Understanding calcium homeostasis in postnatal gonadotropin-releasing hormone neurons using cell-specific Pericam transgenics

The gonadotropin-releasing hormone (GnRH) neurons are the key output cells of a complex neuronal network controlling fertility in mammals. To examine calcium homeostasis in postnatal GnRH neurons, we generated a transgenic mouse line in which the genetically encodable calcium indicator ratiometric Pericam (rPericam) was targeted to the GnRH neurons. This mouse model enabled real-time imaging of calcium concentrations in GnRH neurons in the acute brain slice preparation. Investigations in GnRH-rPericam mice revealed that GnRH neurons exhibited spontaneous, long-duration (~8s) calcium transients. Dual electrical-calcium recordings revealed that the calcium transients were correlated perfectly with burst firing in GnRH neurons and that calcium transients in GnRH neurons regulated two calcium-activated potassium channels that, in turn, determined burst firing dynamics in these cells. Curiously, the occurrence of calcium transients in GnRH neurons across puberty or through the estrous cycle did not correlate well with the assumption that GnRH neuron burst firing was contributory to changing patterns of pulsatile GnRH release at these times. The GnRH-rPericam mouse was also valuable in determining differential mechanisms of GABA and glutamate control of calcium levels in GnRH neurons as well as effects of G-protein-coupled receptors for GnRH and kisspeptin. The simultaneous measurement of calcium levels in multiple GnRH neurons was hampered by variable rPericam fluorescence in different GnRH neurons. Nevertheless, in the multiple recordings that were achieved no evidence was found for synchronous calcium transients. Together, these observations show the great utility of transgenic targeting strategies for investigating the roles of calcium with specified neuronal cell types.     

Ghrelin Decreases Firing Activity of Gonadotropin-Releasing Hormone (GnRH) Neurons in an Estrous Cycle and Endocannabinoid Signaling Dependent Manner

The orexigenic peptide, ghrelin is known to influence function of GnRH neurons, however, the direct effects of the hormone upon these neurons have not been explored, yet. The present study was undertaken to reveal expression of growth hormone secretagogue receptor (GHS-R) in GnRH neurons and elucidate the mechanisms of ghrelin actions upon them. Ca²⁺-imaging revealed a ghrelin-triggered increase of the Ca²⁺-content in GT1-7 neurons kept in a steroid-free medium, which was abolished by GHS-R-antagonist JMV2959 (10µM) suggesting direct action of ghrelin. Estradiol (1nM) eliminated the ghrelin-evoked rise of Ca²⁺-content, indicating the estradiol dependency of the process. Expression of GHS-R mRNA was then confirmed in GnRH-GFP neurons of transgenic mice by single cell RT-PCR. Firing rate and burst frequency of GnRH-GFP neurons were lower in metestrous than proestrous mice. Ghrelin (40nM-4μM) administration resulted in a decreased firing rate and burst frequency of GnRH neurons in metestrous, but not in proestrous mice. Ghrelin also decreased the firing rate of GnRH neurons in males. The ghrelin-evoked alterations of the firing parameters were prevented by JMV2959, supporting the receptor-specific actions of ghrelin on GnRH neurons. In metestrous mice, ghrelin decreased the frequency of GABAergic mPSCs in GnRH neurons. Effects of ghrelin were abolished by the cannabinoid receptor type-1 (CB1) antagonist AM251 (1µM) and the intracellularly applied DAG-lipase inhibitor THL (10µM), indicating the involvement of retrograde endocannabinoid signaling. These findings demonstrate that ghrelin exerts direct regulatory effects on GnRH neurons via GHS-R, and modulates the firing of GnRH neurons in an ovarian-cycle and endocannabinoid dependent manner.     

Dual Somatic Recordings from Gonadotropin-Releasing Hormone (GnRH) Neurons Identified by Green Fluorescent Protein (GFP) in Hypothalamic Slices

