Decreased synaptic activity shifts the calcium dependence of release at the mammalian neuromuscular junction in vivo

Abstract

We examined the mechanism underlying increased quantal content after block of activity at the mouse neuromuscular junction in vivo. We found that, when quantal content was measured in solution containing normal extracellular calcium, block of activity had no effect on either quantal content or the response to repetitive stimulation. However, when quantal content was measured in low extracellular calcium, there was a large increase in quantal content after block of activity. The increase in quantal content was accompanied by increased depression during repetitive stimulation. The explanation for these findings was that there was a shift in the calcium dependence of release after block of activity that manifested as an increase in probability of release in low extracellular calcium. Block of presynaptic P/Q channels eliminated the increase in probability of release. We propose that activity-dependent regulation of presynaptic calcium entry may contribute to homeostatic regulation of quantal content.

Figure 1.

Blocking activity increases quantal content in low, but not normal, extracellular Ca²⁺. A, Average EPCs and quantal content measured in normal calcium after block of activity. Shown are representative EPCs evoked in normal Ca²⁺ (2 mm) and normal Mg²⁺ (0.7 mm) that were obtained from averaging 20-30 nerve stimulations. There was little difference in either EPC amplitude or quantal content between the control and TTX-blocked endplates (p = 0.22 for EPC amplitude; p = 0.15 for quantal content; n = 20 control muscles, 12 TTX-blocked muscles). B, Average EPCs and quantal content measured in low Ca²⁺ (1 mm) and high Mg²⁺ (8 mm) after block of activity. Both the EPC amplitude and the quantal content are much larger after block of synaptic activity (p < 0.01 for both measures; n = 14 control muscles, 10 TTX blocked muscles). Note the difference in calibration for EPC traces in A and B. Control, endplates in which synaptic activity is normal; TTX blocked, endplates in which synaptic activity was blocked for 8-10 d with a TTX cuff around the nerve.

Figure 2.

Repetitive stimulation suggests that probability of release is increased in low, but not normal, calcium after block of synaptic activity. A, The average EPCs from 6-10 trains of 50 Hz pulses are shown for control and TTX-blocked endplates in normal (2 mm Ca²⁺-0.7 mm Mg²⁺) Ca²⁺ and low (1 mm Ca²⁺-0.7 mm Mg²⁺) Ca²⁺. In normal Ca²⁺, there was a similar degree of depression in control and TTX-blocked endplates. In low Ca²⁺, although facilitation occurred in both control and TTX-blocked endplates, the facilitation was not as great in the TTX-blocked endplate and was offset by depression later in the train, such that the 10th pulse had the same amplitude as the first pulse. Calibration shown is the same for normal and low Ca²⁺. Note that the low Ca²⁺ solution used in this study had normal Mg²⁺, as opposed to the solution used in Figure 1.B, Normalized average EPCs during trains of 50 Hz pulses are shown for control (n = 8 muscles) and TTX-blocked (n = 6 muscles) endplates in low and normal extracellular Ca²⁺. In low Ca²⁺, both control and TTX-blocked endplates showed facilitation during repetitive stimulation. However, the degree of facilitation decreased in the TTX-blocked endplates (p < 0.01 for 2nd pulse; p < 0.05 for last pulse). In normal Ca²⁺, there was a similar degree of depression in control and TTX-blocked endplates (p = 0.18 for 2nd pulse; p = 0.57 for 10th pulse).

Figure 3.

There is a leftward shift in the Ca²⁺ dependence of quantal content after block of synaptic activity. The quantal content of control and TTX-blocked endplates is plotted versus calcium concentration on a double-logarithmic plot. Magnesium concentration was 8 mm in all of the solutions used. The slope was 3.9 in control (r = 0.998; p = 0.04) and 3.5 in TTX-blocked (r = 0.995; p = 0.07) endplates. Open squares, TTX-blocked; filled squares, control.

Figure 4.

Increased probability of release is eliminated after block of Ca²⁺ entry through P/Q channels. A1, Average EPCs from representative control (light trace) and TTX-blocked (dark trace) endplates in 1 mm Ca²⁺-8 mm Mg²⁺. A2, Representative average EPCs from control (light trace) and TTX-blocked (dark trace) endplates in 1 mm Ca²⁺-8 mm Mg²⁺ after block of P/Q channels with 1 μm ω-agatoxin GIVA. The light and dark traces are superimposed and cannot be distinguished. The majority of responses were failures, such that the average EPC is ∼10% of the amplitude of an individual MEPC. A3, Representative average EPCs from control (light trace) and TTX-blocked (dark trace) endplates after block of P/Q channels with 1 μm ω-agatoxin GIVA in 2 mm Ca²⁺-8 mm Mg²⁺. B, Quantal content of control and TTX-blocked endplates before and after the P/Q channels were blocked. The concentration of extracellular Ca²⁺ was doubled from 1 to 2 mm after the addition of ω-agatoxin, to return quantal content to the levels seen before block of P/Q channels. Before P/Q channel blockade, quantal content was significantly higher in TTX-blocked endplates (p < 0.01), but, after P/Q channel blockade, there was no significant difference (n = 5 muscles for both groups).