(A) Wiring diagrams of the reproductive circuit (top) and defecation motor circuit (bottom). HSN (green) and VC (blue) neurons synapse onto each other and the
vm2 muscles for egg laying. Data from White J.G. et al. (1986) indicate HSN and VC also make and receive synapses from AVL and DVB, excitatory GABA motor neurons that regulate the contraction of the enteric muscles (em) for defecation. Arrows indicate chemical synapses, and + or - indicates a presumptive excitatory or inhibitory synapse, respectively. Bar-headed lines indicate gap junctions (e.g. electrical synapses).(B) Representative HSN Ca2+ traces at the L4.7-8 larval stage (top) and adults (bottom). Vertical lines indicate the expulsion step of the defecation motor program (DMP); arrowheads indicate adult egg-laying events.(C) Cumulative distribution plots showing instantaneous frequency of the DMP events (
min-1) with no observed HSN Ca2+ transient (black) and those with one or more HSN Ca2+ transients (green) in L4.7-8 (closed circles) and adult animals (open circles). Pound indicates p=0.0058; asterisk indicates p<0.0001 (Kruskal-Wallis test with Dunn's correction for multiple comparisons). Total DMP intervals used for analysis: L4.7-8 DMPs without an HSN Ca2+ transient (n=33); L4.7-8 DMPS with at least one HSN Ca2+ transient (n=62) from 9 animals; Adult DMPs without an HSN Ca2+ transient (n=39); Adult DMPs with at least one HSN Ca2+ transient (n=72) from 11 animals.(D) Scatterplots showing the consequences of HSN optogenetic activation on DMP frequency. L4.7-8 and adult animals expressing Channelrhodpsin-2 (ChR2) in HSN neurons from the wzIs30 transgene were grown in the absence ( -, grey) or presence (+, blue) of all-trans retinal (ATR), illuminated with continuous blue light for two minutes, and the timing of DMP events was recorded. The elapsed time between Expulsion events was used to calculate an instantaneous DMP frequency from each recorded interval (
min-1) from X animals. Error bars show 95% confidence intervals for the mean; * indicates p<0.0001; n.s. indicates p=0.2102 (L4.7-8) or p>0.999 (Adult); one-way ANOVA with Bonferroni correction for multiple comparisons. Total DMP intervals used for analysis from 10 animals: L4.7-8, no ATR (n=43); L4.7-8, plus ATR (n=40); Adult, no ATR (n=67); Adult, plus ATR (n=55).