S and A.A. performed and supervised in vitro experiments in cell/astrocyte cultures and ex vivo analysis of brain tissue; A.B.G, C.I. and P.G.S. performed behavioral experiments and surgical procedures in mice; E.R. and M.G. provided some CB1-KO mice to the group of J.P.B.; D.A and A.P. performed electrophysiological experiments not shown in the manuscript; M.V. and F.J.K performed mouse perfusion and immunohistochemistry experiments; A.C. and L.B. produced some of the viral constructs used (e.g. Syn-mitoCAT); I.B.R, N.P., S.A. and P.G. performed and supervised electron microscopy experiments; M.L.L.R. provided pharmacological tools (HU-Biot); C.J., N.D and L.P provided specific viral constructs to modulate the MCT-2 transporter; C.J. and G.B. provided data and viral vectors regarding mouse retro-orbital injections; B.L and P.V.P. provided important conceptual ideas; A.K.B.S performed in vivo NMR experiments.
Astroglial type-1 cannabinoid (CB ) 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 CB receptors. In particular, brain CB receptors are mostly localized at cells' plasmalemma, but recent evidence indicates their functional presence in mitochondrial membranes. Whether CB receptors are present in astroglial mitochondria has remained unknown. To investigate this issue, we included conditional knock-out mice lacking astroglial CB receptor expression specifically in glial fibrillary acidic protein (GFAP)-containing astrocytes (GFAP-CB -KO mice) and also generated genetic rescue mice to re-express CB receptors exclusively in astrocytes (GFAP-CB -RS). To better identify astroglial structures by immunoelectron microscopy, global CB knock-out (CB -KO) mice and wild-type (CB -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-CB -KO and -WT mice, respectively. Furthermore, double immunogold (for CB ) and immunoperoxidase (for GFAP or hrGFP) revealed that CB receptors are present in astroglial mitochondria from different hippocampal regions of CB -WT, GFAP-CB -RS and GFAPhrGFP-CB -WT mice. Only non-specific gold particles were detected in mouse hippocampi lacking CB receptors. Altogether, we demonstrated the existence of a precise molecular architecture of the CB receptor in astrocytes that will have to be taken into account in evaluating the functional activity of cannabinergic signaling at the tripartite synapse.
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