Evidence of involvement of neurone-glia/neurone-neurone communications via gap junctions in synchronised activity of KNDy neurones.

Pulsatile secretion of gonadotrophin-releasing hormone (GnRH)/luteinising hormone is indispensable for the onset of puberty and reproductive activities at adulthood in mammalian species. A cohort of neurones expressing three neuropeptides, namely kisspeptin, encoded by the Kiss1 gene, neurokinin B (NKB) and dynorphin A, localised in the hypothalamic arcuate nucleus (ARC), so-called KNDy neurones, comprises a putative intrinsic source of the GnRH pulse generator. Synchronous activity among KNDy neurones is considered to be required for pulsatile GnRH secretion. It has been reported that gap junctions play a key role in synchronising electrical activity in the central nervous system. Thus, we hypothesised that gap junctions are involved in the synchronised activities of KNDy neurones, which is induced by NKB-NK3R signalling. We determined the role of NKB-NK3R signalling in Ca2+ oscillation (an indicator of neuronal activities) of KNDy neurones and its synchronisation mechanism among KNDy neurones. Senktide, a selective agonist for NK3R, increased the frequency of Ca2+ oscillations in cultured Kiss1-GFP cells collected from the mediobasal hypothalamus of the foetal Kiss1-green fluorescent protein (GFP) mice. The senktide-induced Ca2+ oscillations were synchronised in the Kiss1-GFP and neighbouring glial cells. Confocal microscopy analysis of these cells, which have shown synchronised Ca2+ oscillations, revealed close contacts between Kiss1-GFP cells, as well as between Kiss1-GFP cells and glial cells. Dye coupling experiments suggest cell-to-cell communication through gap junctions between Kiss1-GFP cells and neighbouring glial cells. Connexin-26 and -37 mRNA were found in isolated ARC Kiss1 cells taken from adult female Kiss1-GFP transgenic mice. Furthermore, 18ß-glycyrrhetinic acids and mefloquine, which are gap junction inhibitors, attenuated senktide-induced Ca2+ oscillations in Kiss1-GFP cells. Taken together, these results suggest that NKB-NK3R signalling enhances synchronised activities among neighbouring KNDy neurones, and that both neurone-neurone and neurone-glia communications via gap junctions possibly contribute to synchronised activities among KNDy neurones.

Lack of pulse and surge modes and glutamatergic stimulation of luteinising hormone release in Kiss1 knockout rats.

Kisspeptin, encoded by the Kiss1 gene, has attracted attention as a key candidate neuropeptide in controlling puberty and reproduction via regulation of gonadotrophin-releasing hormone (GnRH) secretion in mammals. Pioneer studies with Kiss1 or its cognate receptor Gpr54 knockout (KO) mice showed the indispensable role of kisspeptin-GPR54 signalling in the control of animal reproduction, although detailed analyses of gonadotrophin secretion, especially pulsatile and surge-mode of luteinising hormone (LH) secretion, were limited. Thus, in the present study, we have generated Kiss1 KO rats aiming to evaluate a key role of kisspeptin in governing reproduction via pulse and surge modes of GnRH/LH secretion. Kiss1 KO male and female rats showed a complete suppression of pulsatile LH secretion, which is responsible for folliculogenesis and spermatogenesis, and an absence of puberty and atrophic gonads. Kiss1 KO female rats showed no spontaneous LH/follicle-stimulating hormone surge and an oestrogen-induced LH surge, suggesting that the GnRH surge generation system, which is responsible for ovulation, does not function without kisspeptin. Furthermore, challenge of major stimulatory neurotransmitters, such as monosodium glutamate, NMDA and norepinephrine, failed to stimulate LH secretion in Kiss1 KO rats, albeit they stimulated LH release in wild-type controls. Taken together, the results of the present study confirm that kisspeptin plays an indispensable role in generating two modes (pulse and surge) of GnRH/gonadotrophin secretion to regulate puberty onset and normal reproductive performance. In addition, the present study suggests that kisspeptin neurones play a critical role as a hub integrating major stimulatory neural inputs to GnRH neurones, using newly established Kiss1 KO rats, which serve as a useful model for detailed analysis of hormonal profiles.

Enhancement of hippocampal LTP, reference memory and sensorimotor gating in mutant mice lacking a telencephalon-specific cell adhesion molecule.

