Adult Gonadotropin-releasing Hormone Neurons Research Articles

Hydrogen peroxide suppresses excitability of gonadotropin-releasing hormone neurons in adult mouse.

It has been reported that reactive oxygen species (ROS) derived from oxygen molecule reduction can interfere with the cross-talk between the hypothalamic-pituitary-gonadal (HPG) axis and other endocrine axes, thus affecting fertility. Furthermore, ROS have been linked to GnRH receptor signaling in gonadotropes involved in gonadotropin release. There has been evidence that ROS can interfere with the HPG axis and gonadotropin release at various levels. However, the direct effect of ROS on gonadotropin-releasing hormone (GnRH) neuron remains unclear. Thus, the objective of this study was to determine the effect of hydrogen peroxide (H2O2), an ROS source, on GnRH neuronal excitabilities in transgenic GnRH-green fluorescent protein-tagged mice using the whole-cell patch-clamp electrophysiology. In adults, H2O2 at high concentrations (mM level) hyperpolarized most GnRH neurons tested, whereas low concentrations (pM to μM) caused slight depolarization. In immature GnRH neurons, H2O2 exposure induced excitation. The sensitivity of GnRH neurons to H2O2 was increased with postnatal development. The effect of H2O2 on adult female GnRH neurons was found to be estrous cycle-dependent. Hyperpolarization mediated by H2O2 persisted in the presence of tetrodotoxin, a voltage-gated Na+ channel blocker, and amino-acids receptor blocking cocktail containing blockers for the ionotropic glutamate receptors, glycine receptors, and GABAA receptors, indicating that H2O2 could act on GnRH neurons directly. Furthermore, glibenclamide, an ATP-sensitive K+ (KATP) channel blocker, completely blocked H2O2-mediated hyperpolarization. Increasing endogenous H2O2 by inhibiting glutathione peroxidase decreased spontaneous activities of most GnRH neurons. We conclude that ROS can act as signaling molecules for regulating GnRH neuron's excitability and that adult GnRH neurons are sensitive to increased ROS concentration. Results of this study demonstrate that ROS have direct modulatory effects on the HPG axis at the hypothalamic level to regulate GnRH neuron's excitabilities.

Increased GABA transmission to GnRH neurons after intrahippocampal kainic acid injection in mice is sex-specific and associated with estrous cycle disruption

Patients with epilepsy develop reproductive endocrine comorbidities at a rate higher than that of the general population. Clinical studies have identified disrupted luteinizing hormone (LH) release patterns in patients of both sexes, suggesting potential epilepsy-associated changes in hypothalamic gonadotropin-releasing hormone (GnRH) neuron function. In previous work, we found that GnRH neuron firing is increased in diestrous females and males in the intrahippocampal kainic acid (IHKA) mouse model of temporal lobe epilepsy. Notably, GABAA receptor activation is depolarizing in adult GnRH neurons. Therefore, here we tested the hypothesis that increased GnRH neuron firing in IHKA mice is associated with increased GABAergic drive to GnRH neurons. When ionotropic glutamate receptors (iGluRs) were blocked to isolate GABAergic postsynaptic currents (PSCs), no differences in PSC frequency were seen between GnRH neurons from control and IHKA diestrous females. In the absence of iGluR blockade, however, GABA PSC frequency was increased in GnRH neurons from IHKA females with disrupted estrous cycles, but not saline-injected controls nor IHKA females without estrous cycle disruption. GABA PSC amplitude was also increased in IHKA females with disrupted estrous cycles. These findings suggest the presence of an iGluR-dependent increase in feed-forward GABAergic transmission to GnRH neurons specific to IHKA females with comorbid cycle disruption. In males, GABA PSC frequency and amplitude were unchanged but PSC duration was reduced. Together, these findings suggest that increased GABA transmission helps drive elevated firing in IHKA females on diestrus and indicate the presence of a sex-specific hypothalamic mechanism underlying reproductive endocrine dysfunction in IHKA mice.

