This is a renewal application of a research program in its third year of electrophysiological assessment of the role of cannabinoid receptors in brain. In the first 2.5 years of the project an extensive series of studies has been completed in which the effects of cannabinoids have been characterized in cultured hippocampal pyramidal cells. Several types of investigation including patch clamp recordings of whole cell currents and biochemical assessments of cellular mechanisms on the same type of hippocampal neurons have confirmed that cannabinoids modulate voltage dependent potassium A-current (IA) in these neurons. The effect is dramatic in that the capacity of the A-current channel is greatly altered by cannabinoids binding to the receptor. In neurons exposed to several different types of cannabinoid receptor ligands, the voltage dependence of IA was shifted positive by as much as 20 mV, indicating that cannabinoids are capable of profoundly altering the excitability of neurons and axon terminals. Studies went on to verify that, as required, this process was g-protein mediated (Gj) and was sensitive to concentrations of cannabinoid ligand that produced a corresponding increase in low Km GTPase activity in hippocampal slices. A more important finding was that modulation of the voltage dependence of IA was dependent on decreased cellular levels of cAMP produced by the well known inhibitory effect of cannabinoids on adenylyl cyclase. Increasing cellular cAMP either directly (8-bromo-cAMP) or indirectly through stimulation with forskolin, produced a reciprocal negative shift in IA voltage dependence from resting levels. The negative shift in IA produced by forskolin could be reversed by cannabinoid activation of the receptor that inhibited the forskolin stimulated adenylyl cyclase activity. Further studies showed that the cAMP dependent decrease in IA produced by cannabinoid receptor occupancy was likely mediated by alterations in phosphorylation status of the channel, since protein kinase A inhibitors effected a positive shift in IA voltage dependence and at the same time reduced or blocked the effects of cannabinoids on IA Thus the above studies show that cannabinoids alter the responsiveness of hippocampal neurons in culture via a cAMP dependent change in the voltage dependence of potassium A-current. Studies proposed in the next phase of the project extend these investigations to 1) the use of the newly discovered receptor antagonist SR141716A (Sanofi), 2) the role of the putative endogenous cannabinoid receptor ligand """"""""anandamide"""""""" in this process. 3) determination of the interactions between decreased conductance in N-type calcium channels and the alteration in voltage dependence of IA produced by cannabinoids, 4) studies of the effects of cannabinoids on IA in cells from slices of adult hippocampus and 5) the use of antisense probes to determine the type of potassium A channel modulated by the cannabinoid receptor.
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