Stuart A. Tobet

The interstitial nuclei of the human anterior hypothalamus: an investigation of variation with sex, sexual orientation, and HIV status.

The interstitial nuclei of the human anterior hypothalamus (INAH1-4) have been considered candidates for homology with the sexually dimorphic nucleus of the preoptic area of the rat. Volumetric sexual dimorphism has been described for three of these nuclei (INAH1-3), and INAH3 has been reported to be smaller in homosexual than heterosexual men. The current study measured the INAH in Nissl-stained coronal sections in autopsy material from 34 presumed heterosexual men (24 HIV- and 10 HIV+), 34 presumed heterosexual women (25 HIV- and 9 HIV+), and 14 HIV+ homosexual men. HIV status significantly influenced the volume of INAH1 (8% larger in HIV+ heterosexual men and women relative to HIV- individuals), but no other INAH. INAH3 contained significantly more neurons and occupied a greater volume in presumed heterosexual males than females. No sex difference in volume was detected for any other INAH. No sexual variation in neuronal size or density was observed in any INAH. Although there was a trend for INAH3 to occupy a smaller volume in homosexual men than in heterosexual men, there was no difference in the number of neurons within the nucleus based on sexual orientation.

Cells containing immunoreactive estrogen receptor-α in the human basal forebrain

The distribution of estrogen receptor protein-α (ER-α)-containing cells in the human hypothalamus and adjacent regions was studied using a monoclonal antibody (H222) raised against ER-α derived from MCF-7 human breast cancer cells. Reaction product was found in restricted populations of neurons and astrocyte-like cells. Neurons immunoreactive for ER-α were diffusely distributed within the basal forebrain and preoptic area, infundibular region, central hypothalamus, basal ganglia and amygdala. Immunoreactive astrocyte-like cells were noted within specific brain regions, including the lamina terminalis and subependymal peri-third-ventricular region. These data are consistent with the location of estrogen receptors in the basal forebrain of other species and the known effects of estrogens on the cellular functions of both neurons and supporting elements within the human hypothalamus and basal forebrain.

Development of the ventromedial nucleus of the hypothalamus.

The ventromedial nucleus of the hypothalamus (VMH) is important in the regulation of female sexual behavior, feeding, energy balance, and cardiovascular function. It is a highly conserved nucleus across species and a good model for studying neuronal organization into nuclei. Expression of various transcription factors, receptors, and neurotransmitters are important for the development of this nucleus and for mapping the position of identified cells within the nucleus. The VMH is subdivided into regions, all of which may project to specific locations to carry out various functions. For example, the ventrolateral quadrant contains a subset of neurons that highly express estrogen receptors. These neurons specifically are involved in the lordosis response pathway through projections to other estrogen receptor containing regions. In development, neurons that form the VMH generate from the proliferative zone surrounding the third ventricle. Neurons then migrate along radial glial fibers to final positions within the nucleus. Migration and positioning of neurons is an important step in setting up connections to and from the VMH and hence in its function. As compared to other developing brain regions, cell death may play a minor role in sculpting the VMH. We review the processes involved in forming a functional nuclear group and some of the factors known to be involved particularly focusing on the positioning of identified neurons within the VMH.

Effects of γ-Aminobutyric AcidA Receptor Manipulation on Migrating Gonadotropin-Releasing Hormone Neurons through the Entire Migratory Route in Vivo and in Vitro1

GnRH neurons originate in the nasal compartment and migrate along vomeronasal fibers over the cribiform plate to the forebrain. Previously, we found γ-aminobutyric acid (GABA) present in GnRH neurons during development. To clarify the influence of GABA across the entire GnRH migration route, we examined the effects of muscimol and bicuculline (GABAA agonist and antagonist) in vivo and in vitro, maintaining the integrity of the nasal-forebrain connection. For in vivo experiments, mice were administered muscimol, bicuculline, or vehicle on days 10–15 of pregnancy and were killed on embryonic day 15 (E15). For in vitro experiments, 250-μm parasagittal slices of whole heads of E13 mice were incubated with muscimol, bicuculline, or vehicle for 2 days. Muscimol inhibited GnRH cell migration and decreased extension of GnRH fibers. Bicuculline treatment led to a disorganized distribution of GnRH cells in the forebrain and a concomitant dissociation of GnRH cells from fibers of guidance. These results suggest that...

