Lateral regions of the rodent striatum reveal elevated glutamate decarboxylase 1 mRNA expression in medium-sized projection neurons.

The GABA-synthesizing enzymes glutamate decarboxylase (GAD)1 and GAD2 are universally contained in GABAergic neurons in the central nervous system of the mouse and rat. The two isoforms are almost identically expressed throughout the brain and spinal cord. By using in situ hybridization, we found that the mouse lateral striatum concentrates medium-sized projection neurons with high-level expression of GAD1, but not of GAD2, mRNA. This was confirmed with several types of riboprobe, including those directed to the 5'-noncoding, 3'-noncoding and coding regions. Immunohistochemical localization of GAD1 also revealed predominant localization of the enzyme in the same striatal region. The lateral region of the mouse striatum, harboring such neurons, is ovoid in shape and extends between interaural +4.8 and +2.8, and at lateral 2.8 and dorsoventral 2.0. This intriguing region corresponds to the area that receives afferent inputs from the primary motor and sensory cortex that are presumably related to mouth and forelimb representations. The lateral striatum is included in the basal ganglia-thalamocortical loop, and is most vulnerable to various noxious stimuli in the neurodegeneration processes involving the basal ganglia. We have confirmed elevated expression of GAD1 mRNA, but not of GAD2 mRNA, also in the rat lateral striatum. Image analysis favored the view that the regional increase is caused by elevated cellular expression, and that the greatest number of medium-sized spiny neurons were positive for GAD1 mRNA. The GAD1 mRNA distribution in the mouse lateral striatum partially resembled those of GPR155 and cannabinoid receptor type 1 mRNAs, suggesting functional cooperation in some neurons.

Histological determination of the areas enriched in cholinergic terminals and M2 and M3 muscarinic receptors in the mouse central auditory system.

Cholinergic projections to auditory system are vital for coupling arousal with sound processing. Systematic search with in situ hybridization and immunohistochemistry indicated that the ventral nucleus of the medial geniculate body and the nucleus of the brachium of the inferior colliculus constituted cholinergic synaptic sites in the brainstem auditory system, containing a significant number of cholinergic axon terminals and m2 receptor-expressing cell bodies.

GPR155: Gene organization, multiple mRNA splice variants and expression in mouse central nervous system.

Emerging evidence suggests that GPR155, an integral membrane protein related to G-protein coupled receptors, has specific roles in Huntington disease and autism spectrum disorders. This study reports the structural organization of mouse GPR155 gene and the generation of five variants (Variants 1-5) of GPR155 mRNA, including so far unknown four variants. Further, it presents the level of expression of GPR155 mRNA in different mouse tissues. The mRNAs for GPR155 are widely expressed in adult mouse tissues and during development. In situ hybridization was used to determine the distribution of GPR155 in mouse brain. The GPR155 mRNAs are widely distributed in forebrain regions and have more restricted distribution in the midbrain and hindbrain regions. The highest level of expression was in the lateral part of striatum and hippocampus. The expression pattern of GPR155 mRNAs in mouse striatum was very similar to that of cannabinoid receptor type 1. The predicted protein secondary structure indicated that GPR155 is a 17-TM protein, and Variant 1 and Variant 5 proteins have an intracellular, conserved DEP domain near the C-terminal.

Laser microdissection combined with immunohistochemistry on serial thin tissue sections: a method allowing efficient mRNA analysis.

Laser microdissection (LMD) with subsequent reverse transcription-PCR analysis is a powerful histochemical technique subserving the molecular characterization of specific cell types. We developed an efficient method for selective sampling of specific cell populations using immunohistochemistry coupled with LMD. The cerebral cortex of adult rats was cut into serial thin sections. Some sections were immunostained for parvalbumin. The adjacent sections were mounted on Cell Support Film for LMD and stained with neutral red. By comparison of the two adjacent sections, neuronal profiles representing parts of parvalbumin-immunopositive somata were identified in the neutral red-stained sections. These neuronal profiles were safely captured with LMD and analyzed on reverse transcription-PCR using extracted RNA. The method presented here can be applied to cell-type-specific characterizations using fixed cells under RNase-free conditions.

Cloning and expression of ligand-gated ion-channel receptor L2 in central nervous system.

An orphan receptor of ligand-gated ion-channel type (L2, also termed ZAC according to the presence of zinc ion for channel activation) was identified by computer-assisted search programs on human genome database. The L2 protein shares partial homology with serotonin receptors 5HT3A and 5HT3B. We have cloned L2 cDNA derived from human caudate nucleus and characterized the exon-intron structure as follows: (1) The L2 protein has four transmembrane regions (M1-M4) and a long cytoplasmic loop between M3 and M4. (2) The sequence is conserved in species including chimpanzee, dog, cow, and opossum. (3) Nine exons form its protein-coding region and especially exon 5 corresponds to a disulfide bond region on the amino-terminal side. Our analysis using multiple tissue cDNA panels revealed that at least two splicing variants of L2 mRNA are present. The cDNA PCR amplification study revealed that L2 mRNA is expressed in tissues including brain, pancreas, liver, lung, heart, kidney, and skeletal muscle while 5HT3A mRNA could be detected in brain, heart, placenta, lung, kidney, pancreas, and skeletal muscle, and 5HT3B mRNA in brain, kidney, and skeletal muscle, suggesting different significance in tissue expression of these receptors. Regional expression of L2 mRNA and protein was examined in brain. The RT-PCR studies confirmed L2 mRNA expression in hippocampus, striatum, amygdala, and thalamus in adult brain. The L2 protein was immunolocalized by using antipeptide antibodies. Immunostained tissue sections revealed that L2-like immunoreactivity was dominantly expressed in the hippocampal CA3 pyramidal cells and in the polymorphic layer of the dentate gyrus. We analyzed the expression of L2 protein in HEK293 cells using GFP fusion protein reporter system. Western blots revealed that L2 protein confers sugar chains on the extracellular side. In transfected HEK293 cells, cellular membranes and intracellular puncta were densely labeled with GFP, suggesting selective dispatch to the final destination.

