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The apparent mean channel open time (tau) of acetylcholine receptors (AChR) at skeletal muscle endplates decreases greater than 3-fold during development. In rat soleus muscles, the change occurs between postnatal days 8 and 18 as channels with long apparent open times (tau = 4.5 msec) disappear while channels with short apparent open times (tau = 1.5 msec) increase in number. We studied the role of innervation in this process by denervating neonatal soleus muscles prior to channel conversion. Tau at the denervated endplates was assayed at various times between days 8 and 18 by using fluctuation analysis. We found that early denervation blocked, or at least delayed, channel conversion. Unexpectedly, there was enhanced extrajunctional ACh sensitivity in the innervated muscles contralateral to the denervated ones. This observation allowed us to compare the apparent open times of junctional AChRs with those of extrajunctional AChRs 200 micron distant in the same innervated fibers. In developing muscles, tau at the extrajunctional sites decreased in parallel with tau at the endplates. Thus, neural regulation of AChR channel gating extends well beyond the endplate boundaries.

The gating properties of acetylcholine receptors (AChRs) change during the development of rat soleus endplates. During the first 3 weeks after birth, the apparent mean channel open time (tau) decreases severalfold and the single-channel conductance (gamma) increases 50%. To better understand this phenomenon, we used a combination of noise analysis, analysis of miniature endplate currents (MEPCs), and single-channel recordings to quantify the relative levels of fast and slow AChR activity at developing soleus endplates. When the same endplates were studied with both noise analysis and MEPC analysis, results obtained with the two techniques were strongly correlated, but MEPC analysis yielded higher estimates of the relative amount of slow channel activity. Experiments designed to examine the distribution of fast and slow channels gave no evidence for a gradient of either channel type within individual endplates; rather, fast and slow channels appeared to be mixed together. However, the relative amount of fast and slow channel activity did vary markedly among different endplates within individual muscles. In agreement with earlier studies, we found a progressive decrease in the relative amount of slow channel activity during the first 3 weeks after birth. However, our data indicate that this process begins sooner than reported previously and takes longer to complete. Some of the same endplates that were studied physiologically were also examined in the electron microscope to test the hypothesis that changes in AChR gating might be related to ultrastructural changes such as the formation of folds. The physiological and ultrastructural results were essentially uncorrelated.

We have examined the physiological effects of antibodies from a highly specific myasthenic serum on acetylcholine receptors at developing rat endplates. The antibodies reduced the amplitude of miniature endplate potentials by 60% in 3- to 6-day-old animals but had no effect after day 14. Between days 7 and 12 the antibodies had an intermediate effect. This is the same period during which acetylcholine receptors with long channel open times (slow channels) disappear and receptors with short open times (fast channels) increase in number. Therefore, we examined the effect of the antibodies at endplates with a mixture of channel types more carefully. At all times tested, both noise analysis and analysis of miniature endplate currents showed that the antibodies reduced slow channel activity selectively. Single-channel recordings indicated that acetylcholine receptors that remained active after antibody treatment had normal gating properties. Thus, the antibodies appeared to silence slow channels selectively.

We have studied the function of acetylcholine (AcCho) receptors (AcChoRs) in rat soleus endplates before and after exposing the muscles to forskolin, a potent activator of adenylate cyclase. AcChoR function was tested by recording the membrane depolarization evoked by pulses of ionophoretically applied AcCho. Brief (2 msec) AcCho pulses delivered at 7 Hz evoked constant responses at untreated endplates. In contrast, after 10-100 microM forskolin was added to the bath, responses to similar pulse trains fell by as much as 80% within 1 sec. AcCho sensitivity recovered completely in less than 1 min after the pulses were stopped but fell again when the pulses were resumed. Similarly, longer (1 sec) ionophoretic AcCho pulses evoked roughly constant responses at control endplates, but after forskolin treatment the depolarization fell by one-half within less than 200 msec. These results indicate that forskolin increases the rate at which AcChoRs desensitize when exposed to agonist. Focal extracellular recordings showed that 20-100 microM forskolin also increased the decay rate of miniature endplate currents, indicating that forskolin may decrease AcChoR channel open time. Inhibitors of cAMP phosphodiesterase increased the potency of forskolin. When used alone, these inhibitors had effects similar to those of forskolin but smaller. Patch-clamp experiments indicated that forskolin at 100 microM may also interact with AcChoR channels directly, but at 20 microM this effect is negligible. Therefore, it is likely that the forskolin effects were mediated primarily by increased levels of intracellular cAMP.

Rat soleus endplates develop some of their characteristic features before birth and others after birth. Specializations appearing before birth include a localized cluster of acetylcholine receptors (AChRs), an accumulation of acetylcholinesterase (AChE) in the synaptic basal lamina, and a cluster of nuclei near the endplate membrane. In contrast, postsynaptic membrane folds are elaborated during the first three weeks after birth. We denervated soleus muscles on postnatal day 1, before folds had appeared, and followed the subsequent development of endplate regions with light and electron microscopy. We found that the denervated endplates initiated fold formation on schedule and maintained their accumulations of AChRs, AChE, and endplate nuclei. However, the endplates stopped fold formation prematurely and eventually lost their rudimentary folds. At about the same time, the junctional AChR clusters were joined by ectopic patches of AChRs. The former endplate regions also became unusually elongated, possibly as a consequence of the lack of membrane folds. Apparently, endplate membranes have only a limited capacity for further development in the absence of both the nerve and muscle activity.

1. The mean channel open time (tau) of ACh receptors in adult developing rat skeletal muscle was estimated by analysis of ACh current fluctuations. 2. At adult end-plates, tau was 1.4 msec at 21 degrees C. Time constants of miniature end-plate current decays (taus) were equally brief. 3. On the other hand, tau and taus were long (4.5 msec, 21 degrees C) at neonatal rat end-plates. These estimates are comparable to values obtained at extrajunctional sites on embryonic rat myotubes grown in culture and on adult denervated muscle fibres. 4. Both tau and taus decreased to values characteristic of adult end-plates during the first 2 weeks after birth. 5. Complex ACh power spectra were obtained at most end-plates during the transition period and at some, miniature synaptic current decays were doubly exponential. End-plates in transition might contain a mixture of slow, neonatal channels and fast, adult channels.

Miniature end-plate currents (m.e.p.c.s.) were recorded extracellularly from individual fibres in neonatal rat soleus muscles for 2-24 h. In agreement with previous studies, the decay phases of m.e.p.c.s at many end-plates were doubly exponential with time constants of approximately 6 ms and approximately 1.5 ms at 21 degrees C. Earlier studies have shown that doubly exponential decays are due to the combined action of embryonic-type acetylcholine (ACh) receptors (AChRs) with long channel open times and adult-type AChRs with brief open times. When individual end-plates with doubly exponential m.e.p.c.s were studied for several hours or more, the relative size of the slow decay component frequently decreased with time. There was no evidence for a corresponding decrease in total m.e.p.c. amplitude. The time constants of the fast and slow components did not change. M.e.p.c. decays were stable at end-plates that were either very mature (small slow decay component) or very immature (small fast decay component). In these cases, the decay phases were virtually singly exponential and the time constant did not change. These data indicate that at end-plates with a mixture of adult-type and embryonic-type channels, the fraction of adult-type AChRs increases with time. This is similar to what occurs at end-plates developing in vivo. The results of ACh noise analysis experiments support this interpretation.