Ultrastructural and physiological studies on the cannabinoid CB1 receptor localized in astroglia.

Abstract

The cannabinoid CB1 receptor-mediated functions in astrocytes are highly dependent on the CB1 receptor distribution in these glial cells relative to neuronal sites, particularly at the nearby synapses under normal or pathological conditions. However, the whole picture of the subcellular CB1 receptor distribution in astroglial compartments remains uncompleted due to the scattered CB1 receptor expression, and therefore difficult to detect, in astrocytes. Our laboratory has in previous studies estimated that about 5-6 % of the total CB1 receptors in the hippocampal CA1 stratum radiatum are localized in astrocytes identified by the marker glial fibrillary acidic protein (GFAP). However, GFAP is a cytoskeleton protein mostly restricted to the astroglial cell bodies and their main branches. This might be distorting the actual proportion and total amount of CB1 receptors in astrocytes. Therefore, the search for alternative astroglial markers to decipher the precise mapping of CB1 receptors in astrocytes is a timely goal in the cannabinoid field. The glutamate aspartate transporter (GLAST) is used as astroglial marker and raises as a good astroglial marker candidate to study in detail the CB1 receptor distribution in astrocytes. To prove this hypothesis, I have used a pre-embedding immunogold method for electron microscopy to compare first the astroglial distribution of GLAST versus GFAP. GLAST labeling was along the plasma membrane of astrocytes, including cell bodies and the smallest astrocytic projections in close contact with neurons, capillaries and other glial cells, covering altogether a much broader labeled area than GFAP. Furthermore, the use of a pre-embedding immunoperoxidase method for electron microscopy served me to assess that almost three times more astroglial area is visualized with GLAST than GFAP, and that GLAST detects four times as much astroglial membranes as GFAP. Finally, a double pre-embedding immunogold/immunoperoxidase method allowed to estimate that about 12 % of the total CB1 receptor particles are localized in GLAST-positive astrocytes, but the value drops to 5-6 % in GFAP-positive astrocytes, as published previously by our laboratory. Once these findings were obtained, I studied in more detail the CB1 receptor localization in astroglial mitochondria. We discovered the presence of functional CB1 receptors in mitochondrial membranes of hippocampal neurons and astrocytes. Accordingly, I used double GLAST-CB1 immunolabeling to analyze in the electron microscope the density of mitochondrial CB1 receptors in neurons and astrocytes in four brain regions: CA1 hippocampus, prefrontal cortex, piriform cortex and nucleus accumbens. The results showed that the CB1 receptor density in astrocytic mitochondria is higher than in neuronal mitochondria. Altogether, despite the lower absolute levels of CB1 receptors in astrocytes than in neurons, the density of mitochondrial CB1 receptors in astrocytes is higher than in neurons in the four brain regions studied. Namely, CB1 receptors are more expressed in astroglial than neuronal mitochondria. Activation of mitochondrial CB1 receptors alters energy production in neurons and can cause memory impairment. Likewise, activation of mitochondrial CB1 receptors in astrocytes interferes with glucose metabolism and lactate production, disrupting neuronal functions and social behavior. In conclusion, the findings that astrocytes of the four brain regions studied contain more CB1 receptors in their mitochondria than the neuronal mitochondria, and that the cannabinoid-induced reduction of oxygen consumption is absent in mitochondria isolated from the forebrain of GFAP-CB1-KO mice, suggest that mitochondrial CB1 receptors in astrocytes play a crucial role in the global effects of cannabinoids on brain mitochondrial respiration