Telencephalin (TLCN) is a cell adhesion molecule selectively expressed in the telencephalon of the mammalian brain. The mutant mice lacking TLCN had no detectable abnormalities in their neural development and synaptic structures. Ablation of TLCN increased the hippocampal long-term potentiation and its saturation level. The TLCN mutation selectively enhanced the performance of the radial maze and water-finding tasks, learning tasks with appetitive reinforcers, but not the contextual fear conditioning and Morris water maze tasks with aversive stimuli for conditioning. Furthermore, the TLCN mutant mice showed an increase of prepulse inhibition of the acoustic startle response. These results suggest that TLCN is a determinant of the dynamic range of synaptic plasticity and plays roles in reward-motivated learning and memory and sensorimotor gating.

A requirement for neuropilin-1 in embryonic vessel formation.

Neuropilin-1 is a membrane protein that is expressed in developing neurons and functions as a receptor or a component of the receptor complex for the class 3 semaphorins, which are inhibitory axon guidance signals. Targeted inactivation of the neuropilin-1 gene in mice induced disorganization of the pathway and projection of nerve fibers, suggesting that neuropilin-1 mediates semaphorin-elicited signals and regulates nerve fiber guidance in embryogenesis. Neuropilin-1 is also expressed in endothelial cells and shown to bind vascular endothelial growth factor (VEGF), a potent regulator for vasculogenesis and angiogenesis. However, the roles of neuropilin-1 in vascular formation have been unclear. This paper reported that the neuropilin-1 mutant mouse embryos exhibited various types of vascular defects, including impairment in neural vascularization, agenesis and transposition of great vessels, insufficient aorticopulmonary truncus (persistent truncus arteriosus), and disorganized and insufficient development of vascular networks in the yolk sac. The vascular defects induced by neuropilin-1 deficiency in mouse embryos suggest that neuropilin-1 plays roles in embryonic vessel formation, as well as nerve fiber guidance.

Enrichment and efficient screening of ES cells containing a targeted mutation: the use of DT-A gene with the polyadenylation signal as a negative selection maker.

Gene targeting in embryonic stem (ES) cells via homologous recombination can occur at very low frequency. In order to enrich homologous recombinants before screening, a negative selection marker, such as thymidine kinase (TK) and diphtheria toxin A fragment (DT-A), has been commonly used. In this study, we developed a negative selection marker using DT-A gene with polyadenylation signal and it was designated DT-ApA. To determine the difference in targeting efficiency of the negative selections, we constructed three different targeting vectors for each negative selection (first, TK at the 3' end, second, TK at both the 5' and 3' ends < 2 X TK >, and third, DT-ApA at the 5' end of the homologous sequences). Gene targeting experiments using these constructs clearly showed that negative selection using DT-ApA was more efficient than that using TK for homologous recombination and that negative selection using DT-ApA was as efficient as that using 2 X TK. Considering the fact that the use of DT-ApA is more convenient for construction of targeting vectors than that of 2 X TK, DT-ApA is an efficient negative selection marker. In addition, we examined long and accurate PCR (LA-PCR) for screening gene targeted clones. The use of LA-PCR with genomic DNAs from ES cell clones facilitated simple detection of homologous recombinants, suggesting that the screening with LA-PCR is compatible with the use of longer homologous sequences of both arms in vector design. Our results indicate that the use of DT-ApA for negative selection together with the application of LA-PCR for screening ensures efficient and time-saving screening for homologous recombinants.

Reduced hippocampal LTP in mice lacking a presynaptic protein: complexin II.

The SNAP receptor (SNARE) complex is a core complex specialized for synaptic vesicle exocytosis, and the binding of SNAPs to the complex is an essential step for neurotransmitter release. Complexin I and II have been identified as SNARE-complex-associated proteins. Importantly, complexins compete with alpha-SNAP for binding to the complex, suggesting that complexins may modulate neurotransmitter release process. To examine this possibility and to understand the physiological function of complexins, we generated complexin II knockout mice. The complexin-II-deficient mice (-/-) were viable and fertile, and appeared normal. Electrophysiological recordings in the mutant hippocampus showed that ordinary synaptic transmission and paired-pulse facilitation, a form of short-term synaptic plasticity, were normal. However, long-term potentiation (LTP) in both CA1 and CA3 regions was impaired, suggesting that complexin II may not be essential for synaptic vesicle exocytosis, but it does have a role in the establishment of hippocampal LTP.

Efficient production of Cre-mediated site-directed recombinants through the utilization of the puromycin resistance gene, pac: a transient gene-integration marker for ES cells.

