Myosin light chain kinase is not a regulator of synaptic vesicle trafficking during repetitive exocytosis in cultured hippocampal neurons

Abstract

The mechanism by which synaptic vesicles (SVs) are recruited to the release site is poorly understood. One candidate mechanism for trafficking of SVs is the myosin-actin motor system. Myosin activity is modulated by myosin light chain kinase (MLCK), which in turn is activated by calmodulin. Ca(2+) signaling in presynaptic terminals, therefore, may serve to regulate SV mobility along actin filaments via MLCK. Previous studies in different types of synapses have supported such a hypothesis. Here, we further investigated the role of MLCK in neurotransmitter release at glutamatergic synapses in cultured hippocampal neurons by examining the effects of two MLCK inhibitors, 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine.HCl (ML-7) and wortmannin. Bath application of ML-7 enhanced short-term depression of EPSCs to repetitive stimulation, whereas it reduced presynaptic release probability. However, ML-7 also inhibited action potential amplitude and voltage-gated Ca(2+) channel currents. These effects were not mimicked by wortmannin, suggesting that ML-7 was not specific to MLCK in hippocampal neurons. When SV exocytosis was directly triggered by a Ca(2+) ionophore, calcimycin, to bypass voltage-gated Ca(2+) channels, ML-7 had no effect on neurotransmitter release. Furthermore, when SV exocytosis elicited by electrical field stimulation was monitored by styryl dye, FM1-43 [N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridinium dibromide], the unloading kinetics of the dye was not altered in the presence of wortmannin. These data indicate that MLCK is not a major regulator of presynaptic SV trafficking during repetitive exocytosis at hippocampal synapses.

Figure 1.

MLCK is enriched in axons. A, Immunolocalization of endogenous MLCK (green) and an axonal marker, phosphorylated neurofilament H (pNF-H) subunit (red), in cultured hippocampal neurons at 14 DIV. MLCK signal is prominent in axons (arrows) but is also present in dendrites at lower levels (arrowhead). B, Immunostaining of MLCK (green) and synaptotagmin I (Syt) (red). C, Magnified view of the boxed region in B. MLCK and synaptotagmin I show partially overlapping localization. Scale bars: A,10 μm; B, 5 μm; C, 1 μm.

Figure 2.

ML-7 reduces release probability but enhances short-term depression. A–C, Effect of ML-7 on EPSC amplitude. Mean EPSC amplitude in autaptic neurons is shown before and during perfusion of 10 μm ML-7 (A; n = 8) or control 0.1% DMSO (B; n = 6), and the extent of change in EPSC amplitude is summarized in C (square symbols). D–G, Effect of ML-7 on short-term depression. Neurons were stimulated by a train of 40 pulses at 20 Hz before and after ML-7 perfusion (n = 8). Short-term depression of EPSC amplitude (D), total charge transfer (E), synchronous release (F), and asynchronous release (G) are shown. Data are normalized to the first response of the train. ML-7 reduced all four parameters examined (*p < 0.05; t test). H–J, Estimation of RRP size and release probability. Neurons were pretreated with 10 μm ML-7 (n = 10) or control 0.1% DMSO (n = 9) for 15 min, followed by patch-clamp recording. Mean total charge transfer elicited by a single AP-evoked release (H) and the estimate of RRP size by a 4 s perfusion of 0.5 m sucrose (I) are shown for each condition. These values were used to estimate release probability defined as follows: [charge transferred by single AP-induced transmission]/[charge of RRP] (J). Square symbols denote the mean values for each measurement. ML-7 treatment decreases the response elicited by a single AP and the release probability (*p < 0.05; t test), whereas the RRP size is unchanged (p > 0.2; t test). Error bars indicate SEM.

Figure 3.

