Yumiko Hatanaka

Differential expression of γ-aminobutyric acid type B receptor-1a and -1b mRNA variants in GABA and non-GABAergic neurons of the rat brain

To understand the heterogeneity of γ‐aminobutyric acid type B receptor (GABABR)‐ mediated events, we investigated expression of GABABR1a and 1b mRNA variants in GABA and non‐GABAergic neurons of the rat central nervous system (CNS), by using nonradioactive in situ hybridization histochemistry and, in combination with GABA immunocytochemistry, double labeling. In situ hybridization with a pan probe, which recognizes a common sequence of both GABABR1a and GABABR1b mRNA variants, demonstrated widespread expression of GABABR1 mRNA at various levels in the CNS. Both GABABR1a and GABABR1b were expressed in the neocortex, hippocampus, dorsal thalamus, habenula, and septum, but only GABABR1a was detected in cerebellar granule cells, in caudate putamen, and most hindbrain structures. A majority of GABA neurons in cerebral cortex showed hybridization signals for both GABABR1a and GABABR1b, whereas those in most subcortical structures expressed either or neither of the two. GABA neurons in thalamic reticular nucleus and caudate putamen hybridized primarily for GABABR1a. Purkinje cells in the cerebellar cortex expressed predominantly GABABR1b. GABA neurons in dorsal lateral geniculate nucleus did not display significant levels of either GABABR1a or GABABR1b mRNAs. These data suggested widespread availability of GABABR‐mediated inhibition in the CNS. The differential but overlapping expression of GABABR1 mRNA variants in different neurons and brain structures may contribute to the heterogeneity of GABABR‐mediated inhibition. Some GABA neurons possessed, but others might lack the molecular machinery for GABABR‐mediated disinhibition, autoinhibition, or both. J. Comp. Neurol. 416:475–495, 2000.

INTRACORTICAL REGIONALITY REPRESENTED BY SPECIFIC TRANSCRIPTION FOR A NOVEL PROTEIN, LATEXIN

The monoclonal antibody (mAb) PC3.1 recognizes a subset of neurons distributed in the infragranular layers of the lateral neocortex of the rat. Immunoaffinity chromatography with mAb PC3.1 showed that this antibody specifically binds a peptide epitope on a 29 kDa protein named latexin. To study the molecular details of the protein, we isolated four independent cDNA clones for latexin from cDNA libraries of the rat cerebral cortex and whole brain using the amino acid sequences of latexin fragments. Analysis of these cDNA clones showed that the predicted primary structure of latexin consists of 223 amino acids, and has no strict homology to any sequences so far known. Western and Northern blots demonstrated that the latexin and its mRNA were expressed predominantly in neural tissues with some expression in non‐neural tissues. The gene that encodes latexin in the rat appeared to have homologues in other mammalian species and in the chick. In situ hybridization showed that latexin mRNA is synthesized in a subset of neurons in the lateral but not the dorsal neocortex, and that the distribution profile of these neurons is quite similar to that of neurons expressing latexin. These results indicate that latexin is a novel class of neuronal protein which represents intracortical regionality, and suggest that the regional specification of the neocortex involves selective parcellation of neurons which express a particular gene.

Analysis of regulatory region of the rat latexin gene

In mammals, two isozymes of topoisomerase II (Topo II) have been identified. Topo IIcl plays a key role in DNA replication. Physiological functions of Topo IIP are still unclear and its expression level is independent of cellular proliferation profiles. We have reported previously that expression of Topo IIP transiently increased in developing cerebellum. Analysis of genomic regions recognized by Topo I@ would certainly facilitate the understanding of its functional significance. To clone the genomic regions targeted by Topo I@, libraries were constructed from DNA fragments recovered by Topo IIP-specific antibodies from the rat cerebellum (10 days after birth) which had been incubated with VP16. Topo II inhibitors such as VP16 are well known to stabilize the covalent complex between Topo II and DNA (cleavable complex). Nucleotide sequencing of 42 clones at the both ends showed that two-thirds of the clones were multicopy repetitive sequences such as LINE (long interspersed nuclear sequence), satellite I and ID. The remaining one-third appeared to be single copy sequences which were derived mostly from noncoding regions. Detailed mapping of the Topo II cleavage sites is under progress.