Enhanced hippocampal acetylcholine release in nociceptin-receptor knockout mice.

Nociceptin (NOC), an endogenous ligand of the opioid receptor-like 1 receptor, is thought to be involved in learning and memory processes. Since acetylcholine (ACh) is involved in hippocampal function, and the hippocampus plays a critical role on the learning and memory function, hippocampal ACh release in NOC-receptor knockout mice was examined using an in vivo microdialysis method. The release of hippocampal ACh was largely increased in the knockout mice. Furthermore, in the knockout mice, an enhanced hippocampal theta rhythm, which is known to be linked to hippocampal memory function, was also observed. Immunohistochemically, in septum, co-existence of NOC receptor with cholinergic, but not with GABAergic neurons, was verified. The findings demonstrate that the NOC receptor is involved in hippocampal cholinergic function.

Mouse homolog of KIAA0143 protein: hearing deficit induces specific changes of expression in auditory brainstem neurons.

Hearing deficit induced by mechanical cochlear damage, intense noise or ototoxic drugs produces a variety of structural and functional changes in the inner ear and the auditory brainstem. In the present study, we identified a novel gene that has activity dependent plasticity in the superior olivary complex by using suppression subtractive hybridization. We cloned a gene that encodes mouse homolog of KIAA0143 protein, one derived from a series of unidentified human genes. This gene termed mKIAA0143 shows differential expression of mRNA in the lateral superior olive between mice with hearing deficit and those with normal hearing ability. The mRNA thus obtained encodes a unique membrane-bound protein that consists of 819 amino acids. The gene locus was mapped using genomic DNA databases to the mouse chromosome 15D1. Green fluorescent protein-tagged mKIAA0143 was expressed in COS-1 cells. It was amply seen in the cellular plasma membrane.

Lack of nociceptin receptor alters body temperature during resting period in mice.

The role of nociceptin (NOC) receptor on body core temperature (Tcore) control was examined using NOC receptor knockout mice. In homozygote NOC receptor-knockout, wild-type, and control C57BL/6J and 129/SV mice, Tcore was continuously recorded under 12:12 h light:dark (LD) and conditions of constant darkness (DD). The Tcore values during the resting period were higher in the NOC receptor-knockout mice than in both wild-type and control mice under both LD and DD conditions. Spontaneous activity during the resting period and plasma cortisol levels were not different between the NOC receptor-knockout and control mice. The findings herein indicate that the NOC receptor is involved in the control of Tcore during the resting period and is independent of light, physical activity and/or cortisol regulation.

Stem cell-derived neural stem/progenitor cell supporting factor is an autocrine/paracrine survival factor for adult neural stem/progenitor cells.

Recent evidence suggests that adult neural stem/progenitor cells (ANSCs) secrete autocrine/paracrine factors and that these intrinsic factors are involved in the maintenance of adult neurogenesis. We identified a novel secretory molecule, stem cell-derived neural stem/progenitor cell supporting factor (SDNSF), from adult hippocampal neural stem/progenitor cells by using the signal sequence trap method. The expression of SDNSF in adult central nervous system was localized to hippocampus including dentate gyrus, where the neurogenesis persists throughout life. In induced neurogenesis status seen in ischemically treated hippocampus, the expression of SDNSF was up-regulated. As functional aspects, SDNSF protein provided a dose-dependent survival effect for ANSC following basic fibroblast growth factor 2 (FGF-2) withdrawal. ANSCs treated by SDNSF also retain self-renewal potential and multipotency in the absence of FGF-2. However, SDNSF did not have mitogenic activity, nor was it a cofactor that promoted the mitogenic effects of FGF-2. These data suggested an important role of SDNSF as an autocrine/paracrine factor in maintaining stem cell potential and lifelong neurogenesis in adult central nervous system.

A subset of nociceptin/orphanin FQ receptor-expressing neurons in the anterior hypothalamic area, as revealed in mice with lacZ reporter gene.

Nociceptin/orphanin FQ (N/OFQ) is an endogenous peptide agonist for the opioid receptor homolog, N/OFQ receptor, and serves for the central control of autonomic functions. Morphological details including the cell types that may account for such N/OFQ functions, however, remain unclear. By using X-gal histochemistry for the detection of receptor-expressing cells at both light and electron microscopic levels, we examined the hypothalamus from the receptor-deficient mice bearing a lacZ insertional mutation in the N/OFQ receptor gene. The N/OFQ receptor reflected by lacZ expression was seen at high levels in the anterior hypothalamic area. With electron microscopy, lacZ expression was observed in a subset of neurons showing large cell size and indented nucleus.