The role of cGMP-dependent signaling pathway in synaptic vesicle cycle at the frog motor nerve terminals

Abstract

The role of cGMP-dependent pathways in synaptic vesicle recycling in motor nerve endings during prolonged high-frequency stimulation was studied at frog neuromuscular junctions using electrophysiological and fluorescent methods. An increase of intracellular cGMP concentration (8-Br-cGMP or 8-pCPT-cGMP) significantly reduced the cycle time for synaptic vesicles through the enhancement of vesicular traffic rate from the recycling pool to the readily releasable pool and acceleration of fast endocytosis. Pharmacological inhibition of soluble guanylate cyclase or protein kinase G slowed down the rate of recycling as well as endocytosis of synaptic vesicles. The results suggest that cGMP-PKG-dependent pathway serves a significant function in the control of vesicular cycle in frog motor terminals.

Figure 1.

Effects of cGMP analogs, inhibitors of guanylate cyclase, and protein kinase G on EPCs amplitude during prolonged high-frequency stimulation. A, Examples of EPCs, recorded during 20 Hz stimulation for 3 min (0, 15, 180 s) in control and after Br-cGMP, 8-pCPT-cGMP, ODQ, and Rp-8-Br-PET-cGMP application. B, EPCs amplitudes during stimulation for 3 min. C, EPCs amplitudes during first 30 s of stimulation at 20 Hz. y-axis, The amplitude of EPCs as a percentage of the amplitude of the first EPC in the train. D, Cumulative curves of EPCs amplitudes during high-frequency stimulation (in nA). Gray squares, Control; light circles, 8-Br-cGMP (100 μm); black circles, 8-pCPT-cGMP (50 μm); light triangles, ODQ (1 μm); black triangles, Rp-8-Br-PET-cGMP (0.5 μm).

Figure 2.

FM1-43 loading during and after high-frequency stimulation. A, A schematic diagram of the staining protocol. Light columns, with solid line, indicate the period of 20 Hz stimulation. Gray columns, with dotted line, show the time of FM1-43 presence for 1 min during (Δt = 1, 2, 3) and after the end of 3 min stimulation (Δt = 4–18). FM1-43 was applied on the neuromuscular preparation for 1 min and then was quickly washed out with Ringer's solution containing ADVASEP-7 (3 μm). B, Time course of FM1-43 loading in motor nerve terminal. The intensity of nerve terminal fluorescence during and after high-frequency stimulation (per minute dye application). The first three points show the dye uptake during stimulation (20 Hz, 3 min) (horizontal line). y-axis, Fluorescence intensity (ΔJ in a.u.). C, A schematic diagram of the staining protocol. Light columns, with solid line, indicate the period of stimulation (3 min 20 Hz). Gray columns, with dotted line, show the time of FM1-43 application. The total FM1-43 loading: dye was presented during (for 3 min) and after (for 15 min) tetanus. Loading during stimulation: dye was presented only during 3 min tetanus. Loading after stimulation: dye was added to chamber after the end of stimulus train for 15 min. D, The comparison of dye amount (as a percentage) captured during and after high-frequency 3 min stimulation. The solid bars represent loading during stimulation while the open bars represent loading after stimulation. The total uptake of dye (when FM1-43 was presented during and after stimulation, see C) was accepted as 100%. When FM1-43 was presented only during or after stimulation, the nerve terminal staining was less than the brightness of terminals with total dye loading.

Figure 3.

FM1-43 destaining kinetics in motor nerve terminals. A, The decay of nerve terminal fluorescence during 20 Hz stimulation. B, The curves of dye destaining during first 30 s of stimulation. y-axis, The relative fluorescence intensity (ΔJ/ΔJ_max), where 100% represents the intensity before tetanus. x-axis, Time in minutes. C, Change in fluorescent images of nerve terminals during tetanus in control and after treatment by 8-pCPT-cGMP or Rp-8-Br-PET-cGMP (0, 30, 90, 900 s of stimulation). Scale bars, 10 μm.

Figure 4.

Calculation of the recycling time of synaptic vesicles. Superposition of the cumulative EPCs amplitude curve and dye loss curve during 20 Hz stimulation (3 min). The dashed line, The cumulative EPCs amplitude curve (in nA) from Fig. 1; dotted line, dye loss curve from Figure 3, which was scaled and superposed with the cumulative EPCs amplitude curve in their initial parts. The average recycle time (t_r) is the point where the deviation between two curves becomes apparent. A, Control; B, 8-Br-cGMP; C, 8-pCPT-cGMP; D, ODQ; F, Rp-8-Br-PET-cGMP. y-axes, Summed EPCs amplitude in nA (ΣEPC, right axis), and the decrease of relative fluorescence intensity (1−ΔJ/ΔJ_max, left axis). x-axis, Time of stimulation in minutes.