Subpopulation Of Gonadotropin-releasing Hormone Neurons Research Articles

Netrin-1 Stimulates Developing GnRH Neurons to Extend Neurites to the Median Eminence in a Calcium- Dependent Manner

Hypothalamic gonadotropin-releasing hormone (GnRH) neurons are required for fertility in all mammalian species studied to date. In rodents, GnRH neuron cell bodies reside in the rostral hypothalamus, and most extend a single long neuronal process in the caudal direction to terminate at the median eminence (ME), the site of hormone secretion. The molecular cues that GnRH neurites use to grow and navigate to the ME during development, however, remain poorly described. Reverse transcription-PCR (RT-PCR) identified mRNAs encoding Netrin-1, and its receptor, DCC, in the fetal preoptic area (POA) and mediobasal hypothalamus (MBH), respectively, from gestational day 12.5 (GD12.5), a time when the first GnRH neurites extend toward the MBH. Moreover, a subpopulation of GnRH neurons from GD14.5 through GD18.5 express the Netrin-1 receptor, DCC, suggesting a role for Netrin-1/DCC signaling in GnRH neurite growth and/or guidance. In support of this notion, when GD15.5 POA explants, containing GnRH neurons actively extending neurites, were grown in three-dimensional collagen gels and challenged with exogenous Netrin-1 (100 ng/ml or 400 ng/ml) GnRH neurite growth was stimulated. In addition, Netrin-1 provided from a fixed source was able to stimulate outgrowth, although it did not appear to chemoattract GnRH neurites. Finally, the effects of Netrin-1 on the outgrowth of GnRH neurites could be inhibited by blocking either L-type voltage-gated calcium channels (VGCCs) with nifedipine (10 µM), or ryanodine receptors with ryanodine (10 µM). This is consistent with the role of Ca2+ from extra- and intracellular sources in Netrin-1/DCC-dependent growth cone motility in other neurons. These results indicate that Netrin-1 directly stimulates the growth of a subpopulation of GnRH neurites that express DCC, provide further understanding of the mechanisms by which GnRH nerve terminals arrive at their site of hormone secretion, and identify an additional neuronal population whose neurites utilize Netrin-1/DCC signaling for their development.

Differential Roles of GnRH-I and GnRH-II Neurons in the Control of the Primate Reproductive Axis

In vertebrates, gonadotropin-releasing hormone (GnRH) represents the primary neuroendocrine link between the brain and the reproductive axis, and in some species up to three different forms of GnRH have been detected. Until recently, it had been assumed that humans and non-human primates only express one form (GnRH-I), but it is now clear they also express a second form (GnRH-II). GnRH-II, like GnRH-I, is highly effective at stimulating gonadotropin release, both in vitro and in vivo, but the neurons that produce GnRH-II are completely distinct from those producing GnRH-I. Moreover, GnRH-II and GnRH-I producing neurons respond very differently to estradiol; specifically, estradiol stimulates GnRH-II gene expression in the former and inhibit GnRH-I gene expression in the latter. Consequently, the negative feedback action of estradiol may be mediated exclusively by the subpopulation of GnRH neurons that express GnRH-I, while the positive feedback action may be mediated exclusively by the subpopulation that expresses GnRH-II. Taken together, these findings raise the possibility that two completely different GnRH neuronal systems participate in the control of primate reproductive physiology. The primary role of GnRH-I neurons is likely to be focused on the maintenance and modulation of tonic pulsatile LH release, whereas the primary role of GnRH-II neurons is likely to be focused on the generation of the preovulatory LH surge. This functional segregation of the primate neuroendocrine reproductive axis lends itself for novel targeted approaches to fertility control and for treatment of human reproductive disorders.

Excitatory Effects of the Puberty-Initiating Peptide Kisspeptin and Group I Metabotropic Glutamate Receptor Agonists Differentiate Two Distinct Subpopulations of Gonadotropin-Releasing Hormone Neurons

Activation of the G-protein-coupled receptor GPR54 by kisspeptins during normal puberty promotes the central release of gonadotropin-releasing hormone (GnRH) that, in turn, leads to reproductive maturation. In humans and mice, a loss of function mutations of GPR54 prevents the onset of puberty and leads to hypogonadotropic hypogonadism and infertility. Using electrophysiological, morphological, molecular, and retrograde-labeling techniques in brain slices prepared from vGluT2-GFP and GnRH-GFP mice, we demonstrate the existence of two physiologically distinct subpopulations of GnRH neurons. The first subpopulation is comprised of septal GnRH neurons that colocalize vesicular glutamate transporter 2 and green fluorescent protein and is insensitive to metabotropic glutamate receptor agonists, but is exquisitely sensitive to kisspeptin which closes potassium channels to dramatically initiate a long-lasting activation in neurons from prepubertal and postpubertal mice of both sexes. A second subpopulation is insensitive to kisspeptin but is uniquely activated by group I metabotropic glutamate receptor agonists. These two physiologically distinct classes of GnRH cells may subserve different functions in the central control of reproduction and fertility.

