Pituitary Adenylate Cyclase-Activating Peptide in the Bed Nucleus of the Stria Terminalis Mediates Stress-Induced Reinstatement of Cocaine Seeking in Rats.

Stressors often contribute to difficulties in maintaining behavior change following a period of abstinence, and may play a significant role in drug relapse. The activation of pituitary adenylate cyclase-activating peptide (PACAP) systems in the bed nucleus of the stria terminalis (BNST) mediates many consequences of chronic stressor exposure. Here we ask whether PACAP is also involved in producing reinstatement in a model of stress-induced relapse to drug taking. Rats self-administered cocaine for 1 h daily over 10 days that was followed by 20 days of extinction training in which lever pressing no longer produced cocaine. In experiment 1, quantitative PCR (qPCR) was performed at several stages to determine transcript levels of PACAP and corresponding receptors. Reinstatement of cocaine seeking was then tested after footshock exposure in different groups of rats that were pretreated with vehicle solution, a PAC1 receptor antagonist (experiment 2), or a PACAP agonist (experiment 3) without footshock. In experiment 1, cocaine self-administration increased BNST PACAP transcript levels similar to what we have previously reported with chronic stress. In experiment 2, intra-BNST infusions of the PAC1/VPAC2 antagonist, PACAP 6-38, prevented footshock-induced reinstatement of extinguished cocaine seeking. In experiment 3, intra-BNST PACAP infusion reinstated previously extinguished cocaine-seeking behavior in the absence of footshock. Cocaine self-administration elevated BNST PACAP, and BNST PACAP receptor activation was necessary and sufficient for stress-induced reinstatement of cocaine seeking. These data suggest that BNST PACAP systems may be viable targets for relapse prevention.

Src family kinase inhibitors blunt PACAP-induced PAC1 receptor endocytosis, phosphorylation of ERK, and the increase in cardiac neuron excitability.

Pituitary adenylate cyclase activating polypeptide (PACAP, Adcyap1) activation of PAC1 receptors ( Adcyap1r1) significantly increases excitability of guinea pig cardiac neurons. This modulation of excitability is mediated in part by plasma membrane G protein-dependent activation of adenylyl cyclase and downstream signaling cascades. However, additional mechanisms responsible for the enhanced excitability are activated following internalization of the PAC1 receptor and endosomal signaling. Src family kinases play critical roles mediating endocytosis of many trophic factor and G protein-coupled receptors. The present study investigated whether Src family kinases also support the PACAP-induced PAC1 receptor internalization, phosphorylation of ERK, and enhanced neuronal excitability. Using human embryonic kidney cells stably expressing a green fluorescent protein-tagged PAC1 receptor, treatment with the Src family kinase inhibitor PP2 (10 µM) markedly reduced the PACAP-induced PAC1 receptor internalization, and in parallel, both PP2 and Src inhibitor 1 (Src-1, 2 µM) reduced ERK activation determined by Western blot analysis. In contrast, Src family kinase inhibitors did not eliminate a PACAP-induced rise in global calcium generated by inositol (1,4,5)-trisphosphate-induced release of calcium from endoplasmic reticulum stores. From confocal analysis of phosphorylated ERK immunostaining, PP2 treatment significantly attenuated PACAP activation of ERK in neurons within cardiac ganglia whole mount preparations. Intracellular recordings demonstrated that PP2 also significantly blunted a PACAP-induced increase in cardiac neuron excitability. These studies demonstrate Src-related kinase activity in PAC1 receptor internalization, activation of MEK/ERK signaling, and regulation of neuronal excitability. The present results provide further support for the importance of PAC1 receptor endosomal signaling as a key mechanism regulating cellular function.