Activity-dependent presynaptic regulation of quantal size at the mammalian neuromuscular junction in vivo

Abstract

Changes in synaptic activity alter quantal size, but the relative roles of presynaptic and postsynaptic cells in these changes are only beginning to be understood. We examined the mechanism underlying increased quantal size after block of synaptic activity at the mammalian neuromuscular junction in vivo. We found that changes in neither acetylcholinesterase activity nor acetylcholine receptor density could account for the increase. By elimination, it appears likely that the site of increased quantal size after chronic block of activity is presynaptic and involves increased release of acetylcholine. We used mice with muscle hyperexcitability caused by mutation of the ClC-1 muscle chloride channel to examine the role of postsynaptic activity in controlling quantal size. Surprisingly, quantal size was increased in ClC mice before block of synaptic activity. We examined the mechanism underlying increased quantal size in ClC mice and found that it also appeared to be located presynaptically. When presynaptic activity was completely blocked in both control and ClC mice, quantal size was large in both groups despite the higher level of postsynaptic activity in ClC mice. This suggests that postsynaptic activity does not regulate quantal size at the neuromuscular junction. We propose that presynaptic activity modulates quantal size at the neuromuscular junction by modulating the amount of acetylcholine released from vesicles.

Figure 6.

MEPC amplitude is similar in TTX-blocked control and TTX-blocked ClC endplates. Cumulative distribution of MEPC amplitudes from TTX-blocked control and TTX-blocked ClC endplates (n = 38 for ClC TTX; n = 66 for TTX control). The holding potential was -45 mV.

Figure 1.

MEPC amplitude is increased after block of synaptic activity. A, Shown are representative average MEPC waveforms from a control endplate and an endplate after block of synaptic activity. Each trace represents the average of >30 MEPCs from the fiber. The MEPC from the endplate in which synaptic activity was blocked is ∼35% larger. B, Cumulative distribution of MEPC amplitudes from control endplates and endplates in which synaptic activity was blocked (n = 85 endplates for control; n = 66 after block of synaptic activity). Error bars represent SEM.

Figure 2.

Decreased activity of acetylcholinesterase is not the primary cause of increased MEPC amplitude. A, Shown in black are average MEPC waveforms from endplates after blockade of cholinesterase activity by neostigmine. Superimposed on the traces in gray are the normalized MEPC traces from Figure 1 representing MEPCs in endplates in which AChE is active. The increase in MEPC amplitude after block of activity persists after block of AChE. Block of AChE caused prolongation of the time to peak and a marked prolongation of the time constant of MEPCs. B, Cumulative distribution of MEPC amplitudes from control and TTX-blocked endplates after blockade of AChE activity by neostigmine (n = 30 endplates for control; n = 20 after block of synaptic activity). The holding potential was -70 mV for this set of experiments rather than -45 mV as in Figure 1. Error bars represent SEM.

Figure 3.

Quantitative immunofluorescence reveals no increase in AChR density after block of synaptic activity. A, Shown are individual confocal sections used to quantitate AChR density through a control endplate and an endplate in which synaptic activity was blocked. B, The relative AChR labeling in control endplates is plotted relative to TTX-blocked control endplates. There is no significant difference in labeling of control endplates versus TTX-blocked endplates (n = 37 endplates from 4 muscles for control; n = 38 endplates from 4 muscles after block of synaptic activity).

Figure 4.

Muscle activity remaining after loss of nerve activity is sufficient to prevent denervation-induced changes in skeletal muscle in ClC mice. A, EMGs from mouse tibialis anterior muscles in vivo during movement under light anesthesia. Records were taken from control muscle, denervated control muscle, and denervated ClC muscle. In control muscle, runs of action potentials represent activation of motor units. After denervation of control muscle, there is no EMG activity; however, after denervation of ClC muscle, there are spontaneous runs of action potentials (arrow). B, Application of acetylcholine to control endplates (junctional response) results in an inward current, whereas application of acetylcholine to the muscle fiber away from the endplate (extrajunctional response) results in no inward current (n = 9 fibers). After block of activity in control fibers (Control TTX), there is an increase in the response to applied acetylcholine both at and away from the endplate (n = 5 fibers). This suggests that the increase in junctional response to applied acetylcholine may be caused by the presence of extrajunctional AChRs. The presence of extrajunctional AChRs also results in a slower decay of the response to applied acetylcholine, likely because of binding of acetylcholine to receptors along the length of the muscle fiber. After block of synaptic activity in ClC fibers (ClC TTX), there is no response to extrajunctional application of ACh (n = 9 fibers), demonstrating that the muscle activity that remains after denervation is sufficient to prevent this denervation-induced change in skeletal muscle. C, Resting potential of control and ClC muscle before and after 1 week of denervation. Each bar shows the mean of three muscles from each group ± SEM. The resting potential decreased after denervation in control muscle (p < 0.01) but not in ClC muscle. Den, Denervated.

Figure 5.

MEPC amplitude is increased at ClC endplates because of a presynaptic mechanism. A, Cumulative distribution of MEPC amplitudes from ClC endplates (n = 99) relative to the control plot from Figure 1C. Holding potential was -45 mV. B, Cumulative distribution of MEPC amplitudes after block of AChE from ClC endplates (n = 60) relative to the control plot from Figure 2 B. The difference in MEPC amplitude persists after block of AChE. Holding potential was -70 mV. C, Shown are bar graphs of MEPC amplitude and the amplitude of the response to applied acetylcholine in 21 control and 24 ClC endplates. Despite a 40% increase in MEPC amplitude in ClC relative to control (p < 0.01), there was no statistically significant difference in the response to applied acetylcholine. D, Quantitative immunofluorescence reveals no increase in AChR density in ClC endplates. The relative AChR labeling in ClC endplates is plotted relative to control endplates. There is a 20% reduction in AChR intensity in ClC endplates relative to control (p < 0.01; n = 38 endplates from 4 muscles for control; n = 32 endplates from 4 muscles for ClC).