ABSTRACT
Endocannabinoid receptors CB1R and CB2R are present in the CNS and modulate synaptic activity. By using an in vitro model, two concentrations of CB1R agonist ACEA at 0.5 and 5 µM doses and CB1R antagonist AM251 at 1 and 10 µM doses were administered in organotypic slice cultures of mouse hippocampus, and their effects on neurons and glial cells were analyzed at different time points. Exposure to low concentrations of ACEA (0.5 µM) did not seem to affect tissue organization, neuronal morphology, or glial response. In contrast, at a higher concentration of ACEA, many neurons in the dentate gyrus exhibited strong caspase-3 immunoreactivity. After treatment with AM251, we observed an increase in caspase-3 immunoreactivity and a downregulation of CB1R expression. Results show that long-term hippocampal slice cultures respond to both CB1R activation and inactivation by changing neuronal protein expression patterns. In the present study, we demonstrate that CB1R agonist ACEA promotes alterations in the neuronal cytoskeleton as well as changes in CB1R expression in organotypic hippocampal slice cultures, and that CB1R antagonist AM251 promotes neuronal death and astroglial reaction.
Subject(s)
Cannabinoids/metabolism , Hippocampus/drug effects , Receptor, Cannabinoid, CB1/antagonists & inhibitors , Receptor, Cannabinoid, CB2/antagonists & inhibitors , Animals , Arachidonic Acids/pharmacology , Caspase 3/metabolism , Cells, Cultured , Cytoskeleton/metabolism , Hippocampus/metabolism , Mice , Mice, Inbred C57BL , Piperidines/pharmacology , Pyrazoles/pharmacology , Receptor, Cannabinoid, CB1/genetics , Receptor, Cannabinoid, CB1/metabolism , Receptor, Cannabinoid, CB2/genetics , Receptor, Cannabinoid, CB2/metabolismABSTRACT
Little is known about the morphological effects of alcoholism on the developing adolescent brain and its consequences into adulthood. We studied here the relationship between two neurotransmitter systems (the serotoninergic and nitrergic) and the astrocytic and neuronal cytoskeleton immediately and long after drinking cessation of a chronic, but low, ethanol administration. Adolescent male Wistar rats were exposed to ethanol 6.6% (v/v) in drinking water for 6 weeks and studied after ending exposure or after a 10-week recovery period drinking water. Control animals received water. Brain sections were processed by immunohistochemistry using antibodies to serotonin (5-HT); glial fibrillary acidic protein (GFAP); astroglial S-100b protein; microtubule associated protein-2 (MAP-2); 200 kDa neurofilaments (Nf-200); and neuronal nitric oxide synthase (nNOS). The mesencephalic dorsal and median raphe nucleus (DRN; MRN) and three prosencephalic areas closely related to cognitive abilities (CA1 hippocampal area, striatum and frontal cortex) were studied by digital image analysis. 5-HT immunoreactivity (-ir) decreased in the DRN and recovered after abstinence and was not changed in the MRN. In the three prosencephalic areas, astrocytes' cell area (GFAP-ir cells) increased after EtOH exposure and tended to return to normality after abstinence, while cytoplasmic astroglial S100b protein-ir, relative area of MAP-2-ir and Nf-200-ir fibers decreased, and later partially recovered. In the striatum and frontal cortex, nNOS-ir decreased only after abstinence. In conclusion, in the adolescent brain, drinking cessation can partially ameliorate the ethanol-induced morphological changes on neurons and astrocytes but cannot fully return it to the basal state.
