PACAP triggers plasticity at autonomic synapses via nAChR induced NOS1 activation

Pituitary adenylate cyclase activating polypeptide (PACAP) is a pleiotropic neuropeptide found throughout the nervous system. In addition to its neurotrophic and neuromodulatory roles, PACAP regulates stress responses and is considered a biomarker for post-traumatic stress disorder. Consistent with these actions, PACAP has relevance to synaptic function in that it localizes to autonomic presynaptic nerve terminals, regulates neuronal excitability, and potentiates the function of neuronal nicotinic acetylcholine receptors (nAChRs). In addition, we previously found that PACAP acts via high-affinity G-protein coupled receptors (PAC₁Rs) to increase the frequency and amplitude of spontaneous nAChR-mediated excitatory postsynaptic currents. This synaptic plasticity triggered by PACAP/PAC₁R signaling resulted from increased quantal ACh release from presynaptic terminals, and required activation of both protein kinase A (PKA) and neuronal nitric oxide synthase (NOS1). We have now examined how PACAP/PAC₁R signaling increases nitric oxide (NO) production relevant to its ability to induce plasticity at nAChR-mediated synapses on ciliary ganglion (CG) neurons. Western blot assays detected abundant NOS1 in CG extracts, and fluorescence microscopy revealed specific NOS1 immunoreactivity confined to CG neurons. Quantitative live-cell imaging with the fluorescent NO indicator 4-Amino-5-Methylamino-2',7'-Difluorofluorescein (DAF-FM), revealed that NO production in CG neurons requires extracellular Ca²⁺ influx, and that PACAP increases NO levels by a mechanism requiring both NOS1 and PKA. Activating nAChRs, which are Ca²⁺-permeable and potentiated by PKA-dependent PACAP/PAC₁R signaling, also increased NO. Interestingly, nAChRs are required for PACAP-induced synaptic plasticity and for NO production since both outcomes were inhibited by prior reversible nAChR blockade. Taken together, these findings indicate that PACAP/PAC₁R signaling coordinates the activity of nAChRs with NOS1 to induce plasticity at autonomic synapses. The central role played by autonomic synapses in visceral functions suggests that such PACAP/PAC₁R triggered effects may have broad relevance, including the homeostatic regulation of stress responses.