Expression And Function Of Cannabinoid Type1Receptor In Astrocytes Of The Hippocampus In Temporal Lobe Epilepsy

BackgroundCannabinoid type1receptor (CB1R) is a G protein coupled receptor, which is widelyexpressed in the central nervous system. Previous studies showed that CB1R plays animportant role in the pathology of epilepsy and neurodegenerative diseases by modulatingsynaptic transmission. Published works regarding the effects of cannabinoids on epilepsyhave been controversial. CB1R agonists display anti epileptic effects in animal models oftemporal lobe epilepsy (TLE). Conversely proconvulsive effects of CB1R agonists weredescribed as well. These contradictions suggested that these might be some differentmechanisms beyond the CB1R on synapse. Recent works have demonstrated thatastrocytes express CB1R and that CB1R in astrocytes is necessary for sustainingepileptiform activity in rat hippocampus. However, it is not clear whether CB1R inastrocytes involves in epileptogenesis. Therefore, we explored the role of CB1R ofastrocytes in TLE. ObjectiveTo investigate characteristics of astrocytic CB1R expression in sclerotic hippocampusfrom temporal lobe epilepsy patients. To investigate spatiotemporal profile and subcellularlocalization of CB1R in astrocytes in epileptogenesis of TLE. To investigate whetherCB1R participates in excitatory mechanism of activated astrocytes.MethodsFirst，the astrocytic CB1R expression was examined in hippocampus specimens from15cases of refractory TLE patients and5cases of traumatic brain injury patients byimmunofluorescence and confocal laser scanning microscope. The relationship betweenthe CB1R expression and clinical characteristics of TLE patients was analysized. Then,astrocytic CB1R expression was examined3days and4weeks after status epilepticus (SE)respectively in TLE rat models by above methods. Subcellular localization of CB1R insclerotic hippocampal astrocytes was examined by immune electron microscopy. Last, thecultured rat astrocytes were stimulated by lipopolysaccharide (LPS) to simulate thechanges in sclerotic hippocampus. The effects of LPS on astrocytes were examined byTUNNEL and MTT. CB1R distribution in astrocytes was detected by immunofluorescenceand confocal laser scanning microscope, and CB1R protein in astrocytes was examined bywestern blot. LPS treated and untreated astrocytes were stimulated by CB1R agonistWIN552122respectively before extracellular glutamate concentration was detected byHPLC MS/MS and intracellular calcium concentration was detected by calcium imagingtechnology.Results(1) CB1R in astrocytes was detected in9cases of TLE patients while CB1R inastrocytes was not detected in6cases of TLE patients and5cases of control;(2) The CB1R expression in astrocytes was related to seizure frequency positively;(3) The increased CB1R expression in hypertrophic astrocytes was found in TLEmodels respectively3days and4weeks after SE while CB1R was absent in astrocytes ofsaline controls; (4) Detection with immune electron microscopy showed that the CB1R expressionwas significantly increased in astrocytes of epileptic rats and modest level of CB1R wasalso detected on the astrocytic membrane of sclerotic hippocampus;(5) LPS activated the cultured astrocytes and then CB1R expression was increasedsignificantly and accompanied by a move away from the area around the nucleus;(6) WIN552122induced much more intracellular calcium release in the LPS treatedastrocyte than in the normal cells. Similarly, the extracellular glutamate concentration wasmuch higher in the LPS treated astrocytes than in the untreated cells after WIN552122administration.ConclusionsThese findings suggest that the level of CB1R expression in astrocytes might bedirectly linked to the frequency of seizure attacks in TLE patients, and the increasedCB1R expression in astrocytes in sclerotic hippocampus might be involved in the cellularbasis of the effects of cannabinoids on epilepsy through enhanced excitatory role ofactivated astrocytes.