Adolescent ethanol intake alters cannabinoid type‐1 receptor localization in astrocytes of the adult mouse hippocampus

Abstract

Cannabinoid type-1 (CB ) receptors are widely distributed in the brain and play important roles in astrocyte function and the modulation of neuronal synaptic transmission and plasticity. However, it is currently unknown how CB receptor expression in astrocytes is affected by long-term exposure to stressors. Here we examined CB receptors in astrocytes of ethanol (EtOH)-exposed adolescent mice to determine its effect on CB receptor localization and density in adult brain. 4-8-week-old male mice were exposed to 20 percent EtOH over a period of 4 weeks, and receptor localization was examined after 4 weeks in the hippocampal CA1 stratum radiatum by pre-embedding immunoelectron microscopy. Our results revealed a significant reduction in CB receptor immunoparticles in astrocytic processes of EtOH-exposed mice when compared with controls (positive astrocyte elements: 21.50 ± 2.80 percent versus 37.22 ± 3.12 percent, respectively), as well as a reduction in particle density (0.24 ± 0.02 versus 0.35 ± 0.02 particles/μm). The majority of CB receptor metal particles were in the range of 400-1200 nm from synaptic terminals in both control and EtOH. Altogether, the decrease in the CB receptor expression in hippocampal astrocytes of adult mice exposed to EtOH during adolescence reveals a long lasting effect of EtOH on astrocytic CB receptors. This deficiency may also have negative consequences for synaptic function.

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

“…The results showed that the proportion and density of the CB 1 receptor immunolabeling (particles/μm) of astrocytic processes in the hippocampus were not significantly different between GFAP‐ CB 1 ‐RS and CB 1 ‐WT. The percentage of immunopositive astrocytes in CA1 of CB 1 ‐WT was in the range of the previous values reported by our group (Bonilla‐Del Río et al, ; Han et al, ), and almost all of the CB 1 receptor labeling was expressed in astrocytic elements in GFAP‐ CB 1 ‐RS. Furthermore, the proportion of astrocytic processes expressing CB 1 receptors and the density of receptor particles were about 34% and 64% higher, respectively, in the mutant mice targeted to express hrGFP in astroglial cells (GFAPhrGFP‐ CB 1 ‐WT) than in GFAP‐ CB 1 ‐RS.…”

“…Furthermore, CB 1 receptor expression increases in astrocytes of the sclerotic hippocampus (Meng et al, ) and blockade of the astroglial CB 1 receptors modulates the intracellular calcium signaling dampening epileptiform activity (Coiret et al, ). In addition, a strong decrease in CB 1 receptors in adult mouse CA1 astrocytes has been recently observed after adolescent drinking‐in‐the‐dark ethanol intake patterns (Bonilla‐Del Río et al, ). Taken together, the subcellular compartmentalization of the CB 1 receptor in astrocytes suggests the existence of specific and precise distribution of the receptor that seems to be crucial for the functional role of the CB 1 receptor at the tripartite synapse (Araque et al, ; Araque, Castillo, Manzoni, & Tonini, ; Belluomo et al, ; Han et al, ; Metna‐Laurent & Marsicano, ; Navarrete & Araque, ; Oliveira da Cruz et al, 2015; Perez‐Alvarez et al, ).…”

“…As a first step, we determined the CB 1 receptor expression and distribution in the conditional mutant mice in order to draw the level of agreement with the CB 1 receptor expression pattern in the CB 1 ‐WT mice. The combined pre‐embedding immunogold and immunoperoxidase method applied in this study has been previously proven to be an excellent approach for the localization of the CB 1 receptor in astrocytes (Bonilla‐Del Río et al, ; Bosier et al, ; Han et al, ) and mitochondria (Bénard et al, ; Hebert‐Chatelain et al, a,b, ). Specificity control experiments of the CB 1 receptor antibodies were carried out in CB 1 ‐KO and STOP‐ CB 1 mice (carrying a loxP‐flanked stop cassette inserted into the sequences of the 5'UTR of the CB 1 receptor).…”

“…We estimated that 10%–15% of the total CB 1 receptor labeling in the hippocampus is localized at mitochondrial membranes (Bénard et al, ; Bonilla‐Del Río et al, ; Hebert‐Chatelain et al, ), and this percentage is increased in muscle and heart (Mendizabal‐Zubiaga et al, ). Yet, about 22% of the mitochondrial sections in axon terminals and somatodendritic domains contain CB 1 receptors (Hebert‐Chatelain et al, a,b).…”

“…The CB 1 receptor‐mediated astrocyte functions are highly dependent on the CB 1 receptor distribution in astrocytes relative to close neuronal compartments, particularly at synapses (Bonilla‐Del Río et al, ). However, the low CB 1 receptor expression in astrocytes (Bosier et al, ; Han et al, ; Kovács et al, ; Rodriguez, Mackie, & Pickel, ) and mitochondria (Bénard et al, ; Hebert‐Chatelain et al, a; b; 2016) constrains a consolidated picture of the subcellular CB 1 receptor distribution in the astroglial compartments that holds the anatomical substrate for a functional interaction with the nearby synapses under normal or pathological conditions (Bonilla‐Del Río et al, ). Yet, whether intracellular CB 1 receptors exist in astroglial mitochondria has remained unknown.…”