Fig. 6. Loss of
slo-1 in C. elegans activated M4-TB synapses. (A) Pceh-28::
snb-1::gfp transgenic C. elegans animals showed punctate expression of SNB-1::GFP, a synaptic vesicle marker, along M4 axons in the isthmus and terminal bulb. White arrowhead points to the M4 cell body. Black arrow points to punctae in the TB. (B) A schematic of C. elegans EPGs (electropharyngeograms), a current recording from pharyngeal muscles during pumping, where positive and negative spikes correspond to muscle depolarizations and repolarizations, respectively. Neuronal stimulation by MC causes small positive spikes, which either remain as single positive spikes, i.e. MC EPSPs, if the MC stimulation did not induce muscle action potentials, or are followed by large positive and negative spikes if full muscle action potential was triggered. Inhibition by the M3 neurons sometimes occurs, which contributes to pharyngeal muscle repolarization during action potentials, and is seen as small trains of negative spikes on EPGs. Illustrated in the schematic are one full action potential on the left, and one single positive spike (resulting from an MC EPSP) on the right. The neurogenic spikes (i.e. due to MC and M3) are drawn in black, whereas myogenic spikes are drawn in gray. If MC and M3 are inactivated, then the resulting EPGs contain only myogenic spikes. (C) An example of a single positive spike due to an MC EPSP in wild-type C. elegans EPG, indicated by the black arrow. (D)
eat-2;
eat-4 EPGs lacked MC and M3 spikes, including single positive spikes (
eat-2 and
eat-4 remove MC and M3 functions, respectively). (E)
eat-2;
eat-4;
slo-1 EPGs contained single positive spikes, indicated by the black arrows. (F) Quantification of single positive spikes (SPS) in wild-type and
slo-1 mutant backgrounds, with and without MC/M3 function, either by laser ablations or the
eat-2 and
eat-4 mutations. (G) Laser ablation of M4 drastically reduced single positive spikes (SPS) in
eat-2;
eat-4;
slo-1 animals, whereas ablation of another C. elegans neuron, M5, did not. Ablation of M4 and M5 entirely eliminated single positive spikes, suggesting slight activity by M5 as well. (H) The
eat-5 mutation allows the TB to pump independently of the corpus (Starich et al., 1996), and
slo-1 increased TB pumping in the
eat-5 background. (I) Perinuclear expression of Pslo-1::SLO-1::GFP in the M4 neuron. The white arrows point to the M4 nucleus. The white arrowhead points to GFP signal surrounding the M4 nucleus. (J)
eat-2 encodes a nicotinic channel subunit specific to the MC-corpus neuromuscular junction, whereas
eat-18 is required for the surface expression of all pharyngeal nicotinic channels, including in the terminal bulb.
eat-2;
eat-4;
slo-1 animals had single positive spikes, but
eat-18;
eat-4;
slo-1 animals did not. (K) EPGs of
eat-2;
eat-4;
slo-1 animals were recorded in standard conditions, followed by subsequent addition of d-tubocurarine, a nicotinic channel antagonist, to 100 umol l-1, or control saline (see Materials and methods for details). 100 umol l-1 d-tubocurarine decreased single positive spikes in
eat-2;
eat-4;
slo-1 animals (compared to before treatment), whereas control treatment did not (compared to before treatment). Scale bars in C-E indicate 100 pA and 100 ms. Asterisks indicate statistically significant differences from controls by two-tailed t-test (**P<0.01); see Materials and methods for the statistical analysis performed in Fig. 6K. Dagger in G indicates statistical significance compared to M4 ablation by two-tailed U-test (P<0.05).