Abstract
Numerous studies have investigated the roles of the type 1 cannabinoid receptor (CB1) in glutamatergic and GABAergic neurons. Here, we used the cell-type-specific CB1 rescue model in mice to gain insight into the organizational principles of plasma membrane targeting and Gαi/o protein signalling of the CB1 receptor at excitatory and inhibitory terminals of the frontal cortex and hippocampus. By applying biochemical fractionation techniques and Western blot analyses to synaptosomal membranes, we explored the subsynaptic distribution (pre-, post-, and extra-synaptic) and CB1 receptor compartmentalization into lipid and non-lipid raft plasma membrane microdomains and the signalling properties. These data infer that the plasma membrane partitioning of the CB1 receptor and its functional coupling to Gαi/o proteins are not biased towards the cell type of CB1 receptor rescue. The extent of the canonical Gαi/o protein-dependent CB1 receptor signalling correlated with the abundance of CB1 receptor in the respective cell type (glutamatergic versus GABAergic neurons) both in frontal cortical and hippocampal synaptosomes. In summary, our results provide an updated view of the functional coupling of the CB1 receptor to Gαi/o proteins at excitatory and inhibitory terminals and substantiate the utility of the CB1 rescue model in studying endocannabinoid physiology at the subcellular level.
Highlights
To determine the purity of the synaptosomal fraction (SYN), Western blot assays were carried out using antibodies raised against several proteins that have been used as markers of specific sub-cellular compartments
To gain insight into the organizational principles of plasma membrane location and Gαi/o protein signalling of the CB1 receptor at glutamatergic and GABAergic terminals of the mouse frontal cortex and hippocampus, the use of a highly specific anti-CB1 receptor antibody is mandatory
A specific extra band at ~35 kDa was clearly recognized with CB1-Immunogenes and CB1-Go-Af450 antibodies, whereas it was hardly detectable with the CB1-Rb-Af380 antibody in most experiments
Summary
CB1 receptors [5,6,7] This cell-type selective CB1 receptor expression has made it possible to define the contributions of both glutamatergic and GABAergic CB1 receptor to the tetrad effects of ∆9 -tetrahydrocannabinol [8]. This genetic rescue approach has revealed functions of CB1 receptor subpopulations that remain undetected when relying solely on a conditional knockout approach [8,9]. We have addressed cell-type specificity of the functional CB1 receptor coupling to Gαi/o proteins in hippocampal homogenates of conditional knockout mice [10]. The cell type-specific effects on agonist efficacy at the CB1 receptor observed in conditional mutant mouse lines prompted us to focus on CB1 receptors located at nerve terminals, a physiologically relevant location, and to the proximal components of the signalling machinery in this subsynaptic compartment, the
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