For every frequency tested, the cumulative QC (ΣQC) for KO NMJs was reduced by more than 50% of the WT (2,426 ± 178 versus 1,150 ± 73 at 10 Hz; 6,548 ± 449 versus 3,289 ± 289 at 30 Hz; 12,727 ± 1,159 versus 6,413 ± 446 at 100 Hz, estimated vesicle number in WT versus CSP-α KO). In contrast, the spH fluorescence responses of CSP-α KO synapses were above the electrophysiological measurements and closer to WT values (32.2 ± 3.7 versus 44.5 ± 6.9 at 10 Hz; 102 ± 13.3 versus 142.1 ± 17.6 at 30 Hz; 233.2 ± 25.8 versus 363.2 ± 41.8 at 100 Hz fluorescence units increase [ΔF] in WT versus CSP-α KO), indicating a disproportionate increase INCB024360 supplier in ΔF.

Literally, that could apparently mean that the membrane inserted was specifically brighter at the CSP-α KO terminals compared to controls. However, the hypothesis we pursued was that such a relationship between ΔF and ΣQC arose from a surplus of membrane leftover at the cell surface upon exocytosis followed by an inefficient compensatory endocytosis. If that were the case, we should expect the recovery of fluorescence to be slowed down at mutant synapses. The recovery phase was well fitted to a single exponential decay with longer time constants for stronger stimulations (Figure S3C). Surprisingly, however, at different frequency stimulations and postnatal ages, the kinetic recovery in the mutants was faster than in the controls (Figures S3C and S3D and Table S1).

That observation was unexpected and apparently contradictory with an impairment in endocytosis. On the other hand, because mutant synapses release less synaptic vesicles, it Dolutegravir order could then happen that the endocytosis load was therefore less

in the CSP-α KO. To clarify that, we analyzed the kinetics of recovery in WT and mutant recordings having a similar cumulative quantal content (6,039 ± 127 for WT and 5,611 ± 170 for KO, n = 17 and 14, respectively) (Figure S3E). We found that in such a subset of recordings, with a similar endocytic load, the fluorescence recovery in the mutants was slower than in the controls (17.2 ± 1.5 s for WT versus 24.5 ± 2.7 s Rutecarpine for KO, p = 0.022 Student’s t test) (Figures S3F and S3G). That observation also supported the notion that endocytosis depended on the amount of exocytosed vesicles, and endocytosis was impaired at synapses lacking CSP-α. Next, we carried out additional tests to further examine endocytosis in CSP-α KO terminals. At any moment, the spH fluorescence signal yields the simultaneous balance between exo- and endocytosis. Complete fluorescence contribution due to exocytosis is measurable through the blocking of the V-type ATPase required for vesicle reacidification following endocytosis (Nicholson-Tomishima and Ryan, 2004). We used folimycin, a membrane-permeant blocker of the V-type ATPase (Sara et al., 2005) to trap vesicles in the alkaline and hence fluorescent state after fusion.