Shigeharu Saitoh

Distribution of hippocalcin mRNA and immunoreactivity in rat brain

Distribution of hippocalcin in rat brain was analysed by in situ hybridization and immunohistochemical methods. Hippocalcin mRNA and immunoreactivity were expressed more intensely in the pyramidal cells of the hippocampus, intensely in the Purkinje cells of the cerebellum, moderately in the dentate granule cells and pyramidal cells of cerebral cortex layers II-VI and weakly in the large neuronal cells of the caudate-putamen. Some discrepancies in the localization of hippocalcin mRNA and immunoreactivity were noted in the mamillary nuclei, anterior part of the thalamus and the septal nuclei. In most cell types, hippocalcin immunoreactivity was localized in the cytoplasm and plasma membrane of cell bodies and dendrites.

Immunohistochemical localization of neural visinin-like Ca2+-binding protein 2 in adult rat brain

The distribution of neural visinin-like Ca(2+)-binding protein 2 (NVP2) in adult rat brain was analyzed by immunoblot and immunohistochemical methods. NVP2 immunoreactivity was expressed intensely in the pyramidal cells of the CA1 and 2 regions of Ammons horn, the granule cells of the dentate gyrus and the pyramidal cells of the cerebral cortex layers II to VI, moderately in the pyramidal-shaped cells of the anterior olfactory nucleus and large spindle-shaped cells of the globus pallidus, and weakly in neurons in the nucleus accumbens and the anterior and dorsomedial thalamus. In most cell types, NVP2 immunoreactivity was located in the cytoplasm and plasma membrane of the cell bodies and dendrites.

EXPRESSION OF HIPPOCALCIN IN THE DEVELOPING RAT BRAIN

Expression of hippocalcin in the developing rat brain was investigated by a combination of Northern blot, in situ hybridization, immunoblot and immunohistochemical methods. In the hippocampus, hippocalcin mRNA and immunoreactivity first appeared in the CA3 pyramidal cells on embryonic day 19 (E19) and postnatal day 1 (P1), respectively, and extended throughout Ammons horn. After P14, the hippocampal pyramidal cells, especially in the CA1 region, maintained the highest expression level among the brain regions. The dentate granule cells expressed a small amount of hippocalcin mRNA and immunoreactivity from P7 and maintained a low level through the developmental stages. In the cerebral cortex, hippocalcin mRNA and immunoreactivity appeared in the pyramidal cells of the piriform cortex from P1 and P4, respectively. Their expression extended throughout the cerebral cortex and reached the maximum level on P14, and then declined gradually with age to half of the maximum level by adults. In the cerebellum, a few Purkinje cells expressed a small amount of hippocalcin mRNA and immunoreactivity on P7. Their expression became evident in most of the Purkinje cells on P14 and increased gradually by P28. Then, their expression declined with age; however, the immunoreactivity was concentrated in the cell bodies and proximal segments of the dendrites in adults. These results suggest that the expression of hippocalcin mRNA and protein is strictly controlled by both the cell type and the developmental process and that hippocalcin plays a role in neuronal differentiation in the early stages of development and may relate to other neuronal function in the adult brain.

Molecular cloning of a novel calcium-binding protein structurally related to hippocalcin from human brain and chromosomal mapping of its gene.

A cDNA clone (hHLP2) encoding a novel calcium-binding protein structurally related to hippocalcin has been isolated from the human hippocampus cDNA library. The primary structure consists of 193 amino acids, and contains three EF-hand structures and a possible NH2-terminal myristoylation site. A single transcript at a position corresponding to 1.7 kilobases was detected only in the brain. The hHLP2 gene was mapped to human chromosome 2.

The development of neural visinin-like Ca2+-binding protein 2 immunoreactivity in the rat neocortex and hippocampus

Neural visinin-like Ca(2+)-binding protein 2 (NVP2) immunoreactivity in the rat neocortex and hippocampus was barely detectable by immunoblot analysis on postnatal day 1 (P1), but increased during postnatal weeks 2-3, reaching a plateau on P28. Immunohistochemical analysis revealed moderate immunoreactivity firstly on P7 in some subsets of the hippocampal interneurons and in the hippocampal pyramidal cells and dentate granule cells. Immunoreactivity of the interneurons decreased during postnatal weeks 2-3 and disappeared by P28. In contrast, immunoreactivity of the cortical and hippocampal pyramidal cells and dentate granule cells abruptly increased during postnatal week 2. The distinctly immunoreactive cells were distributed throughout the neocortex, especially in the cortical plate, and the stratum pyramidale of Ammons horn and granular layer of the dentate gyrus on P14. Immunoreactivity was homogeneously concentrated in the cell bodies and proximal dendrites at this stage, whereas thereafter immunoreactivity in the neuropil gradually increased, and underwent a relative decrease in the cell bodies. By P28, the higher and granular immunoreactivity in the neuropil covered whole layers of the neocortex, Ammons horn and the dentate gyrus, the same as in adults. Differential expression of NVP2 in different neuron populations may reflect the differential functional consequences for neuronal development.

Hippocalcin expression in the brain of the Snell dwarf mutant mouse.

To determine factors contributing to the expression of the brain-derived protein, hippocalcin, we mapped its distribution in the brain of Snell pituitary dwarf mutant mice (dw) by immunohistochemical and immunoblot methods. Our findings are as follows. (1) In the hippocampus, hippocalcin immunoreactivity was found in the cell body and dendrites of pyramidal neurons of the normal controls and dw mice, although the intensity of immunoreactivity in the dw mice was lower. (2) In the cerebellum, hippocalcin immunoreactivity was strongly expressed in the Purkinje cell body of both the control and dw mice. However, the Purkinje cell dendrites were found to be more intensely stained in the dw mice than in the normal controls. (3) In the dw cerebral cortex, the pyramidal neurons of layers II to VI strongly expressed hippocalcin, whereas its expression in the controls was weak. (4) The amount of hippocalcin in the dw hippocampus was less than in the normal controls, whereas the amount in the dw cerebral cortex and cerebellum was greater. These results indicate that the developmental expression of hippocalcin in the dw brain is affected by the retarded maturation of the neuronal network due to the deficient hormonal state (the lack of growth and thyroid hormones).