Changes in Dendritic Architecture: Not Your “Usual Suspect” in Control of the Onset of Puberty in Male Rats

Until the recent past, the search for the underlying drive for the pubertal increase in gonadotropin-releasing hormone (GnRH) hormone from the GnRH-containing neurons in the hypothalamus was largely focused on extrinsic factors. The most recent evidence however indicates changes in the structure of GnRH neurons themselves may contribute to this fundamental event in development. Based on our studies in males, dendritic architecture is not static from birth until adulthood. Instead, dendrites undergo a dramatic remodeling during the postnatal period which is independent of testosterone and occurs before the pubertal increase in GnRH release. First, the number of dendrites emanating from somata is reduced between infancy and adulthood. Moreover, a dendrite of adult GnRH neurons invariability arises at angle of 180°from the axon as opposed to the extraordinary variability in location during infancy. In fact, in some neurons from infants, no dendrite even resides in the adult location. Thus, there is a spatially selective remodeling of primary dendrites. Secondly, dendrites of GnRH neurons from infants were highly branched prior to assuming the compact morphology of adults. Finally, other morphological aspects of GnRH neurons such as total dendritic length, the numbers of dendrite branches and the lengths of higher order branches were significantly greater in infants than adults, indicating a consolidation of dendritic arbors. Activity in multi-compartment models of GnRH neurons, suggest the impact of structure on neuronal activity is exerted with both active and passive dendrites. Thus, passive properties make a defining contribution to function. Accordingly, changes in morphology alone are likely to have functional consequences for the pattern of activity in GnRH neurons. Our findings suggest structural remodeling of dendrites during the postnatal period likely facilitates repetitive action potentials and thus, GnRH release at the time of puberty.

A mathematical model of adult GnRH neurons in mouse brain and its bifurcation analysis

GnRH neurons are hypothalamic neurons that secrete gonadotropin-releasing hormone (GnRH) which stimulates the release of gonadotropins, one of the crucial hormones for sexual development, fertility and maturation. A mathematical model was built to help elucidate the mechanisms underlying electrical bursting and synchronous [Ca 2+] transients in GnRH neurons ( Lee et al., 2010). The model predicted that bursting in GnRH neurons (at least of the short-bursting type) requires the existence of a [Ca 2+]-dependent slow after-hyperpolarisation current (s I AHP-UCL), and this predicted current was found experimentally. GnRH behaviour under a wide range of conditions (inhibition of Na + channels, IP 3 receptors, [Ca 2+]-dependent K + channels, or Ca 2+ pumps, or in the presence of zero extracellular [Ca 2+]) is successfully reproduced by the model. In this paper, a simplified version of the previous model, with the same qualitative behaviour, is constructed and studied using timescale separation techniques and bifurcation analysis.

The intimate relationship of gonadotropin‐releasing hormone neurons with the polysialylated neural cell adhesion molecule revisited across development and adult plasticity

The neurohormone gonadotropin-releasing hormone (GnRH) is critical for all the aspects of reproductive life in vertebrates. GnRH is secreted by a small number of neurons dispersed within the preoptic-hypothalamic region. These neurons are derived from the embryonic olfactory pit. They then migrate along olfactory, vomeronasal and terminal nerves to their final destination. Classical approaches to study the regulation of GnRH secretion during the reproductive cycle have focused on the various neuronal inputs on GnRH neurons and their regulation by ovarian steroids. However, it is well known that steroids will change the microenvironment of neuronal networks and can induce plasticity and functional changes. In this review, we will focus on the intimate relationship of developing and adult GnRH neurons with the polysialylated form of neural cell adhesion molecule (PSA-NCAM), a major molecular actor in the morphogenesis and adult plasticity of the nervous system. We will first recapitulate the spatiotemporal relationship between PSA-NCAM and migrating GnRH neurons during embryogenesis of various vertebrate species and discuss its importance for GnRH neuron development as shown by various loss of function studies. In the adult, we will review the relationships between PSA-NCAM and GnRH neurons across various physiological states, and open the discussion to the use of new model systems that can help to unravel the function and mechanism of action of PSA-NCAM on GnRH neuronal network activity and GnRH release.