Gonadotropin-Releasing Hormone (GnRH) is a small neuropeptide that regulates pituitary release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). These gonadotropins are essential for the regulation of reproductive function. The GnRH-containing neurons are distributed diffusely throughout the hypothalamus and project to the median eminence where they release GnRH from their axon terminals into the hypophysiotropic portal system (1). In the portal capillaries, GnRH travels to the anterior pituitary gland to stimulate release of gonadotropins into systemic circulation. GnRH release is not continuous but rather occurs in episodic pulses. It is well established that the intermittent manner of GnRH release is essential for reproduction (2, 3).Coordination of activity of multiple GnRH neurons probably underlies GnRH pulses. Total peptide content in GnRH neurons is approximately 1.0 pg/cell (4), of which 30% likely comprises the releasable pool. Levels of GnRH during a pulse (5, 6), suggest multiple GnRH neurons are probably involved in neurosecretion. Likewise, single unit activity extracted from hypothalamic multi-unit recordings during LH release indicates changes in activity of multiple neurons (7). The electrodes with recorded activity during LH pulses are associated with either GnRH somata or fibers (8). Therefore, at least some of this activity arises from GnRH neurons.The mechanisms that result in synchronized firing in hypothalamic GnRH neurons are unknown. Elucidating the mechanisms that coordinate firing in GnRH neurons is a complex problem. First, the GnRH neurons are relatively few in number. In rodents, there are 800-2500 GnRH neurons. It is not clear that all GnRH neurons are involved in episodic GnRH release. Moreover, GnRH neurons are diffusely distributed (1). This has complicated our understanding of coordination of firing and has made many technical approaches intractable. We have optimized loose cell-attached recordings in current-clamp mode for the direct detection of action potentials and developed a recording approach that allows for simultaneous recordings from pairs of GnRH neurons.     

Converse regulatory functions of estrogen receptor-alpha and -beta subtypes expressed in hypothalamic gonadotropin-releasing hormone neurons

Estradiol (E(2)) acts as a potent feedback molecule between the ovary and hypothalamic GnRH neurons, and exerts both positive and negative regulatory actions on GnRH synthesis and secretion. However, the extent to which these actions are mediated by estrogen receptors (ERs) expressed in GnRH neurons has been controversial. In this study, Single-cell RT-PCR revealed the expression of both ERalpha and ERbeta isoforms in cultured fetal and adult rat hypothalamic GnRH neurons. Both ERalpha and ERbeta or individual ERs were expressed in 94% of cultured fetal GnRH neurons. In adult female rats at diestrus, 68% of GnRH neurons expressed ERs, followed by 54% in estrus and 19% in proestrus. Expression of individual ERs was found in 24% of adult male GnRH neurons. ERalpha exerted marked G(i)-mediated inhibitory effects on spontaneous action potential (AP) firing, cAMP production, and pulsatile GnRH secretion, indicating its capacity for negative regulation of GnRH neuronal function. In contrast, increased E(2) concentration and ERbeta agonists increase the rate of AP firing, GnRH secretion, and cAMP production, consistent with ERbeta-dependent positive regulation of GnRH secretion. Consonant with the coupling of ERalpha to pertussis toxin-sensitive G(i/o) proteins, E(2) also activates G protein-activated inwardly rectifying potassium channels, decreasing membrane excitability and slowing the firing of spontaneous APs in hypothalamic GnRH neurons. These findings demonstrate that the dual actions of E(2) on GnRH neuronal membrane excitability, cAMP production, and GnRH secretion are mediated by the dose-dependent activation of ERalpha and ERbeta expressed in hypothalamic GnRH neurons.     

Expression of a functional g protein-coupled receptor 54-kisspeptin autoregulatory system in hypothalamic gonadotropin-releasing hormone neurons

The G protein-coupled receptor 54 (GPR54) and its endogenous ligand, kisspeptin, are essential for activation and regulation of the hypothalamic-pituitary-gonadal axis. Analysis of RNA extracts from individually identified hypothalamic GnRH neurons with primers for GnRH, kisspeptin-1, and GPR54 revealed expression of all three gene products. Also, constitutive and GnRH agonist-induced bioluminescence resonance energy transfer between Renilla luciferase-tagged GnRH receptor and GPR54 tagged with green fluorescent protein, expressed in human embryonic kidney 293 cells, revealed heterooligomerization of the two receptors. Whole cell patch-clamp recordings from identified GnRH neurons showed initial depolarizing effects of kisspeptin on membrane potential, followed by increased action potential firing. In perifusion studies, treatment of GT1-7 neuronal cells with kisspeptin-10 increased GnRH peak amplitude and duration. The production and secretion of kisspeptin in cultured hypothalamic neurons and GT1-7 cells were detected by a specific RIA and was significantly reduced by treatment with GnRH. The expression of kisspeptin and GPR54 mRNAs in identified hypothalamic GnRH neurons, as well as kisspeptin secretion, indicate that kisspeptins may act as paracrine and/or autocrine regulators of the GnRH neuron. Stimulation of GnRH secretion by kisspeptin and the opposing effects of GnRH on kisspeptin secretion indicate that GnRH receptor/GnRH and GPR54/kisspeptin autoregulatory systems are integrated by negative feedback to regulate GnRH and kisspeptin secretion from GnRH neurons.     

