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.     

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.     

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.     

Axl and Tyro3 modulate female reproduction by influencing gonadotropin-releasing hormone neuron survival and migration

GnRH neurons must undergo a complex and precise pattern of neuronal migration to appropriately target their projections to the median eminence to trigger gonadotropin secretion and thereby control reproduction. Using NLT GnRH cells as a model of early GnRH neuronal development, we identified the potential importance of Axl and Tyro3, members of the TAM (Tyro3, Axl, and Mer) family of receptor tyrosine kinases in GnRH neuronal cell survival and migration. Silencing studies evaluated the role of Tyro3 and Axl in NLT GnRH neuronal cells and suggest that both play a role in Gas6 stimulation of GnRH neuronal survival and migration. Analysis of mice null for both Axl and Tyro3 showed normal onset of vaginal opening but delayed first estrus and persistently abnormal estrous cyclicity compared with wild-type controls. Analysis of GnRH neuronal numbers and positioning in the adult revealed a total loss of 24% of the neuronal network that was more striking (34%) when considered within specific anatomical compartments, with the largest deficit surrounding the organum vasculosum of the lamina terminalis. Analysis of GnRH neurons during embryogenesis identified a striking loss of immunoreactive cells within the context of the ventral forebrain compartment (36%) and not more rostrally. Studies using caspase 3 cleavage as a marker of apoptosis showed that Axl(-/-), Tyro3(-/-) double-knockout mice had increased cell death in the nose and dorsal forebrain, supporting the underlying mechanism of cell loss. Together these data suggest that Axl and Tyro3 mediate the survival and appropriate targeting of GnRH neurons to the ventral forebrain, thereby contributing to normal reproductive function and cyclicity in the female.     

Development of a transgenic green fluorescent protein lineage marker for steroidogenic factor 1

Knockout mice lacking steroidogenic factor 1 (SF-1, officially designated Nr5a1) have a complex phenotype that includes adrenal and gonadal agenesis, impaired expression of pituitary gonadotropins, and structural abnormalities of the ventromedial hypothalamic nucleus. To explore further how SF-1 regulates endocrine function, we used bacterial artificial chromosome transgenesis to develop a lineage marker for SF-1-expressing cells. A genomic fragment containing 50 kb of the mouse Nr5a1 gene was used to target enhanced green fluorescent protein (eGFP) in transgenic mice. These sequences directed eGFP to multiple cell lineages that express SF-1, including steroidogenic cells of the adrenal cortex, testes, and ovaries, neurons of the ventromedial hypothalamic nucleus, and reticuloendothelial cells of the spleen. Despite the proven role of SF-1 in gonadotrope function, eGFP was not expressed in the anterior pituitary. These experiments show that 50 kb of the mouse Nr5a1 gene can target transgenic expression to multiple cell lineages that normally express SF-1. The SF-1/eGFP transgenic mice will facilitate approaches such as fluorescence-activated cell sorting of eGFP-positive cells and DNA microarray analyses to expand our understanding of the multiple actions of SF-1 in endocrine development and function.     

Definition of estrogen receptor pathway critical for estrogen positive feedback to gonadotropin-releasing hormone neurons and fertility

The mechanisms through which estrogen regulates gonadotropin-releasing hormone (GnRH) neurons to control mammalian ovulation are unknown. We found that estrogen positive feedback to generate the preovulatory gonadotropin surge was normal in estrogen receptor beta knockout (ERbeta) mutant mice, but absent in ERalpha mutant mice. An ERalpha-selective compound was sufficient to generate positive feedback in wild-type mice. As GnRH neurons do not express ERalpha, estrogen positive feedback upon GnRH neurons must be indirect in nature. To establish the cell type responsible, we generated a neuron-specific ERalpha mutant mouse line. These mice failed to exhibit estrogen positive feedback, demonstrating that neurons expressing ERalpha are critical. We then used a GnRH neuron-specific Pseudorabies virus (PRV) tracing approach to show that the ERalpha-expressing neurons innervating GnRH neurons are located within rostral periventricular regions of the hypothalamus. These studies demonstrate that ovulation is driven by estrogen actions upon ERalpha-expressing neuronal afferents to GnRH neurons.     