Emerging methodologies for the study of hypothalamic gonadotropin-releasing-hormone (GnRH) neurons

Gonadotropin-releasing-hormone (GnRH) neurons form part of a central neural oscillator that controls sexual reproduction through intermittent release of the GnRH peptide. Activity of GnRH neurons, and by extension release of GnRH, has been proposed to reflect intrinsic properties and synaptic input of GnRH neurons. To study GnRH neurons, we used traditional electrophysiology and computational methods. These emerging methodologies enhance the elucidation of processing in GnRH neurons. We used dynamic current-clamping to understand how living GnRH somata process input from glutamate and GABA, two key neurotransmitters in the neuroendocrine hypothalamus. In order to study the impact of synaptic integration in dendrites and neuronal morphology, we have developed full-morphology models of GnRH neurons. Using dynamic clamping, we have demonstrated that small-amplitude glutamatergic currents can drive repetitive firing in GnRH neurons. Furthermore, application of simulated GABAergic synapses with a depolarized reversal potential have revealed two functional subpopulations of GnRH neurons: one population in which GABA chronically depolarizes membrane potential (without inducing action potentials) and a second population in which GABAergic excitation results in slow spiking. Finally, when AMPA-type and GABA-type simulated inputs are applied together, action potentials occur when the AMPA-type conductance occurs during the descending phase of GABAergic excitation and at the nadir of GABAergic inhibition. Compartmental computer models have shown that excitatory synapses at >300 microns from somtata are unable to drive spiking with purely passive dendrites. In models with active dendrites, distal synapses are more efficient at driving spiking than somatic inputs. We then used our models to extend the results from dynamic current clamping at GnRH somata to distribute synaptic inputs along the dendrite. We show that propagation delays for dendritic synapses alter synaptic integration in GnRH neurons by widening the temporal window of interaction for the generation of action potentials. Finally, we have shown that changes in dendrite morphology can modulate the output of GnRH neurons by altering the efficacy of action potential generation in response to after-depolarization potentials (ADPs). Taken together, the methodologies of dynamic current clamping and multi-compartmental modeling can make major contributions to the study of synaptic integration and structure-function relationships in hypothalamic GnRH neurons. Use of these methodological approaches will continue to provide keen insights leading to conceptual advances in our understanding of reproductive hormone secretion in normal and pathological physiology and open the door to understanding whether the mechanisms of pulsatile GnRH release are conserved across species.

μ‐opioid receptor mRNA expression in identified hypothalamic neurons

It has been known for a number of years that mu-opioid receptor agonists (e.g., morphine, beta-endorphin, and enkephalin) inhibit luteinizing hormone (LH), vasopressin (VP), and oxytocin (OT) release and stimulate prolactin secretion in rodents and primates by an action at the level of the brain. Also, electrophysiological studies have established that hypothalamic neurons, including gonadotropin-releasing hormone (GnRH), VP, OT, beta-endorphin, and dopamine neurons, are responsive to mu-receptor activation. Although mu-receptor expression has been demonstrated in the hypothalamus, there have been few studies localizing these receptors in neurosecretory neurons. Therefore, we sought to document mu-opioid receptor mRNA expression in immunocytochemically identified hypothalamic neurons. The brains from both female and male guinea pigs were examined by using in situ hybridization and immunocytochemistry. The studies revealed that mu-receptor mRNA was expressed in different diencephalic regions including the preoptic area, the bed nuclei stria terminalis, the paraventricular nucleus thalamus, and the anterior hypothalamus, as well as the supraoptic (SON), paraventricular (PVH), ventromedial, dorsomedial, and arcuate nuclei of the hypothalamus. Importantly, mu-opioid receptors were expressed in subpopulations of GnRH neurons (33.25 +/- 4.6% and 33.6 +/- 3.7% in females and males, respectively), dopamine neurons (51.7 +/- 5.8% to 75.0 +/- 2.6%, depending on neuronal location), beta-endorphin neurons (68.3.0 +/- 4.4%), and VP neurons (41-70%, depending on neuronal location). Because mu-opioid receptors couple via G-proteins to activate inwardly rectifying potassium channels and to inhibit calcium channels, the presence of these receptors is likely to play a major role in directly controlling the excitability of hypothalamic neurons.

Developmental aspect of the gonadotropin-releasing hormone system

Gonadotropin-releasing hormone (GnRH) regulates the hypothalamo-pituitary-gonadal (HPG) axis in all vertebrates studied. GnRH neurons that regulate the HPG axis are primarily derived from progenitor cells in the nasal compartment (NC) and migrate along olfactory system derived fibers across the cribriform plate to destinations in the forebrain. Across their long and uncommon migratory route many factors are likely important for their successful development. Several classes of molecules are being studied for their potential influences on migration, including those related to cell surface interactions (membrane receptors, adhesion molecules, extracellular matrix (ECM) molecules, etc.) and those related to communication across distances (neurotransmitters, peptides, chemoattractant or repellent molecules). Of the classes of molecules associated with cell surface interactions, glycoconjugates with terminal galactose, are temporally and spatially expressed on olfactory fibers that guide GnRH neurons and may play role(s) in migration. Of the molecules associated with communication across distances, the neurotransmitter gamma-aminobutyric acid (GABA) is associated with the GnRH migration pathway and influences the position and organization of GnRH neurons in vitro and in vivo. Furthermore, galactose-containing glycoconjugates and GABA are associated with GnRH neurons in species ranging from humans to lamprey. In mice and rats, GABA is found transiently within a subpopulation of GnRH neurons as they migrate through the NC. One of the key elements in considering regulators of GnRH neuron migration is the diversity of GnRH synthesizing cells. For example, only subpopulations of GnRH neurons also contain GABA, specific GABA receptors, or select glycoconjugates. Similarly, treatments that influence GnRH neuronal migration may only affect specific subsets and not the entire population. It is likely that we will not be able to characterize the migration of all GnRH neurons by a single factor. By combining molecular inquiries with genetic models, single cell analyses, and an in vitro migration model, we are beginning to decipher one of the most critical events in the establishment of the reproductive axis.