Whole-cell electrophysiology of gonadotropin-releasing hormone neurons that express green fluorescent protein in the terminal nerve of transgenic medaka (Oryzias latipes)

Gonadotropin-releasing hormone (GnRH) controls reproduction in vertebrates. Most studies have focused on the population of GnRH neurons in the hypothalamus that ultimately controls gonadal function. However, all vertebrates studied to date have two to three anatomically distinct populations of GnRH neurons that express different forms of this hormone. The purpose of the present study was to develop a new model for studying the population of GnRH neurons in the terminal nerve (TN) associated with the olfactory bulb and then to characterize their pattern of action potential firing to provide a foundation for understanding the role of these neurons in regulating reproduction. A stable line of transgenic medaka (Oryzias latipes) was generated in which a DNA construct containing the salmon GnRH (Gnrh3) promoter linked to green fluorescent protein (GFP) was expressed in TN-GnRH3 neurons. This population of GnRH neurons is located at or near the ventral surface of the brain, making them ideally situated for electrophysiological analysis. Whole-cell and loose-patch recordings in current-clamp mode were performed on these neurons from excised, intact brains of adult males in which afferent and efferent neural connections remained intact. All TN-GnRH3-GFP neurons that we recorded showed a beating pattern of spontaneous action potential firing. Action potentials were blocked by tetrodotoxin, indicating they are generated by a voltage-sensitive Na+ current; however, an oscillation in subthreshold membrane potential persisted. The present results indicate that this transgenic fish will provide an excellent model for studying the cell physiology of an extrahypothalamic population of GnRH neurons.     

Dendrites determine the contribution of after depolarization potentials (ADPs) to generation of repetitive action potentials in hypothalamic gonadotropin releasing-hormone (GnRH) neurons

The impact of structure in modulating synaptic signals originating in dendrites is widely recognized. In this study, we focused on the impact of dendrite morphology on a local spike generating mechanism which has been implicated in hormone secretion, the after depolarization potential (ADP). Using multi-compartmental models of hypothalamic GnRH neurons, we systematically truncated dendrite length and determined the consequence on ADP amplitude and repetitive firing. Decreasing the length of the dendrite significantly increased the amplitude of the ADP and increased repetitive firing. These effects were observed in dendrites both with and without active conductances suggesting they largely reflect passive characteristics of the dendrite. In order to test the findings of the model, we performed whole-cell recordings in GnRH neurons and elicited ADPs using current injection. During recordings, neurons were filled with biocytin so that we could determine dendritic and total projection (dendrite plus axon) length. Neurons exhibited ADPs and increasing ADP amplitude was associated with decreasing dendrite length, in keeping with the predictions of the models. Thus, despite the relatively simple morphology of the GnRH neuron’s dendrite, it can still exert a substantial impact on the final neuronal output. This work was supported by HD-45436 to KJS and by NCRR P20 RR16481 to Nigel Cooper.     

Impaired episodic LH secretion in female mice with GFP in GnRH neurons

The ability to assess the activity of gonadotropin-releasing hormone (GnRH) neurons has been greatly enhanced by transgenic animal models with targeted expression of green fluorescent protein (GFP). However, it has yet to be demonstrated that the GnRH system continues to exhibit a full range of normal physiological functions in the presence of such genetic manipulation. Accordingly, we have used repetitive blood sampling via indwelling venous catheters to define LH secretory patterns in normal and transgenic mice. Transgenic females proved to be reproductively competent as defined by fecundity, appropriate cyclic changes in vaginal cytology in intact adult females, and spontaneous LH surges as well as surges in response to steroid or mating stimuli. The expression of c-fos following such steroid treatment and mating in ovariectomized transgenics was similar to the expression previously reported in nontransgenic mice. Likewise, the percentage of retrogradely labeled GnRH neurons was similar to that reported in nontransgenic mice. However, episodic LH secretion, an index of GnRH pulse generator activity, was dramatically compromised in ovariectomized female transgenics compared with C57BL6 controls of both sexes and castrated transgenic males. Taken together, these findings suggest that the GnRH pulse generator is selectively impaired in ovariectomized females in which GnRH neurons express GFP.     