Immortalized retinal neurons derived from SV40 T-antigen-induced tumors in transgenic mice

Immortalized retinal neurons have been established in tissue culture from retinal tumors arising in transgenic mice. The mice carry the SV40 T-antigen under the control of 5' flanking sequences from the human phenylethanolamine N-methyltransferase (PNMT) gene in order to target oncogene expression to adrenergic cell types. The retinal cultures contain a proliferation population of T-antigen-positive cells with a neuronal morphology that includes formation of extensive neuritic processes. We identified the cells as amacrine-derived neurons by immunofluorescence using the cell-specific monoclonal antibodies VC1.1 and HPC-1. The cells also express all three neurofilament subunits and GAP-43. These results indicate that CNS neurons can be transformed in transgenic animals to generate cultured cells with many properties of mature 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.     

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.     

Estrogen enhances expression of the complement C5a receptor and the C5a-agonist evoked calcium influx in hormone secreting neurons of the hypothalamus

In the present study we examined presence of the complement C5a receptor (C5aR) in hypothalamic neurosecretory neurons of the rodent brain and effect of estrogen on C5aR expression. Whole cell patch clamp measurements revealed that magnocellular neurons in the supraoptic and paraventricular nuclei of hypothalamic slices of the rats responded to the C5aR-agonist PL37-MAP peptide with calcium ion current pulses. Gonadotropin-releasing hormone (GnRH) producing neurons in slices of the preoptic area of the mice also reacted to the peptide treatment with inward calcium current. PL37-MAP was able to evoke the inward ion current of GnRH neurons in slices from ovariectomized animals. The amplitude of the inward pulses became higher in slices obtained from 17beta-estradiol (E2) substituted mice. Calcium imaging experiments demonstrated that PL37-MAP increased the intracellular calcium content in the culture of the GnRH-producing GT1-7 cell line in a concentration-dependent manner. Calcium imaging also showed that E2 pretreatment elevated the PL37-MAP evoked increase of the intracellular calcium content in the GT1-7 cells. The estrogen receptor blocker Faslodex in the medium prevented the E2-evoked increase of the PL37-MAP-triggered elevation of the intracellular calcium content in the GT1-7 cells demonstrating that the effect of E2 might be related to the presence of estrogen receptor. Real-time PCR experiments revealed that E2 increased the expression of C5aR mRNA in GT1-7 neurons, suggesting that an increased C5aR synthesis could be involved in the estrogenic modulation of calcium response. These data indicate that hypothalamic neuroendocrine neurons can integrate immune and neuroendocrine functions. Our results may serve a better understanding of the inflammatory and neurodegeneratory diseases of the hypothalamus and the related neuroendocrine and autonomic compensatory responses.     

Calcium receptor stimulates chemotaxis and secretion of MCP-1 in GnRH neurons in vitro: potential impact on reduced GnRH neuron population in CaR-null mice

