Transient expression of neuropeptide W in postnatal mouse hypothalamus--a putative regulator of energy homeostasis.

Neuropeptide B and W (NPB and NPW) are cognate peptide ligands for NPBWR1 (GPR7), a G protein-coupled receptor. In rodents, they have been implicated in the regulation of energy homeostasis, neuroendocrine/autonomic responses, and social interactions. Although localization of these peptides and their receptors in adult rodent brain has been well documented, their expression in mouse brain during development is unknown. Here we demonstrate the transient expression of NPW mRNA in the dorsomedial hypothalamus (DMH) of postnatal mouse brain and its co-localization with neuropeptide Y (NPY) mRNA. Neurons expressing both NPW and NPY mRNAs begin to emerge in the DMH at about postnatal day 0 (P-0) through P-3. Their expression is highest around P-14, declines after P-21, and by P-28 only a faint expression of NPW and NPY mRNA remains. In P-18 brains, we detected NPW neurons in the region spanning the subincertal nucleus (SubI), the lateral hypothalamic (LH) perifornical (PF) areas, and the DMH, where the highest expression of NPW mRNA was observed. The majority of these postnatal hypothalamic NPW neurons co-express NPY mRNA. A cross of NPW-iCre knock-in mice with a Cre-dependent tdTomato reporter line revealed that more than half of the reporter-positive neurons in the adult DMH, which mature from the transiently NPW-expressing neurons, are sensitive to peripherally administrated leptin. These data suggest that the DMH neurons that transiently co-express NPW and NPY in the peri-weaning period might play a role in regulating energy homeostasis during postnatal development.

Roles of the glutamate receptor epsilon2 and delta2 subunits in the potentiation and prepulse inhibition of the acoustic startle reflex.

We examined the regulation of the acoustic startle response in mutant mice of the N-methyl-D-aspartate (NMDA)- and delta-subtypes of the glutamate receptor (GluR) channel, which play important roles in neural plasticity in the forebrain and the cerebellum, respectively. Heterozygous mutant mice with reduced GluRepsilon2 subunits of the NMDA receptor showed strongly enhanced startle responses to acoustic stimuli. On the other hand, heterozygous and homozygous mutation of the other NMDA receptor GluRepsilon subunits exerted no, or only small effects on acoustic startle responses. The threshold of the auditory brainstem response of the GluRepsilon2-mutant mice was comparable to that of the wild-type littermates. The primary circuit of the acoustic startle response is a relatively simple oligosynaptic pathway located in the lower brainstem, whilst the expression of GluRepsilon2 is restricted to the forebrain. We thus suggest that the NMDA receptor GluRepsilon2 subunit plays a role in the regulation of the startle reflex. Ablation of the cerebellar Purkinje cell-specific delta2 subunit of the GluR channel exerted little effect on the acoustic startle response but resulted in the enhancement of prepulse inhibition of the reflex. Because inhibition of the acoustic startle response by a weak prepulse is a measure of sensorimotor gating, the process by which an organism filters sensory information, these observations indicate the involvement of the cerebellum in the modulation of sensorimotor gating.

Purkinje cell-specific and inducible gene recombination system generated from C57BL/6 mouse ES cells.

Spatiotemporally restricted gene targeting is needed for analyzing the functions of various molecules in a variety of biological phenomena. We have generated an inducible cerebellar Purkinje cell-specific gene targeting system. This was achieved by establishing a mutant mouse line (D2CPR) from a C57BL/6 mouse ES cell line, which expressed a fusion protein consisting of the Cre recombinase and the progesterone receptor (CrePR). The Purkinje cell-specific expression of CrePR was attained by inserting CrePR into the glutamate receptor delta2 subunit (GluRdelta2) gene, which was expressed specifically in the Purkinje cells. Using the transgenic mice carrying the Cre-mediated reporter gene, we showed that the antiprogesterone RU486 could induce recombinase activity of the CrePR protein specifically in the mature cerebellar Purkinje cells of the D2CPR line. Thus this mutant line will be a useful tool for studying the molecular function of mature Purkinje cells by manipulating gene expression in a temporally restricted manner.

Analysis of stability and catalytic properties of two tryptophanases from a thermophile.

Two tryptophanases, Tna1 and Tna2, both of which were cloned from the thermophile Symbiobacterium thermophilum, differ in their enzymatic properties, such as thermal stability, catalytic efficiency and activation energy of catalysis, despite the great similarity (92%) in their amino acid sequences. Chimeric tryptophanases were constructed by recombination of the two genes to try to elucidate the molecular basis for the difference. The stability of each chimeric enzyme was roughly proportional to the content of amino acid residues from Tna1. Three regions, tentatively named regions 2, 4 and 5, which contained the amino acid residues 70-129, 192-298 and 299-453, respectively, were especially important for the increase in thermal stability. Site-directed mutagenesis revealed that V104 in region 2 and Y198 in region 4 of Tna1 were involved in the increase in thermal stability of Tna1. Amino acid residues contributing to the higher catalytic efficiency of Tna1 were similarly analyzed, using the chimeric tryptophanases, and found to be located in region 5. Site-directed mutagenesis revealed that I383 and G395 in Tna1, which were presumably located close to the putative active center, played an active role in the increase of catalytic efficiency of Tna1. The activation energy of catalysis was proportional to the content of amino acid residues from Tna2, suggesting the amino acid residues responsible for the difference were dispersed over the whole molecule.

Impairment of suckling response, trigeminal neuronal pattern formation, and hippocampal LTD in NMDA receptor epsilon 2 subunit mutant mice.

Multiple epsilon subunits are major determinants of the NMDA receptor channel diversity. Based on their functional properties in vitro and distributions, we have proposed that the epsilon 1 and epsilon 2 subunits play a role in synaptic plasticity. To investigate the physiological significance of the NMDA receptor channel diversity, we generated mutant mice defective in the epsilon 2 subunit. These mice showed no suckling response and died shortly after birth but could survive by hand feeding. The mutation hindered the formation of the whisker-related neuronal barrelette structure and the clustering of primary sensory afferent terminals in the brainstem trigeminal nucleus. In the hippocampus of the mutant mice, synaptic NMDA responses and longterm depression were abolished. These results suggest that the epsilon 2 subunit plays an essential role in both neuronal pattern formation and synaptic plasticity.

A female case of the Leach-Nyhan syndrome.

The classical Lesch-Nyhan syndrome has the deficiency of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) activity as the result of mutation in the structural gene for the enzyme located on the X chromosome and is believed to be of X-linked recessive or sex-linked mode of inheritance. This is the first report of a girl who showed typical clinical features and biochemical characteristics of the classical Lesch-Nyhan syndrome. Her mother was not a heterozygote for a deficiency of HGPRT. Possible genetic mechanisms responsible for this case were discussed.