Effects of kisspeptin1 on electrical activity of an extrahypothalamic population of gonadotropin-releasing hormone neurons in medaka (Oryzias latipes)

Kisspeptin (product of the kiss1 gene) is the most potent known activator of the hypothalamo-pituitary-gonadal axis. Both kiss1 and the kisspeptin receptor are highly expressed in the hypothalamus of vertebrates, and low doses of kisspeptin have a robust and long-lasting stimulatory effect on the rate of action potential firing of hypophysiotropic gonadotropin releasing hormone-1 (GnRH1) neurons in mice. Fish have multiple populations of GnRH neurons distinguished by their location in the brain and the GnRH gene that they express. GnRH3 neurons located in the terminal nerve (TN) associated with the olfactory bulb are neuromodulatory and do not play a direct role in regulating pituitary-gonadal function. In medaka fish, the electrical activity of TN-GnRH3 neurons is modulated by visual cues from conspecifics, and is thought to act as a transmitter of information from the external environment to the central nervous system. TN-GnRH3 neurons also play a role in sexual motivation and arousal states, making them an important population of neurons to study for understanding coordination of complex behaviors. We investigated the role of kisspeptin in regulating electrical activity of TN-GnRH3 neurons in adult medaka. Using electrophysiology in an intact brain preparation, we show that a relatively brief treatment with 100 nM of kisspeptin had a long-lasting stimulatory effect on the electrical activity of an extrahypothalamic population of GnRH neurons. Dose-response analysis suggests a relatively narrow activational range of this neuropeptide. Further, blocking action potential firing with tetrodotoxin and blocking synaptic transmission with a low Ca(2+)/high Mg(2+) solution inhibited the stimulatory action of kisspeptin on electrical activity, indicating that kisspeptin is acting indirectly through synaptic regulation to excite TN-GnRH3 neurons. Our findings provide a new perspective on kisspeptin's broader functions within the central nervous system, through its regulation of an extrahypothalamic population of GnRH neurons involved in multiple neuromodulatory functions.     

Hormone secretion in transgenic rats and electrophysiological activity in their gonadotropin releasing-hormone neurons

Expression of GFP in GnRH neurons has allowed for studies of individual GnRH neurons. We have demonstrated previously the preservation of physiological function in male GnRH-GFP mice. In the present study, we confirm using biocytin-filled GFP-positive neurons in the hypothalamic slice preparation that GFP-expressing somata, axons, and dendrites in hypothalamic slices from GnRH-GFP rats are GnRH1 peptide positive. Second, we used repetitive sampling to study hormone secretion from GnRH-GFP transgenic rats in the homozygous, heterozygous, and wild-type state and between transgenic and Wistar males after ~4 yr of backcrossing. Parameters of hormone secretion were not different between the three genetic groups or between transgenic males and Wistar controls. Finally, we performed long-term recording in as many GFP-identified GnRH neurons as possible in hypothalamic slices to determine their patterns of discharge. In some cases, we obtained GnRH neuronal recordings from individual males in which blood samples had been collected the previous day. Activity in individual GnRH neurons was expressed as total quiescence, a continuous pattern of firing of either low or relatively high frequencies or an intermittent pattern of firing. In males with both intensive blood sampling (at 6-min intervals) and recordings from their GnRH neurons, we analyzed the activity of GnRH neurons with intermittent activity above 2 Hz using cluster analysis on both data sets. The average number of pulses was 3.9 ± 0.6/h. The average number of episodes of firing was 4.0 ± 0.6/h. Therefore, the GnRH pulse generator may be maintained in the sagittal hypothalamic slice preparation.     

Mechanisms of the modulation of pacemaker activity by GnRH peptides in the terminal nerve-GnRH neurons

According to our working hypothesis, the terminal nerve (TN)-gonadotropin releasing hormone (GnRH) system functions as a neuromodulatory system that regulates many long-lasting changes in animal behaviors. We have already shown by using in vitro whole brain preparations of a small fish (dwarf gourami) that the pacemaker activities of TN-GnRH neurons are modulated biphasically by salmon GnRH, which is the same molecular species of GnRH produced by TN-GnRH neurons themselves; the modulation consists of initial transient decrease and late increase of firing frequency. In the present study, we investigated the possible involvement of Ca2+ release from intracellular store and voltage dependent Ca2+ currents in the modulation of pacemaker activities. Pharmacological blockade of Ca2+ release from intracellular stores or apamin-sensitive Ca(2+)-activated K+ current inhibited the initial transient decrease of firing frequency by sGnRH. On the other hand, bath application of Ca2+ channel blockers Ni2+ or La3+ slowed down the pacemaker frequency and attenuated the rate of the late increase of pacemaker frequency by GnRH. Furthermore, voltage-clamp experiments suggested that low-voltage-activated (LVA) Ca2+ current and hihg-voltage-activated (HVA) Ca2+ current were present in the TN-GnRH neurons, and bath application of GnRH shifted the activation threshold of HVA Ca2+ current to more negative potentials. These results suggest that (1) sGnRH induces Ca2+ release from intracellular stores and activates apaminsensitive Ca(2+)-activated K+ current so that it decreases the frequency of pacemaker activity in the initial phase, (2) some kinds of Ca2+ currents contribute to the generation and modulation of pacemaker activities, and (3) HVA Ca2+ current is facilitated by sGnRH so that it increases the frequency of pacemaker activity in the late phase.     

