Anatomical characterization of the cannabinoid CB₁ receptor in cell‐type–specific mutant mouse rescue models

Abstract

Type 1 cannabinoid (CB ) receptors are widely distributed in the brain. Their physiological roles depend on their distribution pattern, which differs remarkably among cell types. Hence, subcellular compartments with little but functionally relevant CB receptors can be overlooked, fostering an incomplete mapping. To overcome this, knockin mice with cell-type-specific rescue of CB receptors have emerged as excellent tools for investigating CB receptors' cell-type-specific localization and sufficient functional role with no bias. However, to know whether these rescue mice maintain endogenous CB receptor expression level, detailed anatomical studies are necessary. The subcellular distribution of hippocampal CB receptors of rescue mice that express the gene exclusively in dorsal telencephalic glutamatergic neurons (Glu-CB -RS) or GABAergic neurons (GABA-CB -RS) was studied by immunoelectron microscopy. Results were compared with conditional CB receptor knockout lines. As expected, CB immunoparticles appeared at presynaptic plasmalemma, making asymmetric and symmetric synapses. In the hippocampal CA1 stratum radiatum, the values of the CB receptor-immunopositive excitatory and inhibitory synapses were Glu-CB -RS, 21.89% (glutamatergic terminals); 2.38% (GABAergic terminals); GABA-CB -RS, 1.92% (glutamatergic terminals); 77.92% (GABAergic terminals). The proportion of CB receptor-immunopositive excitatory and inhibitory synapses in the inner one-third of the dentate molecular layer was Glu-CB -RS, 53.19% (glutamatergic terminals); 2.30% (GABAergic terminals); GABA-CB -RS, 3.19% (glutamatergic terminals); 85.07% (GABAergic terminals). Taken together, Glu-CB -RS and GABA-CB -RS mice show the usual CB receptor distribution and expression in hippocampal cell types with specific rescue of the receptor, thus being ideal for in-depth anatomical and functional investigations of the endocannabinoid system. J. Comp. Neurol. 525:302-318, 2017. © 2016 Wiley Periodicals, Inc.

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

“…The GFAP‐ CB 1 ‐RS mouse expressing CB 1 receptors exclusively in astrocytes described here, together with the Glu‐ CB 1 ‐RS rescue mouse expressing the receptor only in dorsal telencephalic glutamatergic neurons (de Salas‐Quiroga et al, ; Lange et al, ; Ruehle et al, ; Soria‐Gómez et al, ) and the GABA‐ CB 1 ‐RS rescue mouse expressing the CB 1 receptor only in GABAergic neurons (de Salas‐Quiroga et al, ; Lange et al, ) that were recently characterized anatomically (Gutiérrez‐Rodríguez et al, ; Remmers et al, ), suggest that the regulation of the CB 1 receptor expression in astrocytes, glutamatergic neurons and GABAergic neurons may be independent of each others. The present demonstration that the GFAP‐ CB 1 ‐RS in the hippocampus maintains the normal CB 1 receptor expression and distribution in astrocytes make these mutants ideal suited for the study of the astroglial CB 1 receptor function, as shown for Glu‐ CB 1 ‐RS (de Salas‐Quiroga et al, ; Gutiérrez‐Rodríguez et al, ; Lange et al, ; Ruehle et al, ; Soria‐Gómez et al, ) and GABA‐ CB 1 ‐RS mice (de Salas‐Quiroga et al, ; Gutiérrez‐Rodríguez et al, ; Lange et al, ; Remmers et al, ). In fact, these rescue strategies have the advantage of the restoration and visualization of existing CB 1 receptor levels in locations with sparse CB 1 receptors (as the astrocytes and astroglial mitochondria), allowing a more comprehensive functional characterization of the (endo)cannabinoid system based on the precise cellular and subcellular localization of the CB 1 receptor.…”

“…The high CB 1 receptor expression in the hippocampus is unevenly distributed between subcellular compartments of GABAergic and glutamatergic synaptic terminals, astrocytes and neuronal mitochondria (Bénard et al, ; Gutiérrez‐Rodríguez et al, ; Han et al, ; Hebert‐Chatelain et al, a,b, ; Katona & Freund, ; Lu & Mackie, ; Marsicano & Lutz, ; Steindel et al, ). However, no information is available to date whether the CB 1 receptor localizes in astroglial mitochondria as it does in mitochondria of hippocampal GABAergic and glutamatergic neurons (Bénard et al, ; Hebert‐Chatelain et al, a,b, ).…”

“…This system is widely distributed in the central and peripheral nervous system (Katona & Freund, ; Lu & Mackie, ), and also in peripheral organs (Piazza, Cota, & Marsicano, ), where the CB 1 receptors are also localized in mitochondria of striated and heart muscles (Mendizabal‐Zubiaga et al, ). The eCB system regulates brain functions by acting on different cell types and cellular compartments (Busquets‐Garcia, Bains, & Marsicano, ; Gutiérrez‐Rodríguez et al, ; Katona & Freund, ; Lu & Mackie, ). The activation of CB 1 receptors in astrocytes promotes astroglial differentiation (Aguado et al, ) and mediates neuron‐astrocyte communication that plays a role in synaptic plasticity, memory and behavior (Araque et al, ; Gómez‐Gonzalo et al, ; Han et al, ; Metna‐Laurent & Marsicano, ; Navarrete & Araque, ; Navarrete, Diez, & Araque, ; Oliveira da Cruz, Robin, Drago, Marsicano, & Metna‐Laurent, 2015).…”

“…These effects are due to intra‐mitochondrial Gαi protein activation by mtCB 1 receptors that leads to the inhibition of soluble adenylyl cyclase and, consequently, to the decrease in intra‐mitochondrial protein kinase A (PKA) activity (Hebert‐Chatelain et al, ). New tools based on genetic rescue strategies have proven to be useful to dissect the sufficiency of the CB 1 receptors expressed in specific cell types for a particular brain function (de Salas‐Quiroga et al, ; Gutiérrez‐Rodríguez et al, ; Lange et al, ; Remmers et al, ; Ruehle et al, ; Soria‐Gómez et al, ). Importantly, knock‐in mice with cell type‐specific rescue of CB 1 receptors in dorsal telencephalic glutamatergic neurons (Glu‐ CB 1 ‐RS) or GABAergic neurons (GABA‐ CB 1 ‐RS) showed that the distribution pattern and the subcellular CB 1 receptor localization is maintained as it is observed in the wild‐type hippocampus (Gutiérrez‐Rodríguez et al, ; Remmers et al, ).…”

Hippocampal CB1 Receptors Control Incidental Associations

“…Male CB 1 R-floxed mice (Marsicano et al, 2003), CB 1 R full knockout (CB 1 R-KO) mice (Marsicano et al, 2003), conditional knockout mice lacking CB 1 R in forebrain GABAergic Dlx5/6 positive neurons (GABA-CB 1 R-KO) (Bellocchio et al, 2010;Monory et al, 2006), mice expressing Cre recombinase under the regulatory elements of the Dlx5/6 gene (GABA-Cre) or the parvalbumin (PV) gene (PV-Cre) and their wild-type littermates mice were obtained, maintained and genotyped as described before (Bellocchio et al, 2010;Courtin et al, 2014;Marsicano et al, 2003;Monory et al, 2006). The rescue line (GABA-CB 1 R-KO) was generated as described before (Busquets-Garcia et al, 2016;Gutié rrez-Rodríguez et al, 2017;Ruehle et al, 2013;Soria-Gó mez et al, 2014). All the mice used in this study were 9-10 weeks old at the beginning of the experiments.…”