High-resolution Immunoelectron Microscopy Techniques for Revealing Distinct Subcellular Type 1 Cannabinoid Receptor...

Activation of type 1 cannabinoid (CB₁) receptors by endogenous, exogenous (cannabis derivatives) or synthetic cannabinoids (i.e., CP 55.940, Win-2) has a wide variety of behavioral effects due to the presence of CB₁ receptors in the brain. In situ hybridization and immunohistochemical techniques have been crucial for defining the CB₁ receptor expression and localization at the cellular level. Nevertheless, more advanced methods are needed to reveal the precise topography of CB₁ receptors in the brain, especially in unsuspected sites such as other cell types and organelles with low receptor expression (e.g., glutamatergic neurons, astrocytes, mitochondria). High-resolution immunoelectron microscopy provides a more precise detection method for the subcellular localization of CB₁ receptors in the brain. Herein, we describe a single pre-embedding immunogold method for electron microscopy based on the use of specific CB₁ receptor antibodies and silver-intensified 1.4 nm gold-labeled Fab' fragments, and a combined pre-embedding immunogold and immunoperoxidase method that employs biotinylated secondary antibodies and avidin-biotin-peroxidase complex for the simultaneous localization of CB₁ receptors and protein markers of specific brain cells or synapses (e.g., GFAP, GLAST, IBA-1, PSD-95, gephyrin). In addition, a post-embedding immunogold method is also described and compared to the pre-embedding labeling procedure. These methods provide a relatively easy and useful approach for revealing the subcellular localization of low amounts of CB₁ receptors in glutamatergic synapses, astrocytes, neuronal and astrocytic mitochondria in the brain.