Gonadotropin-releasing hormone (GnRH) neuron migration: Initiation, maintenance and cessation as critical steps to ensure normal reproductive function

GnRH neurons follow a carefully orchestrated journey from their birth in the olfactory placode area. Initially, they migrate along with the vomeronasal nerve into the brain at the cribriform plate, then progress caudally to sites within the hypothalamus where they halt and send projections to the median eminence to activate pituitary gonadotropes. Many factors controlling this precise journey have been elucidated by the silencing or over-expression of candidate genes in mouse models. Importantly, a number of these factors may not only play a role in normal physiology of the hypothalamic-pituitary-gonadal axis but also be mis-expressed to cause human disorders of GnRH deficiency, presenting as a failure to undergo normal pubertal development. This review outlines the current cadre of candidates thought to modulate GnRH neuronal migration. The further elucidation and characterization of these factors that impact GnRH neuron development may shed new light on human reproductive disorders and provide potential targets to develop new pro-fertility or contraceptive agents.

The interstitial nuclei of the human anterior hypothalamus : an investigation of sexual variation in volume and cell size, number and density

The four interstitial nuclei of the anterior hypothalamus (INAH) have been considered as candidate human nuclei for homology with the much studied sexually dimorphic nucleus of the preoptic area of the rat. Assessment of the INAH for sexual dimorphism has produced discrepant results. This study reports the first systematic examination of all four INAH in the human for sexual variation in volume, neuronal number and neuronal size. Serial Nissl-stained coronal sections through the medial preoptic area and anterior hypothalamus were examined from 18 males and 20 females who died between the ages of 17 and 65 without evidence of hypothalamic pathology or infection with the human immunodeficiency virus. A computer-assisted image-analysis system and commercial stereology software package were employed to assess total volume, neuronal number and mean neuronal size for each INAH. INAH3 occupied a significantly greater volume and contained significantly more neurons in males than in females. No sex differences in volume were detected for any of the other INAH. No sexual variation in neuronal size or packing density was observed in any nucleus. The present data corroborate two previous reports of sexual dimorphism of INAH3 but provide no support for previous reports of sexual variation in other INAH.

Ontogeny of Sex Differences in the Mammalian Hypothalamus and Preoptic Area

1. There are numerous sites in the nervous system where steroid hormones dramatically influence development. Increasing interest in mechanisms in neural development is providing avenues for understanding how gonadal steroids alter the ontogeny of these regions during sexual differentiation. 2. An increasing number of researchers are examining effects of gonadal steroids on neurite outgrowth, cell differentiation, cell death, cell migration, and synaptogenesis. The interrelated timing of these events may be a key aspect influenced by gonadal steroids throughout development. 3. The preoptic area and hypothalamus are characteristically heterogeneous in terms of cell type (e.g., different neuropeptides) and cell derivation. Perhaps a major reason for the ontogeny of sexual differences in the preoptic area and hypothalamus lies in the convergence of many different cell types from diverse sources (i.e., proliferative zones surrounding the lateral and third ventricles, and the olfactory placodes) that can be influenced in an interactive manner by gonadal steroid mechanisms. 4. The characterization of multiple mechanisms (e.g., trophic, migratory, apoptotic, fate, etc.,) that contribute to permanent changes in brain structure and ultimately function is essential for unraveling the process of sexual differentiation.

Netrin 1‐mediated chemoattraction regulates the migratory pathway of LHRH neurons

Luteinizing hormone‐releasing hormone (LHRH) neurons migrate from the vomeronasal organ (VNO) to the forebrain in all mammals studied. In mice, the direction of LHRH neuron migration is dependent upon axons that originate in the VNO, but bypass the olfactory bulb and project caudally into the basal forebrain. Thus, factors that guide this unique subset of vomeronasal axons that comprise the caudal vomeronasal nerve (cVNN) are candidates for regulating the migration of LHRH neurons. We previously showed that deleted in colorectal cancer (DCC) is expressed by neurons that migrate out of the VNO during development [Schwarting et al. (2001) J. Neurosci., 21, 911–919]. We examined LHRH neuron migration in Dcc−/− mice and found that trajectories of the cVNN and positions of LHRH neurons are abnormal. Here we extend these studies to show that cVNN trajectories and LHRH cell migration in netrin 1 (Ntn1) mutant mice are also abnormal. Substantially reduced numbers of LHRH neurons are found in the basal forebrain and many LHRH neurons migrate into the cerebral cortex of Ntn1 knockout mice. In contrast, migration of LHRH cells is normal in Unc5h3rcm mutant mice. These results are consistent with the idea that the chemoattraction of DCC+ vomeronasal axons by a gradient of netrin 1 protein in the ventral forebrain guides the cVNN, which, in turn, determines the direction of LHRH neuron migration in the forebrain. Loss of function through a genetic deletion in either Dcc or Ntn1 results in the migration of many LHRH neurons to inappropriate destinations.

