Subcellular and Synaptic Localization of Muscarinic Receptors in Neurons Using High-Resolution Electron Microscopic Preembedding Immunogold Technique

Abstract

The function of a G protein-coupled receptor in the modulation of neuronal activity is highly dependent on its availability on the cell surface, on its distribution among different subcellular compartments and in relationship with the presynaptic afferents. Therefore, investigation of the precise localization of GPCRs is required to clarify their contribution to neuronal function, and can be achieved only by immunoelectron microscopy. Here, we describe the high-resolution electron microscopic preembedding immunogold technique that we have developed to analyze the subcellular and synaptic distribution of two acetylcholine muscarinic receptors (MR), M₂ and M₄ MRs in neurons in vivo. We have shown that M₂MR and M₄MR are mostly located at the plasma membrane where they are in a right position to interact with acetylcholine to modulate neuronal function. The synaptic and extrasynaptic localization of M₂MR suggests that the effect of acetylcholine might be mediated through a synaptic as well as diffuse type of transmission. The demonstration that M₂MR are present at the postsynaptic membrane beneath glutamatergic terminals provides a direct argument in favor of a co-release of ACh and glutamate. Finally, we have shown that muscarinic receptors are subject to an intraneuronal trafficking when they are stimulated and that this trafficking is different according to the duration of the stimulation (acute versus chronic).