Epac-mediated modulation of neurotransmitter release from cultured hippocampal neurons

Abstract

cAMP regulates secretory processes through both protein kinase A (PKA)-independent and PKA-dependent signaling pathways. However, their relative contributions to fast neurotransmission are unclear at present, although the cAMP mediated increase in neurotransmitter release could be attributed to an increase of vesicular release probability and to the enhancement of the number of releasable vesicles in various neuronal preparations. The present study analysed the contribution of the novel cAMP receptor Epac to the cAMP related modulation of neurotransmission. Neurotransmitter release from cultured murine or rat hippocampal excitatory autaptic neurons was monitored during low and high frequency stimulation in the presence or absence of Epac-Selective Cyclic AMP Analogs (ESCAs) using the patch-clamp technique. ESCA-induced Epac activation increased both the action potential-evoked excitatory postsynaptic current (EPSC) amplitudes during 0.2 Hz frequency stimulation and the number of spontaneous fusion events (mEPSCs) from autaptic excitatory Dentate Gyrus-neurons. The magnitude of potentiation depended on the average EPSC amplitude recorded during the first two minutes, on the cell culture age, and on the presynaptic calcium ion concentration. ESCA1 (8-pCPT-2 -O-Me-cAMP)-mediated Epac activation accounted on average for 38% of the forskolin-induced increase in evoked EPSC amplitudes and for 100% of the forskolin-induced increase in mEPSC frequency. Forskolin is generally believed to enhance intracellular cAMP levels by stimulation of adenylyl cyclase activity. The mEPSC amplitude and the number of readily releasable vesicles remained unchanged upon brief ESCA1 treatment (1.2 minutes). In conclusion, ESCA1 application increased vesicular release probability. Finally, the data show that ESCA1- and phorbol esters-stimulated signaling pathways interact. The observed coupling of both signaling pathways was dependent on protein kinase C activity.