Voltage-gated Ca2+ channel mRNAs and T-type Ca2+ currents in rat gonadotropin-releasing hormone neurons

Gonadotropin-releasing hormone (GnRH) neurons play a pivotal role in the neuroendocrine regulation of reproduction. We have previously reported that rat GnRH neurons exhibit voltage-gated Ca(2+) currents. In this study, oligo-cell RT-PCR was carried out to identify subtypes of the alpha(1) subunit of voltage-gated Ca(2+) channels in adult rat GnRH neurons. GnRH neurons expressed mRNAs for all five types of voltage-gated Ca(2+) channels. For T-type Ca(2+) channels, alpha(1H) was dominantly expressed in GnRH neurons. Electrophysiological analysis in acute slice preparations revealed that GnRH neurons from adult rats exhibited T-type Ca(2+) currents with fast inactivation kinetics (~20 ms at -30 mV) and a time constant of recovery from inactivation of ~0.6 s. These results indicate that rat GnRH neurons express subtypes of the alpha(1) subunit for all five types of voltage-gated Ca(2+) channel, and that alpha(1H) was the dominant subtype in T-type Ca(2+) channels.

Effects of human growth hormone on gonadotropin-releasing hormone neurons in mice

PurposeRecombinant human growth hormone (rhGH) has been widely used to treat short stature. However, there are some concerns that growth hormone treatment may induce skeletal maturation and early onset of puberty. In this study, we investigated whether rhGH can directly affect the neuronal activities of of gonadotropin-releasing hormone (GnRH).MethodsWe performed brain slice gramicidin-perforated current clamp recording to examine the direct membrane effects of rhGH on GnRH neurons, and a whole-cell voltage-clamp recording to examine the effects of rhGH on spontaneous postsynaptic events and holding currents in immature (postnatal days 13-21) and adult (postnatal days 42-73) mice.ResultsIn immature mice, all 5 GnRH neurons recorded in gramicidin-perforated current clamp mode showed no membrane potential changes on application of rhGH (0.4, 1 µg/mL). In adult GnRH neurons, 7 (78%) of 9 neurons tested showed no response to rhGH (0.2-1 µg/mL) and 2 neurons showed slight depolarization. In 9 (90%) of 10 immature neurons tested, rhGH did not induce any membrane holding current changes or spontaneous postsynaptic currents (sPSCs). There was no change in sPSCs and holding current in 4 of 5 adult GnRH neurons.ConclusionThese findings demonstrate that rhGH does not directly affect the GnRH neuronal activities in our experimental model.

Rapid actions of oestrogen on gonadotropin‐releasing hormone neurons; from fantasy to physiology?

Oestradiol (E2) exerts critical homeostatic feedback effects upon gonadotropin-releasing hormone (GnRH) neurons to maintain fertility. In the female, E2 has both negative and positive feedback actions to suppress and stimulate GnRH neuron activity at different times of the ovarian cycle. This review summarizes reported rapid E2 effects on native embryonic and adult GnRH neurons and attempts to put them into a physiological perspective. Oestrogen has been shown to rapidly modulate multiple processes in embryonic and adult GnRH neurons including intracellular calcium levels, electrical activity and specific second messenger pathways, as well as GnRH secretion itself. Evaluation of in vivo data suggests that there is no essential role for rapid E2 actions in the positive feedback mechanism but that they may comprise part of the negative feedback pathway. Adult GnRH neurons are only likely to be exposed to E2 from the gonads via the circulation with appropriate physiological E2 concentrations in the rodent being 10-50 pM for negative feedback ranging up to 400 pM for positive feedback. Although most studies to date have examined the effects of supraphysiological E2 levels on GnRH neurons, there is accumulating evidence that rapid E2 actions may have a physiological role in suppressing GnRH neuron activity.

Cetrorelix, a Gonadotropin-Releasing Hormone Antagonist, Induces the Expression of Melatonin Receptor 1a in the Gonadotropin-Releasing Hormone Neuronal Cell Line GT1–7