ML-7 reduces the excitability of neurons independently of MLCK activity. A, A plot of I–V relationship in neurons pretreated with 0.1% DMSO (n = 7), 10 μm ML-7 (n = 8), or 10 μm wortmannin (n = 6). B, Representative traces of APs elicited by current injection (0.4 nA; 100 ms) in neurons pretreated with DMSO or ML-7. C, Number of AP spikes generated during current injection in neurons pretreated with DMSO (n = 8), ML-7 (n = 10), or wortmannin (n = 7). The number of APs is significantly reduced in the presence of ML-7 but not in wortmannin (*p < 0.05, **p < 0.01; Dunnett's test, DMSO vs ML-7). D, Representative traces of APs induced by repetitive 2 ms, 2 nA current injections at 20 Hz in neurons pretreated with DMSO or ML-7. E, Summary of the peak AP amplitude change during repetitive stimulation. Data are normalized to the first peak. AP amplitude is substantially decreased in the presence of ML-7 (n = 7), whereas wortmannin (n = 7) is without effect relative to DMSO control (n = 9) (**p < 0.01; Dunnett's test, DMSO vs ML-7). F, G, Summary of the peak amplitude and half-maximal width of the first AP. ML-7 decreases the peak amplitude (*p < 0.05; Dunnett's test, DMSO vs ML-7) and increases half-maximal width of the first AP (**p < 0.01; Dunnett's test, DMSO vs ML-7). Error bars indicate SEM.

Figure 4.

ML-7 reduces VGCC current. A, Overlay of representative VGCC current traces induced by a 30 ms voltage step to 0 mV from −80 mV in neurons pretreated with 0.1% DMSO or 10 μm ML-7. B, Summary of VGCC current density induced by a voltage step to indicated membrane potentials in neurons pretreated with 0.1% DMSO (n = 9), 10 μm ML-7 (n = 10), or 10 μm wortmannin (n = 7). Solid lines are cubic spline interpolation. ML-7 decreases VGCC currents relative to DMSO, whereas wortmannin has no effect (*p < 0.05, **p < 0.01; Dunnett's test, DMSO vs ML-7). Error bars indicate SEM.

Figure 5.

ML-7 does not inhibit Ca²⁺ ionophore-evoked release and the recovery of RRP after depletion by hypertonic solution. A, Example traces of responses elicited by perfusion of 20 μm calcimycin onto an entire microisland in autaptic neurons pretreated with 0.1% DMSO or 10 μm ML-7. B, Summary of charge transfer measurements of calcimycin-evoked responses in neurons pretreated with 0.1% DMSO (n = 6) or 10 μm ML-7 (n = 7). C, Cumulative plot of charge transfer elicited by calcimycin. D, RRP recovery after depletion by hypertonic solution. Pairwise application of hypertonic sucrose solution to local dendritic area of neurons pretreated with DMSO (n = 5), ML-7 (n = 5), or wortmannin (n = 4) was used to monitor the extent of RRP recovery (supplemental Fig. 5A, available at www.jneurosci.org as supplemental material). Each puff to deplete the RRP was for 4 s, and the interpulse interval was 3.6 s. The plot shows the normalized charge transfer ratios of the second puff response to the first puff. ML-7 treatment shows no effect on the extent recovery of the RPP at a time when one-half of the vesicles are refilled in control neurons. Wort, Wortmannin. Error bars indicate SEM.

Figure 6.

Wortmannin does not inhibit FM1-43 unloading rate. A, Experimental scheme of FM1-43 loading, drug treatment, and FM1-43 unloading by field stimulation. Wort, Wortmannin; Adv-7, Advasep-7. B, Representative images of FM1-43 in neurons treated with 0.1% DMSO, 10 μm ML-7, or 10 μm wortmannin at indicated times corresponding to the start of the unloading stimulus train. Scale bar, 4 μm. C, Summary of the time course of FM1-43 unloading in neurons treated with DMSO (n = 80 puncta, 4 coverslips), 10 μm ML-7 (n = 60 puncta, 3 coverslips), or 10 μm wortmannin (n = 80 puncta, 4 coverslips). Data were normalized to the average FM1-43 fluorescence of the first six frames captured before the unloading stimulus. Error bars indicate SEM.