The factors controlling the migration of mammalian gonadotropin-releasing hormone (GnRH) neurons from the nasal placode to the hypothalamus are not well understood. We studied whether the extracellular calcium-sensing receptor (CaR) promotes migration/chemotaxis of GnRH neurons. We demonstrated expression of CaR in GnRH neurons in the murine basal forebrain and in two GnRH neuronal cell lines: GT1-7 (hypothalamus derived) and GN11 (olfactory bulb derived). Elevated extracellular Ca(2+) concentrations promoted chemotaxis of both cell types, with a greater effect in GN11 cells. This effect was CaR mediated, as, in both cell types, overexpression of a dominant-negative CaR attenuated high Ca(2+)-stimulated chemotaxis. We also demonstrated expression of a beta-chemokine, monocyte chemoattractant protein-1 (MCP-1), and its receptor, CC motif receptor-2 (CCR2), in the hypothalamic GnRH neurons as well as in GT1-7 and GN11 cells. Exogenous MCP-1 stimulated chemotaxis of both cell lines in a dose-dependent fashion; the effect was greater in GN11 than in GT1-7 cells, consistent with the higher CCR2 mRNA levels in GN11 cells. Activating the CaR stimulated MCP-1 secretion in GT1-7 but not in GN11 cells. MCP-1 secreted in response to CaR stimulation is biologically active, as conditioned medium from GT1-7 cells treated with high Ca(2+) promoted chemotaxis of GN11 cells, and this effect was partially attenuated by a neutralizing antibody to MCP-1. Finally, in the preoptic area of anterior hypothalamus, the number of GnRH neurons was approximately 27% lower in CaR-null mice than in mice expressing the CaR gene. We conclude that the CaR may be a novel regulator of GnRH neuronal migration likely involving, in part, MCP-1.     

Targeted expression of a dominant-negative fibroblast growth factor (FGF) receptor in gonadotropin-releasing hormone (GnRH) neurons reduces FGF responsiveness and the size of GnRH neuronal population

Increasing evidence suggests that fibroblast growth factors (FGFs) are neurotrophic in GnRH neurons. However, the extent to which FGFs are involved in establishing a functional GnRH system in the whole organism has not been investigated. In this study, transgenic mice with the expression of a dominant-negative FGF receptor mutant (FGFRm) targeted to GnRH neurons were generated to examine the consequence of disrupted FGF signaling on the formation of the GnRH system. To first test the effectiveness of this strategy, GT1 cells, a GnRH neuronal cell line, were stably transfected with FGFRm. The transfected cells showed attenuated neurite outgrowth, diminished FGF-2 responsiveness in a cell survival assay, and blunted activation of the signaling pathway in response to FGF-2. Transgenic mice expressing FGFRm in a GnRH neuron-specific manner exhibited a 30% reduction in GnRH neuron number, but the anatomical distribution of GnRH neurons was unaltered. Although these mice were initially fertile, they displayed several reproductive defects, including delayed puberty, reduced litter size, and early reproductive senescence. Overall, our results are the first to show, at the level of the organism, that FGFs are one of the important components involved in the formation and maintenance of the GnRH system.     

Increased inferior olivary neuron and cerebellar granule cell numbers in transgenic mice overexpressing the human Bcl-2 gene

Neuron-target interactions during development are critical for determining the final numbers of neurons in the nervous system. To investigate the role of Purkinje cells and programmed cell death in the regulation of afferent neuron numbers, we have counted olivary neurons and granule cells in two lines of transgenic mice (NSE73a and NSE71) that overexpress a human gene for bcl-2 (Hu-bcl-2) in Purkinje cells and olivary neurons, but not in granule cells. Bcl-2 overexpression in vivo reduces naturally occurring neuronal cell death and cell death following axotomy, target removal, or ischemia. Olivary neuron numbers in NSE73a and NSE71 transgenic mice are significantly increased compared to controls by 28% and 27%, respectively, while granule cell numbers are only increased in NSE73a mice (29% above controls). We have previously shown that Purkinje cell number is increased by 43% in NSE73a transgenics and by 23% in NSE71 transgenics. The ratio of Purkinje cells to olivary neurons is not significantly different between the control and transgenic mice, while the ratio of granule cells to Purkinje cells is significantly decreased in the NSE71 transgenic mice compared to controls and NSE73a transgenics. The increased numbers of olivary neurons suggest that bcl-2 overexpression rescues these neurons from programmed cell death. The increase in granule cell number in only one transgenic line is discussed with respect to hypotheses that Purkinje cells regulate both granule cell progenitor proliferation and the survival of differentiated granule cells. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 502–516, 1997