along with astrocytes (GFAP+) and other neuronal cell types (NSE+). Based on the topographical association of astrocytes and neurons, particularly NPY neurons, we propose that the aggregate culture system can serve as a model to study the role of paracrine interactions in the regulation of the expression of NPY.