Biochemical and physiological assessment of neural visinin-like calcium-binding protein 3 in rodent cerebellum.

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Neural visinin-like calcium-binding protein 3, a member of neuronal calcium sensor protein (NCS) family, is highly expressed in the cerebellum. Expression of NVP3 was assessed by immunoblot and immunohistochemical analyses in rat brain. NVP3 was markedly expressed in the cerebellum, at a concentration of 9.5μM.At 3 months, NVP3 was primarily localized in the Purkinje cells, with intense staining in the cell bodies, dendrites and axons. The cerebellar granule cells and basal nuclear neurons were faintly stained. During development of the cerebellum, NVP3-positive Purkinje cells first appeared on post-natal day 14 (P14). The staining intensity then increased and plateaued on P28. Labeling showed a tendency to accumulate in the dendrites and nerve terminals in a fine granular pattern. During aging process, NVP3 levels decreased by 43% at 12 months and 68% at 24 months, while the levels of NVP1, synaptophysin and drebrin were preferentially preserved. These results suggest that NVP3 is inv … More olved in dendritic arborization and postsynaptic function in cerebellar Purkinje cells and that presynaptic nerve terminals are another functional sites of the protein.To reveal its physiological roles in vivo, we generated NVP3 null mutant (-/-) mice. Firstly, we isolated the mouse NVP3 gene and cDNA.The mouse NVP3 gene contains 4 exons and 3 introns and spans approximately 7 kb. Southern blot analysis of the mouse genomic DNA demonstrated that positive bands exactly coincide with those expected from sequences of the cloned genes, indicating that the mouse NVP3 gene is present as a single copy gene. Then, we constructed NVP3 knockout vector using neomycin-resistance gene and Diphtheria toxin A gene. The vector DNA was introduced into CCE embryonic stem (ES) cells. Positive clones were selected and microinjected into C57BL/6 blastocysts to generate the chimeras. F1 heterozygotes form the chimeras were intercrossed with each other to yield null mutant (-/-) mice. Null mutant mice exhibited no apparent structural abnormality of the cerebellum in light microscopic levels. The lack of NVP3 was not compensated by possible up-regulation of homologous proteins NVP1, 2 and hippocalcin. No apparent abnormalities were also observed in motor activity and day-night cycles. Electrophysiological responses of the cerebellar Purkije cells and motor coordination learning abilities will be investigated in further studies. Less