Subcellular and Synaptic Distribution of Muscarinic Receptors in Neurons by Confocal and Electron Microscopy

The function of a G protein-coupled receptor in the modulation of neuronal activity is highly dependent on its availability at the cell surface, its distribution in different subcellular compartments, and its localization in relationship with the presynaptic afferents. Therefore, investigation of the precise localization of GPCRs is required to clarify their contribution to neuronal function. Immunohistochemistry and confocal and electron microscopies are outstanding tools to achieve this goal. Here, we describe the confocal immunofluorescence approach and high-resolution electron microscopic pre-embedding immunogold technique that we have developed to analyze the subcellular and synaptic distribution of two acetylcholine muscarinic receptors (MR), M2MR and M4MR in neurons in three different models including fixed brain sections, primary culture neurons, and organotypic cultures. We have shown that M2MR and M4MR are mostly located at the plasma membrane where they are in the 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 intraneuronal trafficking when they are stimulated and that this trafficking is different according to the duration of the stimulation (acute versus chronic).