Melatonin has been implicated in the control of the reproductive system, and the modulatory actions of melatonin on gonadotropin-releasing hormone (GnRH) neurons have been assumed to be indirectly mediated through afferent neurons. However, our previous studies demonstrate sexually dimorphic modulation of A-type γ-aminobutyric acid (GABA) receptor (GABA_AR) currents by melatonin in adult rat GnRH neurons and a preferential expression of melatonin 1a receptor (MT1) in male GnRH neurons. Using immortalized GnRH neurons (GT1–7 cells), the present study investigated the mechanism by which the expression of melatonin receptors is regulated in GnRH neurons. Like endogenous GnRH neurons, GT1–7 cells express both GnRH and GnRH receptor mRNAs, indicating that the cells have a self-stimulatory system. A 2-iodomelatonin binding assay and RT-PCR analysis demonstrated that the cells expressed neither MT1 nor MT2. However, treatment of GT1–7 cells with the GnRH antagonist cetrorelix significantly increased 2-iodomelatonin binding and induced a time- and concentration-dependent MT1 mRNA expression. The GABA_AR currents were then measured using a perforated patch-clamp technique to examine whether the treatment with cetrorelix changed the responses to melatonin. Melatonin augmented the GABA_AR currents in GT1–7 cells treated with 1 μM cetrorelix for 24 h, while melatonin decreased the currents in the cells not treated with cetrorelix, probably via receptor-independent processes. The present results suggest that GnRH downregulates the expression of MT1 via an autocrine-paracrine mechanism in GT1–7 cells, and modifies the melatonin-induced modulation of GABA_AR currents. These findings may provide one possible mechanism for the sexually dimorphic responses to melatonin in adult rat GnRH neurons.

GABAA Receptors Mediate Excitation in Adult Rat GnRH Neurons1

Gonadotropin-releasing hormone (GnRH) neurons form the final common pathway for the central regulation of reproduction. Gamma-amino butyric acid (GABA), the main inhibitory neurotransmitter in the adult brain, has long been implicated in playing key roles in the regulation of GnRH neurons. Two groups reported recently that GABA depolarizes GnRH neurons, although one group reported a hyperpolarizing action of GABA. In this study, we investigated the GABA-induced changes in [Ca(2+)](i) of GnRH neurons from GnRH-enhanced green fluorescent protein (GnRH-EGFP) rats both to confirm the depolarizing action of GABA and to further examine the developmental and estrous cycle-dependent modulations of GABA action. GABA increased [Ca(2+)](i) in GnRH neurons at all developmental stages of both sexes. GABA also increased [Ca(2+)](i) in adult female GnRH neurons prepared in the afternoon at each estrous cycle stage. The percentages of neurons with increased [Ca(2+)](i) were 90% in proestrus, 59% in estrus, 84% in diestrus I, and 89% in diestrus II. In GnRH neurons prepared from adult females in the morning, however, the percentage was significantly lower than in those prepared in the afternoon, except in estrus. The percentage was also lower in adult males than in adult females. GABA responses were mimicked by muscimol and blocked by bicuculline. In addition, removal of extracellular Ca(2+) completely suppressed the GABA action, and bumetanide attenuated the response. These results indicate that GABA depolarizes GnRH neurons by activating GABA(A) receptors, thereby activating voltage-gated Ca(2+) channels and facilitating Ca(2+) influx. In addition, the response to GABA is modulated according to the estrous cycle stage, diurnal rhythm, and sex.

Synaptic integration in hypothalamic gonadotropin releasing hormone (GnRH) neurons

The impact of the A-type GABA (GABA-A) receptor in gonadotropin releasing hormone (GnRH) neurons is controversial. In adult GnRH neurons, the GABA-A receptor conductance has been reported to either hyperpolarize or depolarize GnRH neurons. Regardless of whether GABA is inhibitory or excitatory in GnRH neurons, GABAergic input would be integrated with post-synaptic potentials generated by other synaptic inputs. We used dynamic current clamping and compartmental computer modeling to examine the integration of AMPA-type glutamatergic input and GABA-mediated input in both the hyperpolarizing (inhibitory) and depolarizing (excitatory) modes in GnRH neurons from transgenic mice (Mus Musculus) generated on a C57BL6 background. In both living and model neurons, action potentials were most likely a few ms after a maximum in AMPA conductance coincided with a minimum in inhibitory GABA. Excitatory GABA interacted differently with AMPA, with spikes most likely, in both dynamic clamping of living neurons and in model neurons, when a maximum in AMPA coincided with the decay from peak of a maximum in GABA. Distributing synapses along the dendrite maximized the temporal relationship between AMPA and GABA conductances and therefore, the potential for spiking. Thus, these two dominant neurotransmitters could interact in multiple frames to generate action potentials in GnRH neurons.

