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Abstract: Variations in intracellular calcium activity ([Ca2+]i) play crucial roles in information processing in Purkinje neurons such as synaptic plasticity. Although Na+-Ca2+ exchanger (NCX) has been shown to participate in the regulation of homeostasis and secretion in neuronal cells, the physiological role of NCX in Purkinje neurons, such as a role in cerebellar synaptic plasticity, is not well understood. NCX in acutely dissociated rat Purkinje neurons was identified by double staining with anti-calbindin D-28k antibody and anti-NCX antibody. The physiological activity of NCX was examined by measuring transient intracellular Ca2+ changes resulting from the Ca2+ influx via reverse mode of NCX (with 0 mM Na+/2.5 mM Ca2+ solutions) and the efflux via the forward mode of NCX (with 140 mM Na+/0 mM Ca2+ solutions). This transient increase in Ca2+ concentration was not elicited in the cells pretreated with NCX antisense oligodeoxynucleotides. And the Ca2+ influx resulting from the reverse mode of NCX was significantly reduced by 2-[2-[4-(4-nitrobenyloxy) phenyl] ethyl] isothiourea methanesulfonate, while the Ca2+ efflux via forward mode was inhibited by bepridil. The physiological role of NCX in synaptic function was studied by measuring Ca2+ transients induced by α-amino-3-hydroxy-5-methyl-4-isoxazolone-propionate (AMPA) receptor activation. This AMPA-evoked response was decreased with the inhibition of NCX forward mode and also, to less degree, with the inhibition of reverse mode. In antisense oligodeoxynucleotides pretreated cells, the AMPA-evoked response was also reduced, as was the case in NCX-inhibitor treated cells. The inhibition of NCX activity had depressant effects on Ca2+ transients induced by AMPA receptor activation. These results suggest that NCX plays a physiological role in modulating the activity of cerebellar Purkinje neurons, such as synaptic plasticity, via interaction with AMPA receptors in Purkinje neurons. [Copyright &y& Elsevier]

Recent data indicate that most “silent” synapses in the hippocampus are “presynaptically silent” due to low transmitter release rather than “postsynaptically silent” due to “latent” receptors of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid type (AMPARs). That synapses bearing only N-methyl-d-aspartate (NMDAR) receptors do exist is suggested by the decreased number of transmission failures during postsynaptic depolarisation and by the presence of NMDA-mediated excitatory postsynaptic currents (EPSCs) in synapses silent at rest. We tested whether these effects could be due to potentiated transmitter release at depolarised postsynaptic potentials rather than removal of Mg2+ block from NMDARs. Using whole-cell recordings of minimal EPSCs from CA1 and CA3 neurones of hippocampal slices we confirmed decreased incidence of failures at +40 mV as compared with -60 mV. This effect was associated with a gradual increase of EPSC amplitude after switching to +40 mV and with a decrease of paired-pulse facilitation. In initially silent synapses, potentiation of pharmacologically isolated AMPAR-mediated EPSCs was still observed at +40 mV and this persisted after stepping back to -60 mV. All above effects were blocked when the cell was dialysed with the Ca2+ chelator BAPTA (20 mM). These observations are difficult to reconcile with the “latent AMPAR” hypothesis and suggest an alternative explanation, namely that the reduction in failure rates at positive potentials is due to potentiation of transmitter release following Ca2+ influx through NMDARs. Our results suggest that silent synapses can be mainly “presynaptically” rather than “postsynaptically silent” and thus increased transmitter release rather than insertion of AMPARs is a major mechanism of early long-term potentiation maintenance. [Copyright &y& Elsevier]

The CREB transcription factor mediates neuronal plasticity in many systems, but the relationship between CREB levels and CRE-mediated transcription in individual neurons in vivo is unclear. In FVB/N nontransgenic mice, we observed that Purkinje cells showed low basal levels of Ser133-phosphorylated CREB protein yet displayed strong CRE-directed transcription. Transgenic mice overexpressing CREB in Purkinje cells and dentate gyrus granule cells showed a decreased CRE-lacZ signal in the same cells, indicating repression of ATF/CREB family function. Dentate region long-term potentiation was not altered by these changes in CREB expression. CREB transgenic mice demonstrated an inability to perform the rotarod task, without signs of overt ataxia. Our results demonstrate that the level of phosphorylated CREB protein is not a reliable indicator of CRE-mediated function. Furthermore, we conclude that CRE-mediated transcription may be linked to only a subset of cerebellum-mediated motor behaviors and may not be universally required for long-lasting synaptic potentiation. [Copyright &y& Elsevier]