The Gas pathway has been studied as an important modulator of neurotransmission and a molecular substrate for learning and memory in a variety of organisms. Studies of C. elegans Gas pathway mutants have demonstrated the importance of this signaling cascade in locomotion (Reynolds et al, 2005). Loss-of-function mutations in the major Gas effector, adenylyl cyclase (
acy-1), result in severe uncoordinated locomotory phenotypes and low survival rates. Conversely, gain-of-function mutations in this gene confer hyperactivity in worm locomotion. Cyclic AMP (cAMP) generated by ACY-1 binds to the regulatory of subunit of PKA, KIN-2, causing it to disassociate from the catalytic PKA subunit, KIN-1, thereby activating the enzyme. Loss-of-function mutations in
kin-2, which result in constitutively active PKA, mimic the enhanced locomotion seen in
acy-1(g.o.f.) animals. Despite evidence that the Gas pathway converges on the core Gaq pathway that is required for UNC-13-dependent priming of synaptic vesicles, precisely how the Gas pathway regulates synaptic function remains to be resolved.To understand the molecular mechanisms of neuromodulation due to Gas, we have begun a detailed characterization of synapses in
acy-1(neuron null),
acy-1(g.o.f.) and
kin-2 loss-of-function mutants. We first examined their sensitivity to the acetylcholinesterase inhibitor, Dylox, as an initial readout of synaptic function. We found
kin-2 and
acy-1(g.o.f.) mutants to be Dylox-hypersensitive, where as
acy-1 mutants showed wild-type responses to the drug. Despite these apparently strong indications of altered acetylcholine release, maximal evoked synaptic response amplitudes, recorded from the neuromuscular junctions (NMJs) of
acy-1 and
kin-2 mutants were not significantly affected, although a trend toward faster synaptic depression in stimulus trains was observed in
acy-1 mutants. Additional experiments under conditions that lower release probability will be conducted to complete this analysis and to examine the potential role of PKA in the regulation of calcium influx, as recently reported at enteric NMJs (Wang and Sieburth, 2013). However, the lack of an obvious synaptic defect is consistent with the preliminary EM analysis of synaptic vesicle density and docking at the NMJs of these mutants, which appear close to wildtype. One notable difference that we have observed at the ultrastructural levels is a differential change in the number of dense core vesicles (DCV) at
acy-1 and
kin-2 synapses, suggesting that the Gas pathway may preferentially regulate this secretory pathway. This is consistent with previous data, showing that Gas acts in the same pathway as the DCV priming factor, UNC-31(CAPS) (Charlie et al, 2006). Ongoing experiments will complete this analysis and further probe the potential effects of the Gas pathway on peptide release from DCVs.