Stimulation-evoked increases in cytosolic [Ca(2+)] in mouse motor nerve terminals are limited by mitochondrial uptake and are temperature-dependent

Abstract

Increases in cytosolic [Ca(2+)] evoked by trains of action potentials (20-100 Hz) were recorded from mouse and lizard motor nerve terminals filled with a low-affinity fluorescent indicator, Oregon Green BAPTA 5N. In mouse terminals at near-physiological temperatures (30-38 degrees C), trains of action potentials at 25-100 Hz elicited increases in cytosolic [Ca(2+)] that stabilized at plateau levels that increased with stimulation frequency. Depolarization of mitochondria with carbonylcyanide m-chlorophenylhydrazone (CCCP) or antimycin A1 caused cytosolic [Ca(2+)] to rise to much higher levels during stimulation. Thus, mitochondrial Ca(2+) uptake contributes importantly to limiting the rise of cytosolic [Ca(2+)] during repetitive stimulation. In mouse terminals, the stimulation-induced increase in cytosolic [Ca(2+)] was highly temperature-dependent over the range 18-38 degrees C, with greater increases at lower temperatures. At the lower temperatures, application of CCCP continued to depolarize mitochondria but produced a much smaller increase in the cytosolic [Ca(2+)] transient evoked by repetitive stimulation. This result suggests that the larger amplitude of the stimulation-induced cytosolic [Ca(2+)] transient at lower temperatures was attributable in part to reduced mitochondrial Ca(2+) uptake. In contrast, the stimulation-induced increases in cytosolic [Ca(2+)] measured in lizard motor terminals showed little or no temperature-dependence over the range 18-33 degrees C.

Fig. 1.

In a mouse motor nerve terminal, stimulation-induced changes in OG-5N fluorescence are primarily localized to the terminal regions, are uniform over various regions of the same terminal, and are larger at cooler temperatures.A and B each show five pseudocolor fluorescence images at 32°C (A) and 18°C (B) before (a), during (b–d), and after (e) a train of action potentials (100 Hz for 5 sec). Each illustrated image is an average of five consecutive images. C andD plot the corresponding ΔF/F transients averaged over three regions of this terminal (indicated by the open triangles, squares, and diamondsin the pseudocolored insets) and a region of preterminal axon (filled triangles in theinsets). Horizontal bars above indicate the duration of stimulation; intervals below markeda–e indicate the timing of the averaged images shown inA and B. Inset images inC and D were made during intervald. The images in A and Buse the “heat” scale (illustrated at right) to display stimulation-induced fluorescence increases at warm and cool temperatures on the same scale. The insets inC and D use the more conventional spectral scale (blue for low and red for high ΔF/F values).

Fig. 2.

In mouse terminals, the effect of temperature on ΔF/F transients is greater at higher stimulation frequencies. A, B, Superimposed ΔF/F transients from the same terminal produced by 500 stimuli delivered at 100, 50, or 25 Hz, at 31.5°C (A) or 18.2°C (B). C, Average peak ΔF/F amplitude after 500 stimuli as a function of stimulation frequency. Open circlessummarize data recorded at warm temperatures (28–35°C);filled circles represent cooler temperatures (17–24°C). The warm point at 25 Hz came from the terminal illustrated in A; all other points indicate the mean ± SEM for 5–11 ΔF/Ftransients (the SEM for the 50 Hz warm point was smaller than thesymbol). Conversion of the ΔF/F averages plotted inC into estimated increases in cytosolic [Ca²⁺] over an assumed resting value of 0.1 μm (see Materials and Methods) yielded the following values (in μm): at 25 Hz, 0.5 (both temperature ranges); at 50 Hz, 0.6 (warm) and 1.1 (cool); at 100 Hz, 1.0 (warm) and 3.3 (cool).

Fig. 3.