Kisspeptins Modulate the Biology of Multiple Populations of Gonadotropin-Releasing Hormone Neurons during Embryogenesis and Adulthood in Zebrafish (Danio rerio)

Kisspeptin1 (product of the Kiss1 gene) is the key neuropeptide that gates puberty and maintains fertility by regulating the gonadotropin-releasing hormone (GnRH) neuronal system in mammals. Inactivating mutations in Kiss1 and the kisspeptin receptor (GPR54/Kiss1r) are associated with pubertal failure and infertility. Kiss2, a paralogous gene for kiss1, has been recently identified in several vertebrates including zebrafish. Using our transgenic zebrafish model system in which the GnRH3 promoter drives expression of emerald green fluorescent protein, we investigated the effects of kisspeptins on development of the GnRH neuronal system during embryogenesis and on electrical activity during adulthood. Quantitative PCR showed detectable levels of kiss1 and kiss2 mRNA by 1 day post fertilization, increasing throughout embryonic and larval development. Early treatment with Kiss1 or Kiss2 showed that both kisspeptins stimulated proliferation of trigeminal GnRH3 neurons located in the peripheral nervous system. However, only Kiss1, but not Kiss2, stimulated proliferation of terminal nerve and hypothalamic populations of GnRH3 neurons in the central nervous system. Immunohistochemical analysis of synaptic vesicle protein 2 suggested that Kiss1, but not Kiss2, increased synaptic contacts on the cell body and along the terminal nerve-GnRH3 neuronal processes during embryogenesis. In intact brain of adult zebrafish, whole-cell patch clamp recordings of GnRH3 neurons from the preoptic area and hypothalamus revealed opposite effects of Kiss1 and Kiss2 on spontaneous action potential firing frequency and membrane potential. Kiss1 increased spike frequency and depolarized membrane potential, whereas Kiss2 suppressed spike frequency and hyperpolarized membrane potential. We conclude that in zebrafish, Kiss1 is the primary stimulator of GnRH3 neuronal development in the embryo and an activator of stimulating hypophysiotropic neuron activities in the adult, while Kiss2 plays an additional role in stimulating embryonic development of the trigeminal neuronal population, but is an RFamide that inhibits electrical activity of hypophysiotropic GnRH3 neurons in the adult.     

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.     

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.     

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.     

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.     

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.     

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.     

Control of GnRH expression in the olfactory lobe of Octopus vulgaris

In the cephalopod mollusk Octopus vulgaris, the gonadotropic hormone released by the optic gland controls sexual maturity. Several lobes of the central nervous system control the activity of this gland. In one of these lobes, the olfactory lobe, a gonadotropin releasing hormone (GnRH) neuronal system has been described. We assume that several inputs converge on the olfactory lobes in order to activate GnRH neurons and that a glutamatergic system mediates the integration of stimuli on these neuropeptidergic neurons. The presence of N-methyl-d-aspartate (NMDA) receptor immunoreactivity in the neuropil of olfactory lobes and in the fibers of the optic gland nerve, along with the GnRH nerve endings strongly supports this hypothesis. A distinctive role in the control of GnRH secretion has also been attributed, in vertebrates, to nitric oxide (NO). The lobes and nerves involved in the nervous control of reproduction in Octopus contain nitric oxide synthase (NOS). Using a set of experiments aimed at manipulate a putative l-glutamate/NMDA/NO signal transduction pathway, we have demonstrated, by quantitative real-time PCR, that NMDA enhances the expression of GnRH mRNA in a dose-response manner. The reverting effect of a selective antagonist of NMDA receptors (NMDARs), 2-amino-5-phosphopentanoic acid (D-APV), confirms that such an enhancing action is a NMDA receptor-mediated response. Nitric oxide and calcium also play a positive role on GnRH mRNA expression. The results suggest that in Octopusl-glutamate could be a key molecule in the nervous control of sexual maturation.     

