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 (CB1R) 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 α, and CB 1R. In these neurons, application of L-glutamate produced voltage-dependent modulation of N-type Ca2+ channels mediated by activation of CB1R. 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 CB1R. 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. Copyright © 2009 Society for Neuroscience.