Activation of Neuronal Nitric Oxide Release Inhibits Spontaneous Firing in Adult Gonadotropin-Releasing Hormone Neurons: A Possible Local Synchronizing Signal

The activation of nitric oxide (NO) signaling pathways in hypothalamic neurons plays a key role in the control of GnRH secretion that is central to reproductive function. It is unknown whether NO directly modulates the firing behavior of GnRH neurons in the preoptic region of the mature brain. Using patch-clamp recordings from GnRH neurons expressing green fluorescent protein in adult mice brain slices, we demonstrate that the NO precursor, L-arginine (Arg), or the NO donor, diethylamine/NO, induced a robust and reversible reduction in the spontaneous firing activity of GnRH neurons, including bursting activity. The effects of L-Arg were prevented by the NO synthase inhibitor N omega-nitro-L-Arg methyl ester hydrochloride. Histochemical studies revealing a close anatomical relationship between neurons producing NO and GnRH perikarya, together with the loss of the L-Arg-mediated inhibition of GnRH neuronal activity via the selective blockade of neuronal NO synthase, suggested that the primary source of local NO production in the mouse preoptic region was neuronal. Synaptic transmission uncoupling did not alter the effect of NO, suggesting that NO affects the firing pattern of GnRH neurons by acting at a postsynaptic site. We also show that the NO-mediated changes in membrane properties in the GnRH neurons require soluble guanylyl cyclase activity and may involve potassium conductance. By revealing that NO is a direct modulator of GnRH neuronal activity, our results introduce the intriguing possibility that this gaseous neurotransmitter may be used by the sexual brain to modulate burst firing patterns. It may set into phase the bursting activity of GnRH neurons at key stages of reproductive physiology.

Cell Type-Specific Expression of a Genetically Encoded Calcium Indicator Reveals Intrinsic Calcium Oscillations in Adult Gonadotropin-Releasing Hormone Neurons

The gonadotropin-releasing hormone (GnRH) neurons exhibit a unique pattern of episodic activity to control fertility in all mammals. To enable the measurement of intracellular calcium concentration ([Ca2+]i) in adult GnRH neurons in situ, we generated transgenic mice in which the genetically encodable calcium indicator ratiometric Pericam was expressed by approximately 95% of GnRH neurons. Real-time monitoring of [Ca2+]i within adult male GnRH neurons in the acute brain slice revealed that approximately 70% of GnRH neurons exhibited spontaneous, 10-15 s duration [Ca2+]i transients with a mean frequency of 7 per hour. The remaining 30% of GnRH neurons did not exhibit calcium transients nor did a population of non-GnRH cells located within the lateral septum that express Pericam. Pharmacological studies using antagonists to the inositol-1,4,5-trisphosphate receptor (InsP3R) and several calcium channels, demonstrated that [Ca2+]i transients in GnRH neurons were generated by an InsP3R-dependent store-release mechanism and were independent of plasma membrane ligand- or voltage-gated calcium channels. Interestingly, the abolition of action potential-mediated transmission with tetrodotoxin reduced the number of [Ca2+]i transients in GnRH neurons by 50% (p < 0.05), suggesting a modulatory role for synaptic inputs on [Ca2+]i transient frequency. Using a novel transgenic strategy that enables [Ca2+]i to be examined in a specific neuronal phenotype in situ, we provide evidence for spontaneous [Ca2+]i fluctuations in adult GnRH neurons. This represents the initial description of spontaneous [Ca2+]i transients in mature neurons and shows that they arise from an InsP3R-generating mechanism that is further modulated by synaptic inputs.

