The anatomical basis for the generation of nematode movement has been well described. Excitatory cholinergic motor neurons (MNs) make synaptic contacts onto both muscle cells and GABA MNs that, in turn, make inhibitory synaptic contacts onto contralateral musculature. Several subunits of the nicotinic acetylcholine receptor (nAChR) superfamily are expressed in MNs, suggesting cholinergic signaling is important for coordinating MN activity. In previous work, we isolated several loss-of-function alleles of the nAChR subunit gene acr-12 (Barbagallo et al., 2010) and we have found that acr-12 is expressed in both GABA and ACh MNs. We are now studying the functions of acr-12 signaling complexes in the motor circuit. While the movement of acr-12 mutants is overtly normal, closer examination revealed that the amplitude of body bends during runs of forward movement was highly variable. Additionally, body bend amplitude was, on average, reduced and we observed hypersensitivity to the acetylcholine esterase inhibitor aldicarb. Cell specific expression of acr-12 in GABA MNs but not ACh MNs was sufficient for rescuing each of these phenotypes. Analysis of the subcellular localization of the rescuing ACR-12::GFP fusion protein also suggested ACR-12 receptor complexes play distinct roles across the two motor neuron classes. Expression of ACR-12::GFP specifically in GABA MNs revealed fluorescence at discrete sites along neuronal processes, suggesting ACR-12 may cluster at synapses on GABA MNs. In contrast, we observed diffuse fluorescence upon cell specific expression of ACR-12::GFP in ACh MNs. Our behavioral, pharmacological and expression studies point to a key role for acr-12 in the regulation of GABA MN activity. To test this hypothesis directly, we measured the frequency of GABA synaptic events in whole-cell patch clamp recordings from the muscles of acr-12 mutants and observed a roughly 40% reduction in event frequency. In parallel with our loss of function approach we have pursued a gain of function approach. Transgenic animals expressing acr-12(gf) exhibit spontaneous muscle contractions, similar to strains carrying a previously characterized gain of function acr-2 allele (Jospin et al., 2009). Cell specific expression of acr-12(gf) in ACh MNs was sufficient to generate this behavior, and spontaneous contractions were partially suppressed by a loss of function mutation in acr-2. Our analysis of acr-12(lf) and acr-12(gf) strains has revealed an interesting paradox: the loss of function analysis emphasizes a role for acr-12 in GABA MNs while our gain of function work points toward an important role in ACh MNs. We are currently investigating the basis for this inconsistency and we will present our findings.

Affiliation:
- University of Massachusetts Medical School, Worcester, MA.