Cannabinoids have been proposed to exert neuromodulatory and neuroprotective effects [1-4], although the exact mechanisms by which they do so remain to be established. We aimed to characterise the effects of the non-psychotropic plant cannabinoid cannabidiol (CBD) on Ca²⁺ signalling in primary hippocampal cultures by imaging Ca²⁺ responses with the fluorescent marker Fura-2. Hippocampi were dissected and the tissue enzymatically dissociated, from brains of Sprague-Dawley neonates (1-3 days). Cultures were matured for 5-12 days in medium containing 90% minimal essential medium (MEM) and 10% foetal bovine serum (FBS) and loaded with fluorescent marker Fura-2AM (10µM) for 1 hour. Cultures were perfused with HEPES buffered solution (HBS) (containing 0.5µM TTX) at a rate of ~2mls per minute using a gravity perfusion system. [Ca²⁺]_i measurements were calculated as ratio values (from images captured at wavelengths 350nm and 380nm). Application of CBD (1µM) induced a Ca²⁺ response (range: 0.15-1 ratio units) in both neurones (46%; n=23/50) and glia (83%; n=55/66). By varying neuronal excitability via modification of the HBS, response rate and magnitude was altered in high excitability HBS (KCl: 7mM, CaCl₂: 3mM, MgCl₂: 0.5mM; 72/80 neurones; 50/66 glia) but not in low excitability HBS (KCl: 3mM, CaCl₂: 1mM, MgCl₂: 3mM; 50/75 neurones; 69/80 glia). Moreover, further experiments with thapsigargin (2µM; n=178), which depletes intracellular C²⁺+ stores, suggest that the Ca²⁺ signal is evoked via release from these stores since this compound prevented the CBD-induced Ca²⁺ rise. In addition, it was observed that CBD (1µM) was able to lower [Ca²⁺]_i raised after thapsigargin (2µM) application (by 19%).

Ca²⁺ signals evoked with glutamate (5-10µM; n=58) and with the subtype-specific glutamate receptor agonist kainate (10-50µM, n=64) were decreased by 35.4% and 49.5% (P's<0.0001), respectively. However, Ca²⁺ responses induced with N-methyl-D-aspartate (NMDA, 10µM; n=40), were not significantly affected by CBD (1 µM). Interestingly, responses evoked with the metabotropic glutamate receptor group I agonist (S)-3,5-dihydroxyphenylglycine (DHPG; 10-50µM, n=30) were most potently affected by CBD and reduced to 17.8% of control (P<0.0001). This highlights further a potential association between the modulation of intracellular Ca2+ stores by CBD.

In conclusion, our results indicate that CBD is able to modulate intracellular Ca²⁺ concentrations via a mechanism that involves intracellular Ca2+ stores. Together with the observed reduction of glutamate-evoked Ca²⁺ responses, this may contribute crucially to CBD's neuroprotective potential.

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