Activation of A-type gamma-aminobutyric acid receptors excites gonadotropin-releasing hormone neurons

Gamma-aminobutyric acid (GABA), acting through GABA(A) receptors (GABA(A)R), is hypothesized to suppress reproduction by inhibiting GnRH secretion, but GABA actions directly on GnRH neurons are not well established. In green fluorescent protein-identified adult mouse GnRH neurons in brain slices, gramicidin-perforated-patch-clamp experiments revealed the reversal potential (E(GABA)) for current through GABA(A)Rs was depolarized relative to the resting potential. Furthermore, rapid GABA application elicited action potentials in GnRH neurons but not controls. The consequence of GABA(A)R activation depends on intracellular chloride levels, which are maintained by homeostatic mechanisms. Membrane proteins that typically extrude chloride (KCC-2 cotransporter, CLC-2 channel) were absent from the GT1-7 immortalized GnRH cell line and GnRH neurons in situ or were not localized to the proper cell compartment for function. In contrast, GT1-7 cells and some GnRH neurons expressed the chloride-accumulating cotransporter, NKCC-1. Patch-clamp experiments showed that blockade of NKCC hyperpolarized E(GABA) by lowering intracellular chloride. Regardless of reproductive state, rapid GABA application excited GnRH neurons. In contrast, bath application of the GABA(A)R agonist muscimol transiently increased then suppressed firing; suppression persisted 4-15 min. Rapid activation of GABA(A)R thus excites GnRH neurons whereas prolonged activation reduces excitability, suggesting the physiological consequence of synaptic activation of GABA(A)R in GnRH neurons is excitation.     

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.     

Maternal dexamethasone exposure during pregnancy in rats disrupts gonadotropin-releasing hormone neuronal development in the offspring

The migration of gonadotropin-releasing hormone (GnRH) neurons from the olfactory placode to the preoptic area (POA) from embryonic day 13 is important for successful reproduction during adulthood. Whether maternal glucocorticoid exposure alters GnRH neuronal morphology and number in the offspring is unknown. This study determines the effect of maternal dexamethasone (DEX) exposure on enhanced green fluorescent protein (EGFP) driven by GnRH promoter neurons (TG-GnRH) in transgenic rats dual-labelled with GnRH immunofluorescence (IF-GnRH). The TG-GnRH neurons were examined in intact male and female rats at different postnatal ages, as a marker for GnRH promoter activity. Pregnant females were subcutaneously injected with DEX (0.1 mg/kg) or vehicle daily during gestation days 13–20 to examine the number of GnRH neurons in P0 male offspring. The total number of TG-GnRH neurons and TG-GnRH/IF-GnRH neuronal ratio increased from P0 and P5 stages to P47–52 stages, suggesting temporal regulation of GnRH promoter activity during postnatal development in intact rats. In DEX-treated P0 males, the number of IF-GnRH neurons decreased within the medial septum, organum vasculosom of the lamina terminalis (OVLT) and anterior hypothalamus. The percentage of TG-GnRH neurons with branched dendritic structures decreased in the OVLT of DEX-P0 males. These results suggest that maternal DEX exposure affects the number and dendritic development of early postnatal GnRH neurons in the OVLT/POA, which may lead to altered reproductive functions in adults.     

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.     

Effect of environmental temperature on growth- and reproduction-related hormones gene expression in the female blue gourami (Trichogaster trichopterus)

Fish are ectothermic vertebrates, and their gonadal development and spawning are affected by changes in environmental temperature. Recent global temperature changes have increased the importance of studying the effect of temperature on reproduction. The aim of this paper was to study the effect of temperature on oogenesis and hormone gene expression related to reproduction and growth in the blue gourami female maintained under non-reproductive and reproductive conditions. In females under non-reproductive conditions, vitellogenic oocytes, gonadotropin-releasing hormone 3 (GnRH3), β luteinizing hormone (βLH) and growth hormone (GH) mRNA levels were affected by temperature changes. In females maintained under reproductive conditions with non-reproductively active males, a percentage of females in the final oocyte maturation (FOM) stage, pituitary adenylyl cyclase activating polypeptide (PACAP and PRP-PACAP), gonadotropins and GH mRNA levels were affected due to temperature changes. In females maintained under reproductive conditions with reproductively active males, also GnRH3 and insulin-like growth factor 1 (IGF-1) were affected by temperature changes. In conclusion, in blue gourami females, changes in environmental temperature affect oogenesis through changes in brain and pituitary hormone mRNA levels.     

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.     

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.     

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.