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

The behavior of the gonadotropin-releasing hormones (GnRH) neurons controlling fertility is dependent upon cyclic fluctuations in circulating concentrations of estrogen. However, the nature of estrogen action upon these cells has remained controversial due to their dispersed distribution within the brain, and evidence indicating that they do not express nuclear estrogen receptors (ERs) in vivo. We report here an acute brain slice preparation that enables individual living GnRH neurons to be identified within the mouse brain and show, using single cell multiplex RT-PCR, that the greater than 50% of GnRH neurons in adult and prepubertal females contain ERalpha messenger RNA. Approximately 10% of GnRH neurons contained ERbeta transcripts that were always coexistent with ERalpha. Single cell RT-PCR analysis of nonGnRH cells located in the medial preoptic area revealed a similar coexpression pattern of ERalpha and ERbeta transcripts. In contrast, single striatal cells were not found to contain ERbeta despite ERalpha being present in approximately 25% of cells. The analysis of single GnRH neurons in cycling female mice revealed that the detection of ERalpha and ERbeta transcripts was lowest on proestrus (ERalpha, 18% of all GnRH neurons; ERbeta, 0%) compared with diestrus (44% and 6%) and estrus (75% and 19%, respectively). Using a novel approach that enables single cell RT-PCR analysis of GnRH neurons, we present here evidence for the cyclic expression of ERalpha and ERbeta messenger RNAs within prepubertal and adult female GnRH neurons.

Gonadotropin-releasing hormone-I (GnRH-I) is thought to be expressed by a single, highly spatially restricted group of neurons, which originate in the olfactory placode and migrate through the nose into the medial septum and hypothalamus from where they control fertility. Transgenic mice bearing a 13.5 kb GnRH-I-lacZ reporter construct were derived and found to express high levels of ␤-galactosidase mRNA and protein within the septohypothalamic GnRH neurons in a correct temporal and spatial manner. Unexpectedly, low levels of ␤-galactosidase were also present in three further populations of cells within the lateral septum, bed nucleus of the stria terminalis, and tectum. Analysis of wild-type mice with three different GnRH-I antibodies revealed distinct and transient patterns of GnRH-I peptide expression during development in all three of these populations revealed by transgenics. The synthesis of GnRH by cells of the lateral septum was the most persistent and remained until the third postnatal week. Embryonic "small eye" Pax-6 null mice, which fail to develop an olfactory placode, were also examined and shown to have equivalent populations of GnRH-I-immunoreactive cells in the lateral septum, tectum, and bed nucleus of the stria terminalis but none of the migrating cells that form the septohypothalamic GnRH population. These results prove that so-called "ectopic" expression in promoter transgenic lines can reflect authentic developmental patterns of gene expression. They further provide the first demonstration in mammalian brain that multiple neuronal populations of different embryological origin express GnRH-I peptide during embryonic and postnatal development.

To study the function of astrocytes in the adult brain, we have targeted the expression of E. coli nitroreductase (NTR) to the astrocytes of transgenic mice under the control of the GFAP promoter. The astrocytes expressing NTR were selectively ablated after administration of the prodrug CB1954, resulting in motor discoordination. Histological examination showed that the region most affected in the brain was the cerebellum, in which the Bergmann glia were eliminated and the granular neurons had degenerated. Specific effects were also noted on the dendrites of the Purkinje cells, and the junction between these neurons and granular layer was disrupted. Astrocyte ablation was associated with a dramatic decrease in the expression of glutamate transporters, which may account for the degeneration of granular neurons since the excitotoxic effects of glutamate result in a similar phenotype. These results provide the first evidence that astrocytes are important for the survival of neurons in the adult brain in vivo.

Background and purpose:In various models vagus nerve activation has been shown to ameliorate intestinal inflammation, via nicotinic acetylcholine receptors (nAChRs) expressed on immune cells. As the a7 nAChR has been put forward to mediate this effect, we studied the effect of nicotine and two selective a7 nAChR agonists (AR-R17779, (-)-spiro[1-azabicyclo[2.2.2] octane-3,5′-oxazolidin-2′-one and GSK1345038A) on disease severity in two mouse models of experimental colitis. Experimental approach: Colitis was induced by administration of 1.5% dextran sodium sulphate (DSS) in drinking water or 2 mg 2,4,6-trinitrobenzene sulphonic acid (TNBS) intrarectally. Nicotine (0.25 and 2.50 mmol·kg -1

The ky mouse mutant exhibits a primary degenerative myopathy preceding chronic thoraco-lumbar kyphoscoliosis. The histopathology of the ky mutant suggests that Ky protein activity is crucial for normal muscle growth and function as well as the maturation and stabilization of the neuromuscular junction. Muscle hypertrophy in response to increasing demand is deficient in the ky mutant, whereas adaptive fibre type shifts take place. The ky locus has previously been localized to a small region of mouse chromosome 9 and we have now identified the gene and the mutation underlying the kyphoscoliotic mouse. The ky transcript encodes a novel protein that is detected only in skeletal muscle and heart. The identification of the ky gene will allow detailed analysis of the impact of primary myopathy on idiopathic scoliosis in mice and man.

Recessive mutations at the mouse pirouette (pi) locus result in hearing loss and vestibular dysfunction due to neuroepithelial defects in the inner ear. Using a positional cloning strategy, we have identified mutations in the gene Grxcr1 (glutaredoxin cysteine-rich 1) in five independent allelic strains of pirouette mice. We also provide sequence data of GRXCR1 from humans with profound hearing loss suggesting that pirouette is a model for studying the mechanism of nonsyndromic deafness DFNB25. Grxcr1 encodes a 290 amino acid protein that contains a region of similarity to glutaredoxin proteins and a cysteine-rich region at its C terminus. Grxcr1 is expressed in sensory epithelia of the inner ear, and its encoded protein is localized along the length of stereocilia, the actin-filament-rich mechanosensory structures at the apical surface of auditory and vestibular hair cells. The precise architecture of hair cell stereocilia is essential for normal hearing. Loss of function of Grxcr1 in homozygous pirouette mice results in abnormally thin and slightly shortened stereocilia. When overexpressed in transfected cells, GRXCR1 localizes along the length of actin-filament-rich structures at the dorsal-apical surface and induces structures with greater actin filament content and/or increased lengths in a subset of cells. Our results suggest that deafness in pirouette mutants is associated with loss of GRXCR1 function in modulating actin cytoskeletal architecture in the developing stereocilia of sensory hair cells.

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