Cholecystokinin-8S-Induced Intracellular Calcium Signaling in Acutely Isolated Periaqueductal Gray Neurons of the Rat(Pharmacology)

Many behavior studies indicate that cholecystokinin (CCK) is related to nociception and anxiety/panic actions in the midbrain periaqueductal gray (PAG). We previously reported that a sulfated form of CCK octapeptide (CCK-8S) produced excitatory effects at both pre- and postsynaptic loci in PAG neurons using slice preparations and whole-cell patch-clamp recordings. Here, we further examined the detailed mechanism of CCK-8S in acutely isolated PAG neurons of the rat using fura-2-based imaging of intracellular Ca^<2+> concentration ([Ca^<2+>]_i) and whole-cell patch-clamp recordings. Application of 1μM CCK-8S produced an increase of [Ca^<2+>]_i, and its effect did not desensitize. This CCK-8S-induced [Ca^<2+>]_i increase was inhibited by the CCK_2 receptor antagonist L-365260 but not by the CCK_1 receptor antagonist L-364718. In addition, the effect of CCK-8S was eliminated by removing extracellular Ca^<2+>, but not by an addition of the intracellular Ca^<2+> reuptake inhibitor thapsigargin. When simultaneous recordings of [Ca^<2+>]_i imaging and whole-cell patch-clamp were performed, CCK-8S-induced [Ca^<2+>]_i increase was significantly reduced at a membrane holding potential of -60mV while CCK-8S-induced inward current was still observed. Current-voltage plots revealed that CCK-8S-induced inward current reversed near the equilibrium potential for K^+ ions with a decreased membrane conductance. However, CCK-8S produced a significant inhibition on high-voltage-activated Ca^<2+> channel currents. These results suggest that CCK-8S can excite PAG neurons by inhibiting K^+ channels, and CCK-8S-induced [Ca^<2+>]_i increase occurs secondary to depolarization. The evidence presented here expands our understanding of cellular mechanisms for CCK-mediated anti-analgesic and anxiogenic actions in the PAG.