Localization of the cannabinoid type-1 receptor in subcellular astrocyte compartments of mutant mouse hippocampus

Astroglial type-1 cannabinoid (CB₁ ) receptors are involved in synaptic transmission, plasticity and behavior by interfering with the so-called tripartite synapse formed by pre- and post-synaptic neuronal elements and surrounding astrocyte processes. However, little is known concerning the subcellular distribution of astroglial CB₁ receptors. In particular, brain CB₁ receptors are mostly localized at cells' plasmalemma, but recent evidence indicates their functional presence in mitochondrial membranes. Whether CB₁ receptors are present in astroglial mitochondria has remained unknown. To investigate this issue, we included conditional knock-out mice lacking astroglial CB₁ receptor expression specifically in glial fibrillary acidic protein (GFAP)-containing astrocytes (GFAP-CB₁ -KO mice) and also generated genetic rescue mice to re-express CB₁ receptors exclusively in astrocytes (GFAP-CB₁ -RS). To better identify astroglial structures by immunoelectron microscopy, global CB₁ knock-out (CB₁ -KO) mice and wild-type (CB₁ -WT) littermates were intra-hippocampally injected with an adeno-associated virus expressing humanized renilla green fluorescent protein (hrGFP) under the control of human GFAP promoter to generate GFAPhrGFP-CB₁ -KO and -WT mice, respectively. Furthermore, double immunogold (for CB₁ ) and immunoperoxidase (for GFAP or hrGFP) revealed that CB₁ receptors are present in astroglial mitochondria from different hippocampal regions of CB₁ -WT, GFAP-CB₁ -RS and GFAPhrGFP-CB₁ -WT mice. Only non-specific gold particles were detected in mouse hippocampi lacking CB₁ receptors. Altogether, we demonstrated the existence of a precise molecular architecture of the CB₁ receptor in astrocytes that will have to be taken into account in evaluating the functional activity of cannabinergic signaling at the tripartite synapse.