Activation of Gonadotropin-Releasing Hormone Neurons by Kisspeptin as a Neuroendocrine Switch for the Onset of Puberty

We examined the role of kisspeptin and its receptor, the G-protein-coupled receptor GPR54, in governing the onset of puberty in the mouse. In the adult male and female mouse, kisspeptin (10-100 nM) evoked a remarkably potent, long-lasting depolarization of >90% of gonadotropin-releasing hormone (GnRH)-green fluorescent protein neurons in situ. In contrast, in juvenile [postnatal day 8 (P8) to P19] and prepubertal (P26-P33) male mice, kisspeptin activated only 27 and 44% of GnRH neurons, respectively. This developmental recruitment of GnRH neurons into a kisspeptin-responsive pool was paralleled by an increase in the ability of centrally administered kisspeptin to evoke luteinizing hormone secretion in vivo. To learn more about the mechanisms through which kisspeptin-GPR54 signaling at the GnRH neuron may change over postnatal development, we performed quantitative in situ hybridization for kisspeptin and GPR54 transcripts. Approximately 90% of GnRH neurons were found to express GPR54 mRNA in both juvenile and adult mice, without a detectable difference in the mRNA content between the age groups. In contrast, the expression of KiSS-1 mRNA increased dramatically across the transition from juvenile to adult life in the anteroventral periventricular nucleus (AVPV; p < 0.001). These results demonstrate that kisspeptin exerts a potent depolarizing effect on the excitability of almost all adult GnRH neurons and that the responsiveness of GnRH neurons to kisspeptin increases over postnatal development. Together, these observations suggest that activation of GnRH neurons by kisspeptin at puberty reflects a dual process involving an increase in kisspeptin input from the AVPV and a post-transcriptional change in GPR54 signaling within the GnRH neuron.

Dose-dependent switch in response of gonadotropin-releasing hormone (GnRH) neurons to GnRH mediated through the type I GnRH receptor.

Pulsatile release of GnRH provides central control of reproduction. GnRH neuron activity is likely synchronized to produce hormone pulses, but the mechanisms are largely unknown. One candidate for communication among these neurons is GnRH itself. Cultured embryonic and immortalized GnRH neurons express GnRH receptor type I (GnRHR-1), but expression has not been shown in adult GnRH neurons. Using mice that express green fluorescent protein (GFP) in GnRH neurons, we tested whether adult GnRH neurons express GnRHR-1. GFP-positive (n = 42) and -negative neurons (n = 22) were harvested from brain slices, and single-cell RT-PCR was performed with cell contents. Fifty-two percent of the GnRH neurons tested expressed GnRHR-1, but only 9% of non-GnRH hypothalamic neurons expressed GnRHR-1; no false harvest controls (n = 13) were positive. GnRHR-1 expression within GnRH neurons suggested a physiological ultrashort loop feedback role for GnRH. Thus, we examined the effect of GnRH on the firing rate of GnRH neurons. Low-dose GnRH (20 nm) significantly decreased firing rate in 12 of 22 neurons (by 42 +/- 4%, P < 0.05), whereas higher doses increased firing rate (200 nm, five of 10 neurons, 72 +/- 26%; 2000 nm, nine of 13 neurons, 53 +/- 8%). Interestingly, the fraction of GnRH neurons responding was similar to the fraction in which GnRHR-1 was detected. Together, these data demonstrate that a subpopulation of GnRH neurons express GnRHR-1 and respond to GnRH with altered firing. The dose dependence suggests that this autocrine control of GnRH neurons may be not only a mechanism for generating and modulating pulsatile release, but it may also be involved in the switch between pulse and surge modes of release.

Profiling γ-Aminobutyric Acid (GABAA) Receptor Subunit mRNA Expression in Postnatal Gonadotropin-Releasing Hormone (GnRH) Neurons of the Male Mouse with Single Cell RT-PCR

