Molecular Reconstruction of mGluR5a-Mediated Endocannabinoid Signaling Cascade in Single Rat Sympathetic Neurons

Abstract

Endocannabinoids (eCB) such as 2-arachidonylglycerol (2-AG) are lipid metabolites that are synthesized in a postsynaptic neurons and act upon CB(1) cannabinoid receptors (CB(1)R) in presynaptic nerve terminals. This retrograde transmission underlies several forms of short and long term synaptic plasticity within the CNS. Here, we constructed a model system based on isolated rat sympathetic neurons, in which an eCB signaling cascade could be studied in a reduced, spatially compact, and genetically malleable system. We constructed a complete eCB production/mobilization pathway by sequential addition of molecular components. Heterologous expression of four components was required for eCB production and detection: metabotropic glutamate receptor 5a (mGluR5a), Homer 2b, diacylglycerol lipase alpha, and CB(1)R. In these neurons, application of l-glutamate produced voltage-dependent modulation of N-type Ca(2+) channels mediated by activation of CB(1)R. Using both molecular dissection and pharmacological agents, we provide evidence that activation of mGluR5a results in rapid enzymatic production of 2-AG followed by activation of CB(1)R. These experiments define the critical elements required to recapitulate retrograde eCB production and signaling in a single peripheral neuron. Moreover, production/mobilization of eCB can be detected on a physiologically relevant time scale using electrophysiological techniques. The system provides a platform for testing candidate molecules underlying facilitation of eCB transport across the plasma membrane.