Stromal Cell-Derived Factor-1 (Chemokine C-X-C Motif Ligand 12) and Chemokine C-X-C Motif Receptor 4 Are Required for Migration of Gonadotropin-Releasing Hormone Neurons to the Forebrain

Gonadotropin-releasing hormone (GnRH) neurons migrate from the vomeronasal organ (VNO) in the nasal compartment to the basal forebrain in mice, beginning on embryonic day 11 (E11). These neurons use vomeronasal axons as guides to migrate through the nasal mesenchyme. Most GnRH neurons then migrate along the caudal branch of the vomeronasal nerve to reach the hypothalamus. We show here that stromal cell-derived factor-1 [SDF-1, also known as chemokine C-X-C motif ligand 12 (CXCL12)] is expressed in the embryonic nasal mesenchyme from as early as E10 in an increasing rostral to caudal gradient that is most intense at the border of the nasal mesenchyme and the telencephalon. Chemokine C-X-C motif receptor 4 (CXCR4), the receptor for SDF-1, is expressed by neurons in the olfactory epithelium and VNO. Cells derived from these sensory epithelia, including migrating GnRH neurons and ensheathing glial precursors of the migrating mass (MM), also express CXCR4, suggesting that they may use SDF-1 as a chemokine. In support of this, most GnRH neurons of CXCR4−/− mice fail to exit the VNO at E13, and comparatively few GnRH neurons reach the forebrain. There is also a significant decrease in the total number of GnRH neurons in CXCR4−/− mice and an increase in cell death within the VNO relative to controls. The MM is smaller in CXCR4−/− mice, suggesting that some MM cells also require SDF-1/CXCR4 function for migration and survival.

Sex differences in cell migration in the preoptic area/anterior hypothalamus of mice

The preoptic area/anterior hypothalamus (POA/AH) sits as a boundary region rostral to the classical diencephalic hypothalamus and ventral to the telencephalic septal region. Numerous studies have pointed to the regions importance for sex-dependent functions. Previous studies suggested that migratory guidance cues within this region might be particularly unique in their diversity. To better understand the early development and differentiation of the POA/AH, cytoarchitectural, birthdate, immunocytochemical, and cell migration studies were conducted in vivo and in vitro using embryonic C57BL/6J mice. A medial preoptic nucleus became discernible using Nissl stain in males and females between embryonic days (E) E15 and E17. Cells containing immunoreactive estrogen receptor-alpha were detected in the POA/AH by E13, and increased in number with age in both sexes. From E15 to E17, examination of the radial glial fiber pattern by immunocytochemistry confirmed the presence of dual orientations for migratory guidance ventral to the anterior commissure (medial-lateral and dorsal-ventral) and uniform orientation more caudally (medial-lateral). Video microscopy studies followed the migration of DiI-labeled cells in coronal 250-microm brain slices from E15 mice maintained in serum-free media for 1-3 days. Analyses showed significant migration along a dorsal-ventral orientation in addition to medial-lateral. The video analyses showed significantly more medial-lateral migration in males than females in the caudal POA/AH. In vivo, changes in the distribution of cells labeled by the mitotic indicator bromodeoxyuridine (BrdU) suggested their progressive migration through the POA/AH. BrdU analyses also indicated significant movement from dorsal to ventral regions ventral to the anterior commissure. The significant dorsal-ventral migration of cells in the POA/AH provides additional support for the notion that the region integrates developmental information from both telencephalic and diencephalic compartments. The sex difference in the orientation of migration of cells in the caudal POA/AH suggests one locus for the influence of gonadal steroids in the embryonic mouse forebrain.