Comparison of temperature effects on ΔF/F transients in mouse (A, B) and lizard (C,D) terminals. A and C show superimposed ΔF/F transients recorded in a mouse (A) and a lizard (C) terminal during a train of 500 stimuli delivered at 50 (left) or 100 (right) Hz at 31°C (filled circles) or 19°C (open circles). B and D show histograms of average ΔF/F amplitudes recorded after 1 (open bars) or 10 (filled bars) sec of stimulation at 50 Hz in mouse (B) and lizard (D) terminals over the temperature ranges indicated on the abscissa. Eachbar plots the mean ± SEM for 8–20 ΔF/F transients. For the mouse data inB, the difference between amplitudes recorded at 18–23°C and amplitudes recorded at each of the three warmer temperature ranges was significant after both 1 and 10 sec stimulation (Student–Newman–Keuls test). The differences between amplitudes recorded after 1 and 10 sec were significant for all four temperature ranges, and there was a significant linear trend for ΔF/F at 10 sec to increase as temperature decreased (p < 0.001, one-way ANOVA). Lizard values were not significantly different from each other. Conversion of the average ΔF/F values in B and D into estimated increases in cytosolic [Ca²⁺] over an assumed resting value of 0.1 μm yielded the following values (in μm): mouse: 18–23°C, 0.85 ± 0.069 SEM after 1 sec, 2.03 ± 0.34 after 10 sec; 23–28°C, 0.52 ± 0.036 after 1 sec, 1.07 ± 0.15 after 10 sec; 28–33°C, 0.52 ± 0.03 after 1 sec, 0.87 ± 0.12 after 10 sec; 33–38°C, 0.43 ± 0.041 after 1 sec, 0.54 ± 0.04 after 10 sec; lizard: 18–23°C, 0.38 ± 0.02 after 1 sec, 0.43 ± 0.029 after 10 sec; 28–33°C, 0.42 ± 0.037 after 1 sec, 0.44 ± 0.037 after 10 sec.

Fig. 4.

Cooling and 3,4-DAP have differential effects on the time course of ΔF/F transients (A), and EPP generation is reliable throughout stimulus trains (B). A, Three superimposed ΔF/F transients produced by 5 sec of 100 Hz stimulation in a mouse terminal, first at 19°C (open circles), then after heating to 32°C (filled circles), and then after addition of 10 μm 3,4-DAP to prolong the action potential (open triangles). Cooling and 3,4-DAP both increase the amplitude of the ΔF/F transient, but ΔF/F reaches a limiting plateau value in 3,4-DAP, whereas it continues to increase throughout the train at 19°C. Note also that the first ΔF/Fvalue sampled after the onset of stimulation is similar for warm and cool temperatures but is larger in 3,4-DAP. B, Eachtrace shows a sample of five successive EPPs recorded in a muscle fiber at the beginning (a), middle (b), and end (c) of a 1000 stimulus train delivered at 50 Hz at 33°C. Reliable transmission throughout the stimulus train was also verified at lower temperatures (22–25°C) at other terminals in this muscle (data not shown). In this experiment, muscle contractions were blocked using μ-conotoxin GIIIB (2 μg/ml), which blocks muscle (but not axonal) Na⁺ channels. Use of this drug (instead of tubocurare) minimized the rundown of EPP amplitudes usually measured during repetitive stimulation in the presence of nicotinic antagonists. EPPs were recorded using standard intracellular recording techniques, as detailed by David (1999). The downward andupward deflections preceding each EPP are calibrating pulses and stimulus artifacts, respectively. The resting potential was −75 mV.

Fig. 5.

Agents that depolarize mitochondria increase the amplitude of stimulation-induced ΔF/Ftransients in mouse terminals more at warm than at cool temperatures. Superimposed ΔF/F transients produced by 10 sec of 50 (A, B, 33–34.5°C) or 20 (C, 25°C) Hz stimulation were recorded in three terminals in control saline (open circles), 15–20 min after addition of 5 μg/ml oligomycin (open triangles), and after the further addition of 1 μm CCCP (10–15 min exposure; A, C, filled circles) or 2 μm antimycin A1 (6 min exposure;B, filled circles). The warm 50 Hz records in A and the cool 20 Hz records inC were chosen for comparison because the stimulation-induced increases in cytosolic [Ca²⁺] in control saline were similar. The effects of brief CCCP exposures were partially reversible, but more prolonged exposures (>30 min) resulted in a marked increase in resting fluorescence accompanied by failure of action potential conduction at both warm and cold temperatures (data not shown). The effects of antimycin exposure were not reversible. Two control ΔF/Ftransients are shown in B.

Fig. 6.

Mitochondrial membrane potentials in mouse motor terminals do not depolarize during nerve stimulation at warm or cool temperatures (A) but are depolarized by CCCP (B). Plots showF/F_rest, whereF_rest is the fluorescence measured before stimulation or CCCP application. The mitochondrial-localizing dye TMRE (1 μm) was present in all solutions. A, Superimposed records before, during, and after stimulation at 50 Hz for 10 sec at 20°C (open triangles) and at 33°C (inverted open triangles), and during a similar interval without stimulation at 33°C (open circles). The lack of effect of stimulation on mitochondrial membrane potential was confirmed in three additional TMRE-treated terminals and in another terminal loaded with JC-1; these experiments also revealed no consistent effect of temperature changes onF_rest. B, Effect of briefly exposing the same terminal to 1 μm CCCP at 20°C; the decrease in fluorescence indicates mitochondrial depolarization.A, 0.533 sec/image; B, 2.133 sec/image.