The inhibitory transmitters glycine and GABA undergo a developmental shift from depolarizing to hyperpolarizing action (D/H-shift). To analyse this shift in functionally related nuclei of the rat superior olivary complex (SOC), we employed voltage-sensitive dye recordings in auditory brainstem slices. Complementarily, we analysed single neurons in gramicidin perforated-patch recordings. Our results show a differential timing of the D/H-shift in the four SOC nuclei analysed. In the medial superior olive (MSO), the shift occurred at postnatal day (P) 5-9. In the superior paraolivary nucleus (SPN), it occurred between embryonic day (E) 18 and P1. No D/H-shift was observed in the medial nucleus of the trapezoid body (MNTB) until P10. This is in line with the finding that most of the patched MNTB neurons displayed glycine-induced depolarizations between P0-9. While no regional differences regarding the D/H-shift were found within the MSO, SPN, and MNTB, we observed such differences in the lateral superior olive (LSO). All LSO regions showed a D/H-shift at P4-5. However, in the high-frequency regions, hyperpolarizations were large already at P6, yet amplitudes of this size were not present until P8 in the low-frequency regions, suggesting a delayed development in the latter regions. Our physiological results demonstrate that D/H-shifts in SOC nuclei are staggered in time and occur over a period of almost two weeks. Membrane-associated immunoreactivity of the Cl- outward transporter KCC2 was found in every SOC nucleus already at times when glycine was still depolarizing. This implies that the mere presence of KCC2 does not correlate with functional Cl- outward transport.

Synaptic strength is determined by release probability and the size of the readily releasable pool of docked vesicles. Here we describe the effects of blocking myosin light chain kinase (MLCK), a cytoskeletal regulatory protein thought to be involved in myosin-mediated vesicle transport, on synaptic transmission at the mouse calyx of Held synapse. Application of three different MLCK inhibitors increased the amplitude of the early excitatory postsynaptic currents (EPSCs) in a stimulus train, without affecting the late steady-state EPSCs. A presynaptic locus of action for MLCK inhibitors was confirmed by an increase in the frequency of miniature EPSCs that left their average amplitude unchanged. MLCK inhibition did not affect presynaptic Ca(2+) currents or action potential waveform. Moreover, Ca(2+) imaging experiments showed that [Ca(2+)](i) transients elicited by 100-Hz stimulus trains were not altered by MLCK inhibition. Studies using high-frequency stimulus trains indicated that MLCK inhibitors increase vesicle pool size, but do not significantly alter release probability. Accordingly, when AMPA-receptor desensitization was minimized, EPSC paired-pulse ratios were unaltered by MLCK inhibition, suggesting that release probability remains unaltered. MLCK inhibition potentiated EPSCs even when presynaptic Ca(2+) buffering was greatly enhanced by treating slices with EGTA-AM. In addition, MLCK inhibition did not affect the rate of recovery from short-term depression. Finally, developmental studies revealed that EPSC potentiation by MLCK inhibition starts at postnatal day 5 (P5) and remains strong during synaptic maturation up to P18. Overall, our data suggest that MLCK plays a crucial role in determining the size of the pool of synaptic vesicles that undergo fast release at a CNS synapse.

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