The small GTPase RHO-1 is involved in regulation of adult neuronal activity. Expression of constitutively active RHO-1 in cholinergic motorneurons (N::RHO-1*) causes loopy locomotion and an increase in acetylcholine (ACh) release as shown by hypersensitivity to acetylcholinesterase inhibitors. RHO-1 acts in part via inhibition of the DAG kinase, DGK-1 resulting in accumulation of diacylglycerol, which stimulates neurotransmitter release. However, changes in RHO-1 activity in a
dgk-1 null mutant can still alter neuronal activity. Suggesting that in neurons there are RHO-1 effectors in addition to DGK-1. In order to identify other targets of RHO-1 we conducted a genetic screen for suppressors of the N::RHO-1* loopy locomotion. 2000 F1 N::RHO-1* animals mutagenized with EMS were screened for wild type locomotion. As a secondary screen RHO-1* was expressed from a heat-shock promoter that causes both neuronal and non-neuronal phenotypes. We then followed mutants suppressing the loopy locomotion but not the non-neuronal effects. This identified 20 strong N::RHO-1* suppressors. One suppressor,
nz94, has a mutation in
unc-80. UNC-80 along with UNC-79 are important regulators of the neuronal ion channel NCA-1/NCA-2, and mutations in any of these genes suppress the loopy locomotion of N::RHO-1*. Expression of UNC-80 in the cholinergic motor neurons is sufficient to rescue the loopy behavior demonstrating that UNC-80 acts in the same neurons as N::RHO-1* to regulate locomotion. Recent data in our lab suggest RHO-1 regulates the NCA-channel via the PI4P5 lipid kinase PPK-1. A second N::RHO-1* suppressor,
nz110, has a missense mutation in the MHD domain of
unc-31. A putative
unc-31 null mutation (
e928) also suppresses N::RHO-1* loopy locomotion. UNC-31 is required for the release of dense core vesicles (DCV). Our results suggest that RHO-1 regulates both classical neurotransmitter release and release of neuropeptides from DCVs. Interestingly previous
unc-31 mutations are paralyzed in the presence of food whereas our
unc-31 (
nz110) mutants move normally, suggesting the
unc-31 (
nz110) mutation only effects stimulated levels of DCV release. We assumed that suppressors of N::RHO-1* loopy locomotion would also suppress the increased release of ACh. However, many of our N::RHO-1* suppressors resulted in wild type locomotion but did not decrease ACh release. Our current model is that increased ACh release is necessary but not sufficient for loopy locomotion. We believe that an additional RHO-1 regulated signal from the cholinergic motorneurons is required for loopy locomotion. Our genetic screen suggests that the additional signal is released from DCVs, possibly a neuropeptide that acts on the muscle or other neurons.