Estrogen modulates potassium currents and expression of the Kv4.2 subunit in GT1-7 cells

The proper maintenance of reproduction requires the pulsatile secretion of gonadotropin-releasing hormone (GnRH), which is ensured by synchronized periodic firing of multiple GnRH neurons. Both hormone secretion and electrophysiological properties of GnRH cells are influenced by estrogen. The impact of 17beta-estradiol treatment on the function of voltage gated A- and K-type potassium channels, known modulators of firing rate, was therefore examined in our experiments using immortalized GnRH-producing GT1-7 neurons. Whole cell patch clamp recordings showed the absence of the A-type current in GT1-7 cells cultured in estrogen-free medium and after 8h 17beta-estradiol treatment. Exposure of the cells to 17beta-estradiol for 24 and 48 h, respectively, resulted in the appearance of the A-type current. The induction of the A-type current by 17beta-estradiol was dose-related (50 pM to 15 nM range). In contrast, the K-type potassium current was apparent in the estrogen-free environment and 17beta-estradiol administration significantly decreased its amplitude. Co-administration of 17beta-estradiol and estrogen receptor blocker, Faslodex (ICI 182,780; 1 microM) abolished the occurrence of the A-type current. Real-time PCR data demonstrated that expression of the Kv4.2 subunit of the A-type channel was low at 0, 0.5, 2 and 8h, peaked at 24h and diminished at 48 h 17beta-estradiol treatment (15 nM). These data indicate that potassium channels of GT1-7 neurons are regulated by estrogen a mechanism that might contribute to modulation of firing rate and hormone secretion in GnRH neurons.     

Social regulation of the electrical properties of gonadotropin-releasing hormone neurons in a cichlid fish (Astatotilapia burtoni)

Variation in reproductive capacity is common across the lives of all animals. In vertebrates, hypothalamic neurons that secrete GnRH are a primary mediator of such reproductive plasticity. Since social interactions suppress gonadal maturity in the African cichlid fish, Astatotilapia (Haplochromis) burtoni, we investigated whether the electrical properties of GnRH neurons were also socially regulated. Adult A. burtoni males are either territorial (T) and reproductively active or nonterritorial (NT) and reproductively regressed, depending upon their social environment. We compared the basic electrical properties of hypothalamic GnRH neurons from T and NT males using whole-cell electrophysiology in vitro. GnRH neurons were spontaneously active and exhibited several different activity patterns. A small fraction of neurons exhibited episodic activity patterns, which have been described in GnRH neurons from mammals. The type of activity pattern and spontaneous firing rate did not vary with reproductive capacity; however, several basic electrical properties were different. Neurons from T males were larger than those from NT males and had higher membrane capacitance and lower input resistance. In neurons from NT males, action potential duration was significantly longer and after-hyperpolarization characteristics were diminished, which led to a tendency for neurons from NT males to fire less rapidly in response to current injection. We predict this could serve to decrease GnRH release in NT males. These data are the first electrophysiological characterization of hypothalamic GnRH neurons in a nonmammalian species and provide evidence for several changes in electrical properties with reproductive state.     

An Immortal Cell Culture Model of Hypothalamic Gonadotropin-Releasing Hormone Neurons

The neuroendocrine hypothalamus has been the object of intensive study in vivo and in tissue slices. However, using these models it is difficult to approach questions at the molecular and cellular level and to differentiate between direct effects and those mediated by other neurons. By using the regulatory domain of the rat gonadotropin-releasing hormone (GnRH) gene to target expression of the oncogene SV40 T antigen in transgenic mice, we have produced hypothalamic tumors which were cultured to produce clonal cell lines (GT1 cells). These cells express GnRH and many other neuronal markers, but do not express glial cell markers or other hypothalamic hormones. They have a distinctive neuronal phenotype, process the GnRH peptide accurately, and secrete GnRH in a pulsatile pattern. They respond to many neurotransmitters and neuromodulators including activin, norepinephrine, dopamine, nitric oxide, NMDA, and GABA, as well as GnRH itself. Thus, we have immortalized GnRH neurons by targeting oncogenesis to a defined population of neurons using the regulatory region of a gene which is expressed late in the differentiation of that cell lineage. The GT1 cell lines serve as an excellent model for molecular, pharmacological, electrophysiological, and biochemical investigations into the regulation of GnRH and the characteristics of a pure CNS neuronal population. Moreover, their derivation demonstrates the success of targeting tumorigenesis to specific differentiated neurons of the central nervous system in transgenic mice.     

