Syt1^M3 bladder strips are less sensitive to BoNT/B in ex vivo bladder contraction assays.

A. Schematic diagram of ex vivo bladder strip contraction assay. Bladder strips were immersed in aerated Krebs solution. Exogenous pharmacologic agents (i.e., carbachol, BoNT/B) were added directly to the solution. Trains of voltage delivered over a range of frequencies were generated by the electrical field stimulator to trigger bladder contraction, which is detected and measured by the force transducer. B. Bladder weights across WT and KI mice were similar prior to ex vivo contraction assay. ns = not significant (ANOVA). C-D. The nerve-evoked contraction forces of WT (n = 3), Syt1^M3 (n = 5), and Syt2^M3 (n = 4) bladder strips were recorded, with the representative force trace shown in C. A set of frequency-dependent stimulations (enclosed by brackets above tracing) were delivered every 100 min. After a baseline frequency-response, evoked responses at constant frequency (16 Hz) were produced every 15 min. BoNT/B (0.3 nM) was administered at red arrow. The time dependent effects of BoNT/B on nerve evoked responses are shown in frequency-response curves that were plotted in 100 min intervals, shown in D. Incubation with BoNT/B reduced contractions of WT and Syt2^M3 bladders, but did not affect Syt1^M3 bladders. *p<0.05, repeated measures ANOVA. E. Carbachol induces direct muscle contraction independent of neurotransmission. Carbachol does-response curves were generated at the beginning and end of the experiment as a control showing that bladder strips maintained their viability and contractility after incubation with BoNT/B for 300 min. p>0.05, RM ANOVA.