Presynaptic function is altered in snake K+-depolarized motor nerve terminals containing compromised mitochondria

Abstract

Presynaptic function was investigated at K+-stimulated motor nerve terminals in snake costocutaneous nerve muscle preparations exposed to carbonyl cyanide m-chlorophenylhydrazone (CCCP, 2 M), oligomycin (8 g x ml(-1)) or CCCP and oligomycin together. Miniature endplate currents (MEPCs) were recorded at -150 mV with two-electrode voltage clamp. With all three drug treatments, during stimulation by elevated K+ (35 mM), MEPC frequencies initially increased to values > 350 s(-1), but then declined. The decline occurred more rapidly in preparations treated with CCCP or CCCP and oligomycin together than in those treated with oligomycin alone. Staining with FM1-43 indicated that synaptic vesicle membrane endocytosis occurred at some CCCP- or oligomycin-treated nerve terminals after 120 or 180 min of K+ stimulation, respectively. The addition of glucose to stimulate production of ATP by glycolysis during sustained K+ stimulation attenuated the decline in MEPC frequency and increased the percentage of terminals stained by FM1-43 in preparations exposed to either CCCP or oligomycin. We propose that the decline in K+-stimulated quantal release in preparations treated with CCCP, oligomycin or CCCP and oligomycin together could result from a progressive elevation of intracellular calcium concentration ([Ca2+]i). For oligomycin-treated nerve terminals, a progressive elevation of [Ca2+]i could occur as the cytoplasmic ATP/ADP ratio decreases, causing energy-dependent Ca2+ buffering mechanisms to fail. The decline in MEPC frequency could occur more rapidly in preparations treated with CCCP or CCCP and oligomycin together because mitochondrial Ca2+ buffering and ATP production were both inhibited. Therefore, the proposed sustained elevation of [Ca2+]i could occur more rapidly.

Figure 1. MEPC recordings from control and oligomycin- or CCCP-treated endplates during exposure to 35 KP

A, MEPC recordings obtained from oligomycin-treated preparations. Traces 1-3 were recorded from oligomycin-treated endplates after 15, 130 and 182 min in 35 KP solution, respectively. B, recordings obtained from CCCP-treated preparations. Traces 1-3 were recorded from CCCP-treated endplates after 5, 29 and 79 min in 35 KP solution, respectively. C, MEPC recording obtained in a control preparation after 200 min in 35 KP.

Figure 2. MEPC frequency progressively declined at oligomycin-, CCCP- or oligomycin and CCCP-treated twitch endplates during exposure to 35 KP

Mean MEPC frequency recorded in 35 KP for preparations treated with oligomycin (□), CCCP (○) and oligomycin and CCCP together (Δ). Each data point represents the mean ±s.e.m. from at least 3 endplates.

Figure 3. FM1-43 fluorescence at control and CCCP- and oligomycin-treated twitch motor nerve terminals after 120-180 min in 35 KP

A, an example of the uniform FM1-43 fluorescence for a control terminal after 120 min in 35 KP. B-D, examples of FM1-43 staining patterns at CCCP- or oligomycin-treated terminals after 120-180 min in 35 KP. B, an example of an oligomycin-treated nerve terminal (180 min in KP) that did not exhibit FM1-43 fluorescence. C, an example of a CCCP-treated terminal (120 min in KP) that exhibited partial FM1-43 staining. D, an example of an oligomycin-treated terminal (180 min in KP) that exhibited uniform FM1-43 staining. E and F, uniformly stained nerve terminals from a CCCP-treated (E) or an oligomycin-treated (F) preparation exposed to 35 KP containing 15 mm glucose. Calibration bar in F, 40 μm. Note that the myelin sheath of the terminal axon was commonly stained by the dye (A, B, C and E). In all cases, the FM1-43 was present only in the final 6 min of the 35 KP exposure.

Figure 4. Electron micrographs of twitch neuromuscular junctions from control and CCCP-treated muscle preparations exposed to 35 KP

A, a terminal from a control preparation after 120 min in 35 KP. B, a terminal from a CCCP-treated preparation maintained in 35 KP for 90 min. Note that the mitochondrial morphology was similar and that synaptic vesicles were abundant in both examples. M, mitochondria; SV, synaptic vesicles.