Gene targeting in embryonic stem (ES) cells is a powerful tool for generating mice carrying specifically designed mutations in the germline. Puromycin can completely kill ES cells within 24 to 48 h whereas G418 and hygromycin cannot. We have, therefore, proposed that the puromycin N-acetyltransferase ( pac ) gene, may be utilized as a transient gene-integration marker. Using a circular expression vector of cre and pac genes, Cre-mediated mutant cells were effectively enriched by pulse treatment of puromycin without stable integration of their genes. We have thus demonstrated the first application of pac as a transient gene-integration marker for ES cells.

Neuropilin-semaphorin III/D-mediated chemorepulsive signals play a crucial role in peripheral nerve projection in mice.

Neuropilin is a neuronal cell surface protein and has been shown to function as a receptor for a secreted protein, semaphorin III/D, that can induce neuronal growth cone collapse and repulsion of neurites in vitro. The roles of neuropilin in vivo, however, are unknown. Here, we report that neuropilin-deficient mutant mice produced by targeted disruption of the neuropilin gene show severe abnormalities in the trajectory of efferent fibers of the PNS. We also describe that neuropilin-deprived dorsal root ganglion neurons are perfectly protected from growth cone collapse elicited by semaphorin III/D. Our results indicate that neuropilin-semaphorin III/D-mediated chemorepulsive signals play a major role in guidance of PNS efferents.

Cleft palate and decreased brain gamma-aminobutyric acid in mice lacking the 67-kDa isoform of glutamic acid decarboxylase.

In addition to its role as an inhibitory neurotransmitter, gamma-aminobutyric acid (GABA) is presumed to be involved in the development and plasticity of the nervous system. GABA is synthesized by glutamic acid decarboxylase (GAD), but the respective roles of its two isoforms (GAD65 and 67) have not been determined. The selective elimination of each GAD isoform by gene targeting is expected to clarify these issues. Recently we have produced GAD65 -/- mice and demonstrated that lack of GAD65 does not change brain GABA contents or animal behavior, except for a slight increase in susceptibility to seizures. Here we report the production of GAD67 -/- mice. These mice were born at the expected frequency but died of severe cleft palate during the first morning after birth. GAD activities and GABA contents were reduced to 20% and 7%, respectively, in the cerebral cortex of the newborn GAD67 -/- mice. Their brain, however, did not show any discernible defects. Previous pharmacological and genetic investigations have suggested the involvement of GABA in palate formation, but this is the first demonstration of a role for GAD67-derived GABA in the development of nonneural tissue.

Mice lacking the 65 kDa isoform of glutamic acid decarboxylase (GAD65) maintain normal levels of GAD67 and GABA in their brains but are susceptible to seizures.

The gene encoding of the 65 kDa isoform of the gamma-aminobutyric acid (GABA)-synthesizing enzyme, glutamic acid decarboxylase (GAD), GAD65, was targeted in mice by homologous recombination. Viable GAD65 -/- mice were obtained with the expected mendelian frequency and displayed no gross morphological defects. Despite the complete loss of GAD65 mRNA and protein in a homozygous mutant, there was no difference in GABA content in the brains of GAD65 +/+, +/-, and -/- mice. As for the other 67 kDa isoform (GAD67), the levels of mRNA and protein were largely unchanged by the GAD65 mutation. General behavior, including locomotor activity and performance in the Morris water maze task, appeared normal, but seizures were more easily induced by picrotoxin and pentylenetetrazol: the latencies to seizures induced by picrotoxin were shorter and the dose of pentylenetetrazol required for induction of seizures was lower.

Stable production of mutant mice from double gene converted ES cells with puromycin and neomycin.

The antibiotic puromycin is an effective inhibitor of protein synthesis and puromycin N-acetyl transferase gene could be used as a dominant selection marker. We report the effective production of mutant mice from double gene-converted ES cells by selection with G418 and puromycin. We confirmed that (i) puromycin efficiently inhibited the growth of ES cells at a low-dose (0.1 microgram/ml) and for a short time (2 days), independent of G418 selection; (ii) when these selected ES cells were injected into eight-cell stage embryos, the cells produced chimeras with high levels of chimerism; (iii) these chimeric males were fertile and exclusively yielded ES cell-derived offspring; and (iv) each offspring contained both neomycin transferase and puromycin N-acetyl transferase genes.