beta-Phorbol ester-induced enhancement of exocytosis in large mossy fiber boutons of mouse hippocampus

Abstract

beta-Phorbol esters (BPE), synthetic analogues of diacylglycerol (DAG), induce the potentiation of transmission in many kinds of synapses through activating the C(1) domain-containing receptors. However, their effects on synaptic vesicle exocytosis have not yet been investigated. Here, we evaluated the vesicular exocytosis directly from individual large mossy fiber boutons (LMFBs) in hippocampal slices from transgenic mice that selectively express synaptopHluorin (SpH). We found that the activity-dependent increment of SpH fluorescence (DeltaSpH) was enhanced by 4beta-phorbol 12,13-diacetate (PDAc), one of the BPEs, without influencing the recycled component of SpH. These PDAc effects on DeltaSpH were almost completely inhibited by staurosporine, a non-selective antagonist of protein kinases. However, intermittent synaptic transmission was still potentiated through a staurosporine-resistant mechanism. The staurosporine-sensitive cascade may facilitate the vesicle replenishment, thus maintaining the fidelity of transmission at a high level during repetitive firing of the presynaptic neuron.

Fig. 1

Synaptic vesicle dynamics in individual large MF boutons (LMFBs) in the hippocampus. a A synaptopHluorin (SpH) fluorescence image of an acute slice of TV-42 transgenic mouse hippocampus. The MFs were stimulated at the dentate hilus (right) and recorded from the distal CA3 region (left square). b An enlarged view of the stratum lucidum included in the square in a. Individual LMFBs are identifiable by the SpH fluorescence in the plasma membrane. c A sample averaged image of the SpH fluorescence of LMFBs before nerve stimulation. d Similar to c, but the images were sampled near the end of repetitive stimulation of 10 Hz for 10 s. e The digital subtraction of both images (difference image). The magnitude is shown as a pseudocolor rating. f Time-dependent profiles of SpH fluorescence signals of the ROIs indicated in e. The stimulation period is indicated by a red stripe. g Average SpH signal profile of sample records in f. Note that the fluorescence intensity steadily increased during repetitive stimulation, but recovered to the baseline after the cessation of stimulation

Fig. 2

Ca²⁺ sensitivity of ΔSpH. a Sample records of fEPSP evoked by a two-pulse protocol (interval, 100 ms) at 0.033 Hz in an ACSF containing [Ca²⁺]_o at 2.5 mM (top trace), 5 mM (middle trace) or 0 mM (bottom trace). b [Ca²⁺]_o dependence of fEPSP (n = 8 slices). c Sample records of ΔSpH from a LMFB by a train of repetitive stimulation (10 Hz for 10 s); [Ca²⁺]_o at 2.5 mM (top trace), 5 mM (middle trace) or 0 mM (bottom trace). d [Ca²⁺]_o dependence of ΔSpH (n = 12 boutons, 3 slices)

Fig. 3

Enhancement of the activity-dependent increment of SpH fluorescence (ΔSpH) by β-phorbol esters (BPE). a The fEPSP was monitored at 0.033 Hz using a two-pulse protocol (insets left, before and right, after PDAc); the peak amplitude of the first fEPSP (filled circles, mean ± SEM, n = 8 slices) and the fiber volley amplitude (open diamonds, mean ± SEM, n = 3 slices). Each data was normalized to the mean of five values preceding the sampling 1. The numbered red double lines indicate image sampling 1 and 2 when repetitive stimulations were applied at 10 Hz for 10 s. Drugs were bath-applied for the indicated periods. b The difference images of the same region before (left, sampling 1) and after PDAc (10 μM) (right, sampling 2). c The sample ΔSpH traces during MF stimulation of three LMFBs in b (indicated by white arrows) are compared before (sampling 1, black) and after PDAc (sampling 2, blue). d In some LMFBs, the negligibly small ΔSpH before (sampling 1, black) became obvious after PDAc (sampling 2, blue). e Each LMFB was plotted two-dimensionally to the sampling-1 (control) and the sampling-2 (test) values of ΔSpH; vehicle alone (open circles, n = 31 boutons, 5 slices), 4α-phorbol (yellow squares, n = 30 boutons, 6 slices) and PDAc (blue diamonds, n = 65 boutons, 10 slices). The magenta line shows that both are equal. f Cumulative probability plots of the ratio value of the ΔSpH at sampling 2 divided by that at sampling 1 (ratio-2/1 of ΔSpH); vehicle alone (black line), 4α-phorbol (gray line) and PDAc (blue line)

Fig. 4

Effects of BPE on the rate of SpH recycling. a The ΔSpH signals were normalized and averaged before (closed circles) and after 5 μM bafilomycin A1 (open diamonds) (mean ± SEM, n = 30 boutons, 7 slices). b The contribution of SpH recycling through the processes of endocytosis-reacidification was estimated by the subtraction of the ΔSpH before bafilomycin A1 from that after, and was plotted (gray squares, mean ± SEM, n = 30 boutons, 7 slices). c Each ΔSpH signal before (top left) and after PDAc (top right) was normalized to the value at the end of a train of repetitive stimulations (bottom traces) and a regression line (blue) was fitted to the data of 0–5 s after the last stimulation. d Summary (mean ± SEM) of the SpH recycling rate before (open columns) and after vehicle alone (DMSO, left filled column, n = 27 boutons, 4 slices) or PDAc (right filled column, n = 44 boutons, 10 slices)

Fig. 5

Effects of staurosporine. a The effects of PDAc on fEPSPs in the presence of 1 μM staurosporine. The numbered double lines indicate image sampling 1 and 2. Reagents were bath-applied for the indicated periods. b Summary of the effects on the first fEPSP amplitude of the treatments: vehicle alone (first left, n = 5 slices), 4α-phorbol (second left, n = 7 slices), PDAc (third left, n = 8 slices) and staurosporine plus PDAc (right most, n = 12 slices). *P < 0.01 and **P < 0.0005 (Mann–Whitney U-test). c Summary of the effects of PDAc on the paired-pulse ratio of fEPSPs in the absence (left, n = 8 slices) and the presence of staurosporine (right, n = 12 slices). Open column, paired-pulse ratio before PDAc treatment; closed column, 10 minutes after exposure to PDAc. *P < 0.01 (Wilcoxon signed-ranks test). d Average fluorescence profiles of LMFBs (n = 65 boutons, 15 slices) in the presence of staurosporine before (top), after PDAc (middle) and the difference (bottom). e Cumulative probability plots of the ratio-2/1 value of ΔSpH; vehicle alone (black line), PDAc (blue line) and staurosporine plus PDAc (magenta line)