Prolongation of evoked and spontaneous synaptic currents at the neuromuscular junction after activity blockade is caused by the upregulation of fetal acetylcholine receptors

Abstract

It has been shown previously in a number of systems that after an extended block of activity, synaptic strength is increased. We found that an extended block of synaptic activity at the mouse neuromuscular junction, using a tetrodotoxin cuff in vivo, increased synaptic strength by prolonging the evoked endplate current (EPC) decay. Prolongation of EPC decay was accompanied by only modest prolongation of spontaneous miniature EPC (MEPC) decay. Prolongation of EPC decay was reversed when quantal content was lowered by reducing extracellular calcium. These findings suggested that the cause of EPC prolongation was presynaptic in origin. However, when we acutely inhibited fetal-type acetylcholine receptors (AChRs) using a novel peptide toxin (alphaA-conotoxin OIVA[K15N]), prolongation of both EPC and MEPC decay were reversed. We also blocked synaptic activity in a mutant strain of mice in which persistent muscle activity prevents upregulation of fetal-type AChRs. In these mice, there was no prolongation of EPC decay. We conclude that upregulation of fetal-type AChRs after blocking synaptic activity causes modest prolongation of MEPC decay that is accompanied by much greater prolongation of EPC decay. This might occur if acetylcholine escapes from endplates and binds to extrajunctional fetal-type AChRs only during large, evoked EPCs. Our study is the first to demonstrate a functional role for upregulation of extrajunctional AChRs.

Figure 1.

Extended blockade of neuromuscular activity increases synaptic strength because of the prolongation of the EPC. A, Superimposed EPCs from a control endplate (black) and an endplate in which synaptic activity was blocked by a TTX cuff for 8 d (gray). Although the peak amplitude of the EPC is similar, there is prolongation of EPC decay after block of activity. Each EPC is preceded by a stimulus artifact (arrow with long tail). B, Mean EPC amplitude in control endplates (black) and endplates in which activity was blocked for 7–10 d (gray). There is no statistically significant increase in EPC amplitude after block of synaptic activity (p = 0.55). C, Mean EPC integral in control endplates (black) and endplates in which activity was blocked for 7–10 d (gray). There is a statistically significant increase in total charge carried by the EPCs after blocking synaptic activity (p < 0.01). n = 9 control muscles and 6 muscles in which activity was blocked. Error bars represent the SEM.

Figure 2.

The close relationship between EPC decay time constant and MEPC decay time constant is lost after block of synaptic activity but is regained in solution containing low calcium. A, Scatter plot of EPC decay time constant versus MEPC decay time constant for control endplates (open circles) and endplates after block of activity with a TTX cuff (filled squares). EPC and MEPC recordings were obtained in Ringer’s solution containing normal extracellular calcium. There is marked prolongation of EPC decay time constant after blocking activity in endplates that had no prolongation of MEPC decay time constant. B, Scatter plot of EPC decay time constant versus MEPC decay time constant in Ringer’s solution containing low extracellular calcium for control endplates and endplates after block of activity with a TTX cuff. The data from endplates in which activity was blocked essentially overlaps data from control endplates. C, Superimposed EPCs recorded in solution containing low calcium from a control endplate (black) and an endplate in which synaptic activity was blocked by a TTX cuff for 8 d (gray). Although the peak amplitude of the EPC recorded in low calcium is larger after block of activity (Wang et al., 2004), there is little difference in the rate of EPC decay. Each EPC is preceded by a stimulus artifact (arrow). Note the difference in vertical scale relative to Figure 1A. D, The superimposed, normalized EPCs shown in C.

Figure 3.

Prolongation of EPCs after activity blockade is reversed by a toxin against the fetal-type AChR. A, EPC from an endplate in which synaptic activity was blocked for 8 d (gray) and from the same endplate after application of αA-conotoxin OIVA[K15N] to inhibit fetal-type AChRs (black). B, Mean EPC integral of activity-blocked endplates before (gray) and after (black) application of αA-conotoxin OIVA[K15N]. In endplates in which activity was blocked, application of αA-conotoxin OIVA[K15N] caused a significant reduction in EPC integral (p < 0.01). n = 6 muscle fibers. Error bars represent the SEM. C, The response to extrajunctional application of ACh for a muscle fiber in which synaptic activity has been blocked for 8 d (gray) and the same fiber after application of αA-conotoxin OIVA[K15N] to inhibit fetal-type AChRs (black). The extrajunctional response to ACh was completely blocked in all five fibers studied (p < 0.01). The arrow indicates the beginning of a 100 ms pulse of ACh onto the muscle fiber.

Figure 4.

Prolongation of EPC decay does not occur in ClC-1 chloride channel null mice that do not upregulate fetal-type AChRs. A, Superimposed EPCs from an active ClC endplate (black) and a ClC endplate in which synaptic activity was blocked for 8 d (gray). B, Mean EPC integral in active ClC endplates (black) and ClC endplates in which synaptic activity was blocked for 7–10 d (gray). There is no statistically significant difference in EPC integral after block of synaptic activity in ClC endplates (p = 0.17). n = 7 muscles for both active ClC endplates and ClC endplates in which nerve activity was blocked. Error bars represent the SEM.