PACAP/PAC1R signaling modulates acetylcholine release at neuronal nicotinic synapses.

Neuropeptides collaborate with conventional neurotransmitters to regulate synaptic output. Pituitary adenylate cyclase-activating polypeptide (PACAP) co-localizes with acetylcholine in presynaptic nerve terminals, is released by stimulation, and enhances nicotinic acetylcholine receptor- (nAChR-) mediated responses. Such findings implicate PACAP in modulating nicotinic neurotransmission, but relevant synaptic mechanisms have not been explored. We show here that PACAP acts via selective high-affinity G-protein coupled receptors (PAC(1)Rs) to enhance transmission at nicotinic synapses on parasympathetic ciliary ganglion (CG) neurons by rapidly and persistently increasing the frequency and amplitude of spontaneous, impulse-dependent nicotinic excitatory postsynaptic currents (sEPSCs). Of the canonical adenylate cyclase (AC) and phospholipase-C (PLC) transduction cascades stimulated by PACAP/PAC(1)R signaling, only AC-generated signals are critical for synaptic modulation since the increases in sEPSC frequency and amplitude were mimicked by 8-Bromo-cAMP, blocked by inhibiting AC or cAMP-dependent protein kinase (PKA), and unaffected by inhibiting PLC. Despite its ability to increase agonist-induced nAChR currents, PACAP failed to influence nAChR-mediated impulse-independent miniature EPSC amplitudes (quantal size). Instead, evoked transmission assays reveal that PACAP/PAC(1)R signaling increased quantal content, indicating that it modulates synaptic function by increasing vesicular ACh release from presynaptic terminals. Lastly, signals generated by the retrograde messenger, nitric oxide- (NO-) are critical for the synaptic modulation since the PACAP-induced increases in spontaneous EPSC frequency, amplitude and quantal content were mimicked by NO donor and absent after inhibiting NO synthase (NOS). These results indicate that PACAP/PAC(1)R activation recruits AC-dependent signaling that stimulates NOS to increase NO production and control presynaptic transmitter output at neuronal nicotinic synapses. Copyright 2009 Elsevier Inc. All rights reserved.