The present investigation has examined which subunits of the GABA_A receptor are expressed by gonadotropin-releasing hormone (GnRH) neurons in the juvenile and adult male mouse. Cells of defined morphology, located in the medial septum (MS) and rostral preoptic area (POA), were patch-clamped in the acute brain slice preparation and their cell contents extracted. A reverse transcriptase polymerase chain reaction (RT-PCR) procedure using nested primers was used to establish individual GnRH mRNA-expressing cells which were then evaluated for eleven GABA_A receptor (α1–5, β1–3, γ1–3) subunit transcripts. Single and multiple GABA_A receptor subunit mRNAs were detected in approximately 70% of all GnRH neurons. A range of different subunit mRNAs (α1, α2, α5, β1, β2, β3, γ2) were found in juvenile GnRH neurons, with the α1γ2 and α5γ2 combinations encountered most frequently within individual cells. The expression profile in adult GnRH neurons was more extensive than that detected in juveniles with α1, α2, α3, α5, β1, β2, β3, γ1 and γ2 subunits all being detected. The major difference in subunit profile between GnRH neurons located in the MS and POA involved the β subunits. The principal postnatal developmental change was one of increasing overall subunit heterogeneity in maturing POA GnRH neurons. The profile of GABA_A receptor subunit mRNAs detected in male GnRH neurons was quite different to that reported by us for female GnRH neurons in the mouse using the same RT-PCR approach. Together, these findings indicate that postnatal GnRH neurons are likely to express a range of GABA_A receptor subunit mRNAs in a sexually dimorphic and developmentally-regulated manner.

Heterogeneity in the Basic Membrane Properties of Postnatal Gonadotropin-Releasing Hormone Neurons in the Mouse

The electrophysiological characteristics of unmodified, postnatal gonadotropin-releasing hormone (GnRH) neurons in the female mouse were studied using whole-cell recordings and single-cell RT-PCR methodology. The GnRH neurons of adult animals fired action potentials and exhibited distinguishable voltage-current relationships in response to hyperpolarizing and depolarizing current injections. On the basis of their patterns of inward rectification, rebound depolarization, and ability to fire repetitively, GnRH neurons in intact adult females were categorized into four cell types (type I, 48%; type II, 36%; type III, 11%; type IV, 5%). The GnRH neurons of juvenile animals (15-22 d) exhibited passive membrane properties similar to those of adult GnRH neurons, although only type I (61%) and type II (7%) cells were encountered, in addition to a group of "silent-type" GnRH neurons (32%) that were unable to fire action potentials. A massive, action potential-independent tonic GABA input, signaling through the GABA(A) receptor, was present at all ages. Afterdepolarization and afterhyperpolarization potentials (AHPs) were observed after single action potentials in subpopulations of each GnRH neuron type. Tetrodotoxin (TTX)-independent calcium spikes, as well as AHPs, were encountered more frequently in juvenile GnRH neurons compared with adults. These observations demonstrate the existence of multiple layers of functional heterogeneity in the firing properties of GnRH neurons. Together with pharmacological experiments, these findings suggest that potassium and calcium channels are expressed in a differential manner within the GnRH phenotype. This heterogeneity occurs in a development-specific manner and may underlie the functional maturation and diversity of this unique neuronal phenotype.

Late postnatal reorganization of GABA(A) receptor signalling in native GnRH neurons.

The molecular and cellular characteristics of the gonadotropin-releasing hormone (GnRH) neurons have been difficult to ascertain due to their scattered distribution within the basal forebrain. Using morphological criteria coupled with single cell RT-PCR postidentification, we have developed a method for investigating native GnRH neurons in the mouse brain and used it to examine the development of GABA(A) receptor signalling in this phenotype. Following the harvesting of the cytoplasmic contents of individual GnRH neurons, single cell multiplex RT-PCR experiments demonstrated that GABAA receptor alpha1-5, beta1-3 and gamma2 & 3 subunit transcripts were expressed by both neonatal (postnatal day 5) and juvenile (day 15-20) GnRH neurons in a heterogeneous manner. Following puberty, this profile was reduced to a predominant alpha1, alpha5, beta1, gamma2 subunit complement in rostral preoptic area GnRH neurons of the adult female. Whole-cell patch-clamp recordings revealed little difference between juvenile and adult GnRH neurons in their resting membrane potential and spontaneous firing rates. All GnRH neurons were found to be subjected to a tetrodotoxin-insensitive, tonic GABAergic barrage signalling through the GABA(A) receptor. However, marked heterogeneity in the sensitivity of individual juvenile GnRH neurons to GABA was revealed and, in parallel with the change in subunit mRNA profile, this was dramatically reduced in the reproductively competent adult GnRH neurons. These findings provide the first electrical and molecular characterization of the GnRH phenotype and demonstrate a novel pattern of late postnatal reorganization of native GABA(A) receptor gene expression and signalling in the GnRH neuronal population.