Regulation of motoneuron excitability via motor endplate acetylcholine receptor activation

Abstract

Motoneuron populations possess a range of intrinsic excitability that plays an important role in establishing how motor units are recruited. The fact that this range collapses after axotomy and does not recover completely until after reinnervation occurs suggests that muscle innervation is needed to maintain or regulate adult motoneuron excitability, but the nature and identity of underlying mechanisms remain poorly understood. Here, we report the results of experiments in which we studied the effects on rat motoneuron excitability produced by manipulations of neuromuscular transmission and compared these with the effects of peripheral nerve axotomy. Inhibition of acetylcholine release from motor terminals for 5-6 d with botulinum toxin produced relatively minor changes in motoneuron excitability compared with the effect of axotomy. In contrast, the blockade of acetylcholine receptors with alpha-bungarotoxin over the same time interval produced changes in motoneuron excitability that were statistically equivalent to axotomy. Muscle fiber recordings showed that low levels of acetylcholine release persisted at motor terminals after botulinum toxin, but endplate currents were completely blocked for at least several hours after daily intramuscular injections of alpha-bungarotoxin. We conclude that the complete but transient blockade of endplate currents underlies the robust axotomy-like effects of alpha-bungarotoxin on motoneuron excitability, and the low level of acetylcholine release that remains after injections of botulinum toxin inhibits axotomy-like changes in motoneurons. The results suggest the existence of a retrograde signaling mechanism located at the motor endplate that enables expression of adult motoneuron excitability and depends on acetylcholine receptor activation for its normal operation.

Figure 1.

Axotomy increases motoneuron excitability. Data represent mean ± SEM for MG motoneuron rheobase current measurements obtained from four normal, unoperated animals and from four normal animals in which the MG nerve had been crushed 5 d earlier. The crush site was located at the nerve entry into the MG muscle. Each mean value was derived from the data of at least eight motoneurons. Error bars represent SEM.

Figure 2.

Blockade of AChRs increases motoneuron excitability to the same extent as axotomy. Each group of vertical bars show mean ± SEM of MG motoneuron rheobase from individual experiments. Top and bottom dashed lines represent, respectively, the mean of mean values for MG motoneuron rheobase current from control animals and from animals with axotomized MG motoneurons. Botox was injected once daily into the MG muscle for 2 consecutive days, and motoneurons were studied after a 3 d delay. α-Btx was injected once daily into the MG muscle for 4 consecutive days, and motoneurons were studied after an additional 2 d delay. Saline injections followed the same daily pattern used for α-btx. Statistical analysis showed that the effects of α-btx injections were indistinguishable from axotomy effects, whereas botox effects were indistinguishable from the effects of saline injections.

Figure 3.

Effects of botulinum toxin on neuromuscular transmission at MG muscle NMJs. Effects were studied at the same delay as that used to study motoneuron properties after intramuscular injections of botox. A-C, Vertical bars show the mean ± SEM of mean values obtained from individual control experiments (n = 3) and from botox-treated muscles (n = 3). Each experiment includes the data from at least eight muscle fibers. MEPC amplitude was unchanged relative to normal (A), whereas MEPC spontaneous occurrence frequency was significantly depressed (B). Quantal content (mean EPC integral/mean MEPC integral) was also significantly decreased after botox injections. D, At control NMJs, EPC amplitude typically showed depression during high-frequency nerve stimulation (50 Hz). Five days after botox treatment, the same stimulation produced facilitation of EPC amplitudes. Records were retouched to remove stimulus artifacts.

Figure 4.

Effects of α-btx on neuromuscular transmission at MG muscle NMJs. A, Vertical bars show the mean ± SEM of mean EPC amplitudes (holding potential, -50 mV) obtained from individual control experiments (n = 3) and from MG muscle fibers studied 24 h after two daily α-btx injections (n = 2). Each mean includes data from at least eight fibers. B, Comparison of EPC amplitude depression at a stimulus rate of 1/s between EPCs obtained from one control experiment and from one experiment 24 h after two daily α-btx injections. Each line represents data from one muscle fiber. EPC amplitudes are normalized to the amplitude of the first EPC in the sequence. C, Vertical bars show the mean ± SEM of mean amplitudes of the fifth EPC normalized to the first EPC amplitude in sequences obtained at 1/s as in B. Data obtained from MG muscles of two control animals and from MG muscles of two animals 24 h after two daily α-btx injections. D, Vertical bars show the mean ± SEM of mean MEPC amplitudes studied at a holding potential of -120 mV and obtained from a single control experiment and from MG muscles studied 48 h after four daily α-btx injections (n = 2).