Immortalization of hypothalamic GnRH neurons by genetically targeted tumorigenesis

By genetically targeting tumorigenesis to specific hypothalamic neurons in transgenic mice using the promoter region of the gonadotropin-releasing hormone (GnRH) gene to express the SV40 T-antigen oncogene, we have produced neuronal tumors and developed clonal, differentiated, neurosecretory cell lines. These cells extend neurites, express the endogenous mouse GnRH mRNA, release GnRH in response to depolarization, have regulatable fast Na+ channels found in neurons, and express neuronal, but not glial, cell markers. These immortalized cells will provide an invaluable model system for study of hypothalamic neurosecretory neurons that regulate reproduction. Significantly, their derivation demonstrates the feasibility of immortalizing differentiated neurons by targeting tumorigenesis in transgenic mice to specific neurons of the CNS.     

Regulation of Electrical Bursting in a Spatiotemporal Model of a GnRH Neuron

Gonadotropin-releasing hormone (GnRH) neurons are hypothalamic neurons that control the pulsatile release of GnRH that governs fertility and reproduction in mammals. The mechanisms underlying the pulsatile release of GnRH are not well understood. Some mathematical models have been developed previously to explain different aspects of these activities, such as the properties of burst action potential firing and their associated Ca²⁺ transients. These previous studies were based on experimental recordings taken from the soma of GnRH neurons. However, some research groups have shown that the dendrites of GnRH neurons play very important roles. In particular, it is now known that the site of action potential initiation in these neurons is often in the dendrite, over 100 μm from the soma. This raises an important question. Since some of the mechanisms for controlling the burst length and interburst interval are located in the soma, how can electrical bursting be controlled when initiated at a site located some distance from these controlling mechanisms? In order to answer this question, we construct a spatio-temporal mathematical model that includes both the soma and the dendrite. Our model shows that the diffusion coefficient for the spread of electrical potentials in the dendrite is large enough to coordinate burst firing of action potentials when the initiation site is located at some distance from the soma.     

Effect of steroid milieu on gonadotropin-releasing hormone-1 neuron firing pattern and luteinizing hormone levels in male mice

GnRH neuronal function is regulated by gonadal hormone feedback. In males, testosterone can act directly or be converted to either dihydrotestosterone (DHT) or estradiol (E2). We examined central steroid feedback by recording firing of green fluorescent protein (GFP)-identified GnRH neurons in brain slices from male mice that were intact, castrated, or castrated and treated with implants containing DHT, E2, or E2 + DHT. Castration increased LH levels. DHT or E2 alone partially suppressed LH, whereas E2 + DHT reduced LH to intact levels. Despite the inhibitory actions on LH, the combination of E2 + DHT increased GnRH neuron activity relative to other treatments, reflected in mean firing rate, amplitude of peaks in firing rate, and area under the curve of firing rate vs. time. Cluster8 was used to identify peaks in firing activity that may be correlated with hormone release. Castration increased the frequency of peaks in firing rate. Treatment with DHT failed to reduce frequency of these peaks. In contrast, treatment with E2 reduced peak frequency to intact levels. The frequency of peaks in firing rate was intermediate in animals treated with E2 + DHT, perhaps suggesting the activating effects of this combination partially counteracts the inhibitory actions of E2. These data indicate that E2 mediates central negative feedback in males primarily by affecting the pattern of GnRH neuron activity, and that androgens combined with estrogens have a central activating effect on GnRH neurons. The negative feedback induced by E2 + DHT to restore LH to intact levels may mask an excitatory central effect of this combination.     

Gonadotropin-releasing hormone (GnRH) receptor expression and membrane signaling in early embryonic GnRH neurons: role in pulsatile neurosecretion

