Martin G. Todman

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.

Profiling neurotransmitter receptor expression in mouse gonadotropin-releasing hormone neurons using green fluorescent protein-promoter transgenics and microarrays

The definition of neurotransmitter receptors expressed by individual neuronal phenotypes is essential for our understanding of integrated neural regulation. We report here a single-neuron strategy using green fluorescent protein (GFP)-promoter transgenic mice and oligonucleotide microarrays that has enabled us to provide a qualitative profile of the neurotransmitter receptors expressed by the gonadotropin- releasing hormone (GnRH) neurons, critical for the neural regulation of fertility. Acute brain slices were prepared from adult female GnRH-GFP transgenic mice and single GnRH neurons identified and patched. The contents of GnRH neurons underwent reverse transcription and cDNA amplification using the switch mechanism at the 5 end of RNA templates system, and hybridization to mouse gene oligonucleotide arrays. Fifty different neurotransmitter receptor subunit mRNAs were detected in GnRH neurons. Many of the classical amino acid and aminergic receptors were present in addition to 14 distinct, and in most cases novel, neuropeptidergic receptor signaling families. Four of the latter were selected for functional validation with gramicidin-perforated patch-clamp electrophysiology. Galanin, GnRH and neuromedin B were all found to exert direct depolarizing actions upon GnRH neurons whereas somatostatin induced a potent hyperpolarizing response. These studies demonstrate a relatively straightforward approach for transcriptome profiling of specific neuronal phenotypes. The stimulatory actions of GnRH and galanin upon GnRH neurons found here indicate that positive ultrashort feedback loops exist among the GnRH neuronal population.

Critical role for estrogen receptor alpha in negative feedback regulation of gonadotropin-releasing hormone mRNA expression in the female mouse

Estrogen exerts an important regulatory influence upon the functioning of the gonadotropin-releasing hormone (GnRH) neurons. Whether this is mediated by estrogen receptor α (ERα) or ERβ or both ERs is presently unclear. Using female mice with targeted disruptions of ERα and ERβ (αERKO and βERKO, respectively) we have investigated the in vivo role of the two ERs in the negative feedback influence of estrogen upon GnRH mRNA expression. Compared with intact wild-type mice, plasma luteinizing hormone (LH) levels were substantially (p < 0.01) higher in intact αERKO females and increased modestly (p < 0.05) in intact βERKO mice. Three weeks after ovariectomy, LH concentrations were elevated significantly in wild-type (p < 0.01) and βERKO (p < 0.05) mice but not changed in αERKO females. Quantitative analysis of GnRH mRNA expression using in situ hybridization revealed that cellular GnRH mRNA content was greater (p < 0.05) in intact αERKO mice compared with intact wild-type and βERKO mice. Following ovariectomy, GnRH mRNA expression was elevated in wild-type (p = 0.06) and βERKO (p < 0.05) females but not αERKO mice. These data demonstrate that both ERα and ERβ are involved in inhibiting LH levels at times of estrogen-negative feedback in vivo. However, only ERα appears to be critical for the estrogen-negative feedback suppression of GnRH mRNA expression in the female mouse.

Gap junctions in Drosophila: developmental expression of the entire innexin gene family

Invertebrate gap junctions are composed of proteins called innexins and eight innexin encoding loci have been identified in the now complete genome sequence of Drosophila melanogaster. The intercellular channels formed by these proteins are multimeric and previous studies have shown that, in a heterologous expression system, homo- and hetero-oligomeric channels can form, each combination possessing different gating characteristics. Here we demonstrate that the innexins exhibit complex overlapping expression patterns during oogenesis, embryogenesis, imaginal wing disc development and central nervous system development and show that only certain combinations of innexin oligomerization are possible in vivo. This work forms an essential basis for future studies of innexin interactions in Drosophila and outlines the potential extent of gap-junction involvement in development.

Expression of mRNAs Encoding Receptors That Mediate Stress Signals in Gonadotropin-Releasing Hormone Neurons of the Mouse

Neurons that synthesize and secrete gonadotropin-releasing hormone (GnRH) represent the neural control point for fertility modulation in vertebrates. As such GnRH neurons are ideally situated to integrate stress responses on reproduction. By isolating individual GnRH neurons from acute brain slices of adult female GnRH-EGFP transgenic mice and using microarray analyses, we have identified a range of transcripts encoding receptors known to be involved in stress responses in GnRH neurons. Prominent among these were receptors for corticotropin-releasing hormone (CRH), vasopressin, interleukins, prostaglandins, tumor necrosis factor alpha and other inflammatory mediators. We selected 4 of these targets [interleukin 1 receptor accessory protein (IL-1Racc), prostaglandin E2 receptor subtype EP2 (PGER2), CRH receptor type 1 (CRH-R1), and arginine-vasopressin receptor type 1b (AVP-R1b)] for validation using single-cell RT-PCR from individual GnRH neurons. In total, 54% of GnRH neurons (n = 26) were found to express at least 1 of these transcripts. The IL-1Racc, PGER2 and CRH-R1 mRNAs were each detected in approximately 25% of the GnRH neurons tested, but no evidence was found for AVP-R1b transcripts. Overlap was found between the expression of CRH-R1 and PGER2, and IL-1Racc and PGER2 in individual GnRH neurons. Dual immunofluorescence experiments confirmed the expression of CRH-R1/2 in a subpopulation (∼30%) of GnRH neurons. These observations indicate that a variety of different stressors and stress pathways have the capacity to have an impact directly upon a subpopulation of GnRH neurons to influence the reproductive axis.

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 ∼95% of GnRH neurons. Real-time monitoring of [Ca2+]i within adult male GnRH neurons in the acute brain slice revealed that ∼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.

Disruption of Ephrin Signaling Associates with Disordered Axophilic Migration of the Gonadotropin-Releasing Hormone Neurons

Ephrin signaling is involved in repulsive and attractive interactions mediating axon guidance and cell-boundary formation in the developing nervous system. As a result of a fortuitous transgene integration event, we have identified here a potential role for EphA5 in the axophilic migration of gonadotropin-releasing hormone (GnRH) neurons from the nasal placode into the brain along ephrin-expressing vomeronasal axons. Transgene integration in the GNR23 mouse line resulted in a 26 kb deletion in chromosome 5, ∼67 kb 3′ to Epha5. This induced a profound, region-specific upregulation of EphA5 mRNA and protein expression in the developing mouse brain. The GnRH neurons in GNR23 mice overexpressed EphA5 from embryonic day 11, whereas ephrin A3 and A5 mRNA levels in olfactory neurons were unchanged. The GnRH neurons were found to be slow in commencing their migration from the olfactory placode and also to form abnormal clusters of cells on the olfactory axons, prohibiting their migration out of the nose. As a result, adult hemizygous mice had only 40% of the normal complement of GnRH neurons in the brain, whereas homozygous mice had <15%. This resulted in infertility in adult female homozygous GNR23 mice, suggesting that some cases of human hypogonadotropic hypogonadism may result from ephrin-related mutations. These data provide evidence for a role of EphA-ephrin signaling in the axophilic migration of the GnRH neurons during embryogenesis.