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Necdin, a growth suppressor expressed predominantly in postmitotic neurons, interacts with viral oncoproteins and cellular transcription factors E2F1 and p53. In search of other cellular targets of necdin, we screened cDNA libraries from neurally differentiated murine embryonal carcinoma P19 cells and adult rat brain by the yeast two-hybrid assay. We isolated cDNAs encoding partial sequences of mouse NEFA and rat nucleobindin (CALNUC), which are Ca 2؉ -binding proteins possessing similar domain structures. Necdin interacted with NEFA via a domain encompassing two EF hand motifs, which had Ca 2؉ binding activity as determined by 45 Ca 2؉ overlay. NEFA was widely distributed in mouse organs, whereas necdin was expressed predominantly in the brain and skeletal muscle. In mouse brain in vivo, NEFA was localized in neuronal perikarya and dendrites. By immunoelectron microscopy, NEFA was localized to the cisternae of the endoplasmic reticulum and nuclear envelope in brain neurons. NEFAgreen fluorescent protein (GFP) fusion protein expressed in neuroblastoma N1E-115 cells was retained in the cytoplasm and partly secreted into the culture medium. Necdin enhanced the cytoplasmic retention of NEFA-GFP and potentiated the effect of NEFA-GFP on caffeine-evoked elevation of cytosolic Ca 2؉ levels. Thus, necdin and NEFA might be involved in Ca 2؉ homeostasis in neuronal cytoplasm.

Long-term plasticity of synaptic transmission is assumed to underlie the formation of long-term memory. Although the cellular mechanisms underlying short-term plasticity have been analyzed in detail, the mechanisms underlying the transformation from short-term to long-term plasticity remain largely unrevealed. We propose the novel long-lasting phenomenon as a model system for the analysis of long-term plasticity. We previously reported that the repetitive activation of cAMP-dependent protein kinase (PKA) by forskolin application led to an enhancement in synaptic strength coupled with synaptogenesis that lasted more than 3 weeks in cultured rat hippocampal slices. To elucidate whether this long-lasting synaptic enhancement depended on the induction of long-term potentiation (LTP) or on the pharmacological effect of forskolin, we applied glutamate (Glu) and correlated its dose with the production of the long-lasting synaptic enhancement. When the dose of Glu was low (10, 30 muM), only transient excitation or early-phase LTP (E-LTP) was induced by a single application and no long-lasting synaptic enhancement was produced by three applications. When the dose was raised to 100 or 300 muM, late-phase LTP (L-LTP) was induced by a single application and long-lasting synaptic enhancement was produced by three applications. The Glu-produced enhancement was accompanied by an increase in the frequency (but not the amplitude) of miniature EPSC and the number of synaptic structures. The enhancement depended on the interval of repetition and protein synthesis immediately after the Glu applications. These results indicate that the repetitive induction of L-LTP, but not E-LTP or transient excitation, triggers cellular processes leading to the long-lasting synaptic enhancement and the formation of new synapses.

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Short- and long-lasting synaptic plasticity is assumed to be the cellular basis of short- and long-lasting memory, respectively. However, the cellular consequences leading to the long-lasting synaptic plasticity, assumed to include the processes of synapse formation and elimination, remain unknown. Using hippocampal slices maintained stably in culture, we found previously that the repeated induction of long-term potentiation (LTP) triggered a slowly developing long-lasting enhancement in synaptic transmission strength accompanied by synapse formation, which was separate from LTP itself. We recently reported a phenomenon apparently of a mirror-image effect. The repeated activations of metabotropic glutamate receptor (mGluR), which induces long-term depression (LTD), triggered a long-lasting reduction in synaptic strength accompanied by synapse elimination. To clarify whether the reported long-lasting effect was specific to the drugs used previously and whether the effect was specific to mGluR-mediated LTD, we exposed the cultured slices repeatedly to another Group I metabotropic glutamate receptor (mGluR) agonist, an N-methyl-d-aspartate receptor agonist, and a Na+/K+-pump inhibitor. All these treatments resulted in an equivalent long-lasting synaptic reduction/elimination when repeated three times, indicating that the repeated LTD induction leads to synapse elimination. The independence of synapse elimination to the means of LTD induction suggests that the signals leading to short-term plasticity and long-term plasticity are independent. Detailed inspections in the representative case of mGluR activation revealed that the reduction in synaptic strength developed with a approximately 1-week delay from the decrease in the number of synaptic structures. This synapse elimination should be unique as it is activity-dependent rather than inactivity-dependent.