The characteristic pulsatile secretion of GnRH from hypothalamic neurons is dependent on an autocrine interaction between GnRH and its receptors expressed in GnRH-producing neurons. The ontogeny and function of this autoregulatory process were investigated in studies on the properties of GnRH neurons derived from the olfactory placode of the fetal rat. An analysis of immunocytochemically identified, laser-captured fetal rat hypothalamic GnRH neurons, and olfactory placode-derived GnRH neurons identified by differential interference contrast microscopy, demonstrated coexpression of mRNAs encoding GnRH and its type I receptor. Both placode-derived and immortalized GnRH neurons (GT1-7 cells) exhibited spontaneous electrical activity that was stimulated by GnRH agonist treatment. This evoked response, as well as basal neuronal firing, was abolished by treatment with a GnRH antagonist. GnRH stimulation elicited biphasic intracellular calcium ([Ca2+]i) responses, and both basal and GnRH-stimulated [Ca2+]i levels were reduced by antagonist treatment. Perifused cultures released GnRH in a pulsatile manner that was highly dependent on extracellular Ca2+. The amplitude of GnRH pulses was increased by GnRH agonist stimulation and was diminished during GnRH antagonist treatment. These findings demonstrate that expression of GnRH receptor, GnRH-dependent activation of Ca2+ signaling, and autocrine regulation of GnRH release are characteristics of early fetal GnRH neurons and could provide a mechanism for gene expression and regulated GnRH secretion during embryonic migration.     

Interleukin-6 stimulates gonadotropin-releasing hormone secretion from rat hypothalamic cells

There are reports that both interleukin-1 beta and interleukin-6 (IL-6) stimulate the release of adrenocorticotropin through stimulation of hypothalamic corticotropin-releasing factor. We established a primary culture system for hypothalamic neurons producing gonadotropin-releasing hormone (GnRH) and examined whether IL-6 stimulated their GnRH secretion. We demonstrated immunohistochemically that some of these neurons contained GnRH-like immunoreactivity. In primary cultures of these GnRH neurons, we found that the calcium ionophore A23187 stimulated GnRH secretion in a dose- and time-dependent manner. These hypothalamic cells secreted IL-6 spontaneously, producing about 10 ng/l in 24 h, and their IL-6 secretion was significantly stimulated by E2 at 10(-9)-10(-8) mol/l. This stimulatory effect was observed within 3 h. IL-6 also stimulated the release of GnRH in a dose- and time-dependent manner, and these effects of IL-6 were significantly blocked by anti-IL-6 antiserum. These results suggest that the central action of IL-6 on the GnRH neurons may be an important physiological event in the hypothalamus.     

Ion channel properties and episodic activity in isolated immortalized gonadotropin-releasing hormone (GnRH) neurons

The mechanism of periodic gonadotropin-releasing hormone (GnRH) secretion from hypothalamic neurons is difficult to elucidate due to the diffuse distribution of GnRH neurons and the complex interaction of neuronal inputs onto them. Recent use of transgenic techniques allowed construction of an immortalized GnRH neuronal cell line (GT1), which has neuronal markers and secretes GnRH in a periodic fashion. Using the patch-clamp recording technique in the whole-cell and nystatin perforated-patch configuration, the present experiments show that this cell line expressed a tetrodotoxin-sensitive Na channel, two types of Ca channels, three types of outward K channels and a K inward rectifier. The latter current was suppressed in some cells by GnRH or somatostatin. In addition, a gamma-aminobutyric acid (GABA) response, presumably through GABA_A receptors, is recorded. In long-term current-clamp recordings, spontaneous depolarizing activity was found to increase, and then decrease, between 20–35 min after removal of the cells from serum- and steroid-containing medium. In some cases, more than one cycle of activity was seen. Under voltage clamp, an inward current was recorded at similar times, with reversal at about ?15 mV. Thus, two mechanisms of cell interaction, GABA_A responses and feedback through GnRH responses, and one mechanism of endogenous periodic electrical activity were observed in these cells, which could synchronize periodic GnRH release.     

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.     

Progesterone stimulation of in vitro GnRH release from the hypothalamus of the estrogen-primed, ovariectomized rat: serotoninergic role

The ability of ovarian steroids to affect luteinizing hormone secretion is closely related to the influence of these steroids on the activities of several neurotransmitter systems within specific areas of the hypothalamus and associated brain areas. The purpose of this study was to characterize in vitro progestagenic effects on serotonin (5-hydroxytryptamine, 5-HT) and gonadotropin-releasing hormone (GnRH) release from hypothalamic slices from estrogen-primed, ovariectomized rats. Results of this study show that (1) progesterone can stimulate in vitro GnRH and 5-HT release from hypothalamic tissue slices of ovariectomized rats primed with estrogen and (2) the 5-HT receptor antagonist mianserin blocks the ability of progesterone to augment in vitro GnRH release from these tissue slices. This suggests that the influence of progesterone on the estrogen-induced LH surge is, at least in part, via progestagenic release of 5-HT and the subsequent effect of this neurotransmitter on the release of GnRH within the hypothalamus.