Cell-autonomous regulation of AMPA receptor phenotype by spontaneous action potentials in cerebellar stellate cells

BENDER C; SUN L; LIU SQ

NEW ORLEANS

Congreso; Annual Meeting of Society for Neuroscience; 2012

SfN

Cerebellar stellate cells are inhibitory interneurons that fire spontaneous action potentials in the absence of synaptic inputs and impose tonic inhibition onto Purkinje cells. The output of Purkinje cells is also controlled by a feed-forward inhibition that is driven by excitatory synaptic inputs onto stellate cells. Excitatory synaptic transmission in stellate cells is mediated by GluA2-lacking AMPA receptors, which have a large channel conductance and exhibit paired pulse facilitation during a train of synaptic activity, therefore promoting a strong feed forward inhibition. However it is not clear whether GluA2-lacking receptor expression in stellate cells is determined by their characteristic intrinsic membrane properties. To test this possibility, we incubated cerebellar slices in TTX (tetrodotoxin) for 3 hours and then measured EPSCs in the whole-cell configuration. Synaptic AMPAR phenotype was assessed from the I-V relationship of the EPSCs when spermine was included in the pipette solution. We found that blocking the generation of postsynaptic action potentials changed the I-V relationship of the EPSCs from inwardly rectifying to nearly linear, indicating an increase in synaptic GluA2-containing receptors in stellate cells. This TTX-induced switch in GluA2 receptors was prevented by protein synthesis inhibitors, anisomycin and cycloheximide. Protein translation efficiency is attenuated when eukaryotic elongation factor 2 (eEF2) is phosphorylated via eEF2 kinase, a Ca2+/calmodulin-dependent protein kinase, and enhanced by the eEF2 kinase inhibitor, NH125. Following treatment with NH125, the I-V relationship of EPSCs became more linear. To determine whether TTX increased GluA2 expression by reducing eEF2 kinase activity, we examined the expression of phospho-eEF2 in cultured stellate cells (18-21 days in vitro). To our surprise, the phospho-eEF2 immunoreactivity in TTX-treated stellate cells was not different from matched controls. We next examined whether spontaneous action potentials activate PKC, another Ca-dependent signaling pathway. In control stellate cells, we observed prominent PKC-ir in the cell membrane. In contrast, after TTX treatment the cytoplasmic PKC-ir increased whereas membrane PKC-ir diminished. While we have previously shown that PKC can regulate synaptic AMPA receptor subtype in stellate cells, whether PKC mediates the TTX-induced switch remains to be determined.