Calcium-activated proteases are critical for refilling depleted vesicle stores in cultured sensory-motor synapses of Aplysia

The inhibition of calcium-dependent proteinases accelerates the rate of homosynaptic depression and inhibits 5HT-induced facilitation of depressed synapses. To examine the roles of proteinases in the cascade leading to homosynaptic depression and facilitation of highly depressed synapse by 5HT, we compared the rates of synaptic depression and 5-HT-induced facilitation in the presence and absence of proteinase inhibitors; (A) 100 μM calpeptin, (B) 100 μM MG132, (C) 100 μM ALLN, and (D) intracellularly injected lactacystin into the sensory neuron. Examples of EPSPs recorded during synaptic depression induced by 40 stimuli at 0.05 Hz and 5HT-induced facilitation (arrows) in control experiments are shown in aI, bI, cI, and dI, and the solid squares graph in aIII, bIII, cIII, and dIII. The traces shown in aI, bI, cI, and dI depict the first EPSP, the 40^th EPSP, and the second EPSP after 5HT application (42 stim). The effects of protease inhibitors are depicted in aII, bII, cII, and dII and the mean values are shown in aIII, bIII, cIII, and dIII (○). For experiments A, B, and C, the preparations were bathed in the protease inhibitor containing ASW for 15 min before the onset of the experiment. The paired controls were incubated for 15 min in 0.2% DMSO ASW (the vehicle solution of the inhibitors). For the experiment in D, lactacystin was pressure injected into the presynaptic neuron (reaching an estimated concentration of 25 μM) 1 h prior to the experiment. Note that in the presence of (A) calpeptin and (B) MG132, the rate of synaptic depression is significantly accelerated with respect to the control (F_(39,390) = 1.971, P < 0.01 repeated measures one-way ANOVA for calpeptin and F_(39,312) = 2.063, P < 0.001 for MG132) and that the 5-HT-induced facilitation is significantly reduced (F_(4,40) = 8.348, P < 0.001 repeated measures one-way ANOVA for calpeptin and F_(4,32) = 5.909, P < 0.01 for MG132). The 100 μM ALLN (C) does not accelerate the rate of synaptic depression, but inhibits 5HT-induced synaptic facilitation. In contrast, the proteasome inhibitor lactacystin neither affected the rate of synaptic depression nor the facilitation induced by 5HT.