SUMMARY1. Characteristics of receptor-channel activation and desensitization have been compared at voltage-clamped snake slow and twitch fibre end-plates maintained in an isotonic potassium propionate solution.2. Miniature end-plate current (m.e.p.c.) decay was slower and less voltage dependent at slow fibre end-plates than at twitch fibre end-plates. The peak m.e.p.c. amplitude versus voltage relationship and reversal potential were similar at the two end-plate types.3. Acetylcholine-induced noise and m.e.p.c.s were recorded at slow fibre end-plates. At most slow fibres the spectral density was not adequately fitted by a single Lorentzian function. Rather, the observed spectral density was greater at high frequencies than the values predicted using the m.e.p.c. decay rate. The noise could be well described by the sum of two Lorentzian functions, one of which corresponded to a single Lorentzian function with the corner frequency determined by the m.e.p.c. decay rate.4. The shape of the carbachol concentration-peak end-plate current relationship was similar at both slow and twitch fibre end-plates. However, for all concentrations tested, the peak carbachol-induced end-plate current (e.p.c.carb ) value was markedly less at slow fibre end-plates than at twitch fibre end-plates. 5. The onset of desensitization was determined using two methods. The first concerned analysis of the time course of decay of the e.p.c.carb from a peak value during the sustained application of agonist. The second involved a double-perfusion technique in which a 'desensitizing' dose was applied for varying intervals before the application of a second 'test' dose of carbachol. With both methods the development of desensitization at both end-plate types was dependent on carbachol concentration and duration of exposure. At each end-plate type the time course of desensitization onset often exhibited two components; one with a time constant of seconds and a slower component having time constants in the range of tens to hundreds of seconds. E. A. CONNOR AND OTHERS6. The slope of the relationship between carbachol concentration and equilibrium desensitization at slow and twitch fibre end-plates was close to two, suggesting that two molecules of agonist are probably bound during the development of desensitization. However, for all concentrations tested, desensitization developed more rapidly and to a greater extent at twitch fibre end-plates than at slow fibre end-plates.7. The voltage dependence of the 3 min steady-state desensitization produced by 108 jSM-carbachol was very similar (--0.0250 mV-1) at both fibre types. However, the 3 min steady-state level of desensitization was consistently greater at corresponding voltages for twitch fibre end-plates than at slow fibre end-plates. It was also observed at twitch fibre end-plates exposed to 216,M-carbachol that the fast component of desensitization and 3 min steady-state level of desensitization could exhibit different voltage dependencies. This is consistent with the view that the fast and slow ...

Abstract. If skeletal muscles are denervated, the number of mononucleated cells in the connective tissue between muscle fibers increases. Since interstitial cells might remodel extracellular matrix, and since extracellular matrix in nerve and muscle plays a direct role in reinnervation of the sites of the original neuromuscular junctions, we sought to determine whether interstitial cell accumulation differs between junctional and extrajunctional regions of denervated muscle. We found in muscles from frog and rat that the increase in interstitial cell number was severalfold (14-fold for frog, sevenfold for ra0 greater in the vicinity of junctional sites than in extrajunctional regions. Characteristics of the response at the junctional sites of frog muscles are as follows. During chronic denervation, the accumulation of interstitial cells begins within 1 wk and it is maximal by 3 wk. Reinnervation 1-2 wk after nerve damage prevents the maximal accumulation. Processes of the cells form a multilayered veil around muscle fibers but make little, if any, contact with the muscle cell or its basal lamina sheath. The results of additional experiments indicate that the accumulated cells do not originate from terminal Schwann cells or from muscle satellite cells. Most likely the cells are derived from fibroblasts that normally occupy the space between muscle fibers and are known to make and degrade extracellular matrix components. DNERVATION of skeletal muscles results in a marked increase in the number of mononucleated cells in the connective tissue between muscle fibers, particularly in cells that look much like fibroblasts (29,32,33,52). Since fibroblasts and other connective tissue cells make and degrade extracellular matrix constituents (6, 16), such changes raise the possibility that the cells that accumulate after denervation act to remodel the extracellular matrix of muscle. Indeed, several studies have shown that the concentrations of some extracellular matrix molecules are altered by denervation (4, 41,45). This is of particular interest since there is evidence that extracellular matrix components play a direct role in regeneration of the neuromuscular junction. For example, in vivo studies of damaged motor nerves have revealed that regenerating axons preferentially grow through tubes of Schwann cell basal lamina to reinnervate muscle fibers at the original synaptic sites on the myofibers' basal lamina sheaths (17,23,27,35), and in vitro studies have shown that neurites preferentially elongate on laminin, a major constituent of Schwann cell and muscle fiber basal lamina (10,21,26,37,43,46). Moreover, the myofiber basal lamina is known to contain molecules that direct the formation of synaptic apparatus in regenerating axon terminals and myofibers (3,7,12,30,44).In the study described here, we denervated frog and rat muscles and compared the change in number of mononucleated cells in junctional regions with that in extrajunctional regions. We report that for both species the increase in the number of mononucleated ...

Excitatory postsynaptic currents (EPSCs) have been studied in voltage-clamped bullfrog sympathetic ganglion B cells. The EPSC was small, rose to a peak within 1-3 ms, and then decayed exponentially over most of its time-course. For 36 cells at -50 mV (21-23~ peak EPSC size was -6.5 • 3.5 nA (mean • SD), and the mean decay time constant 1" was 5.3 • 0.9 ms. ~" showed a small negative voltage dependence, which appeared independent of temperature, over the range -90 to -30 mV; the coefficient of voltage dependence was -0.0039 • 0.0014 mV -1 (n = 29). The peak current-voltage relationship was linear between -120 and -30 mV but often deviated from linearity at more positive potentials. The reversal potential determined by interpolation was ~-5 mV. EPSC decay T had a O~0 = 3. The commonly used cholinesterase inhibitors, neostigmine and physostigmine, exhibited complex actions at the ganglia. Neostigmine (1 • 10 -s M) produced a time-dependent slowing of EPSC decay without consistent change in EPSC size. In addition, the decay phase often deviated from a single exponential function, although it retained its negative voltage dependence. With 1 • 10 -6 M physostigmine, EPSC decay was slowed but the decay phase remained exponential. At higher concentrations of physostigmine, EPSC decay was markedly prolonged and was composed of at least two decay components. High concentrations of atropine (10 -5 to 10 -4 M) produced complex alterations in EPSC decay, creating two or more exponential components; one decay component was faster and the other was slower than that observed in untreated cells. These results suggest that the time-course of ganglionic EPSC decay is primarily determined by the kinetics of the receptorchannel complex rather than hydrolysis or diffusion of transmitter away from the postsynaptic receptors.

To gain insight into the role of F-actin in the organization of synaptic vesicles at release sites, we examined the synaptic distribution of F-actin by using a unique synaptic preparation of frog target-deprived nerve terminals. In this preparation, imaging of the synaptic site was unobstructed by the muscle fiber cytoskeleton, allowing for the examination of hundreds of synaptic sites in their entirety in whole mounts. At target-deprived synaptic sites F-actin was distributed in a ladder-like pattern and was colocalized with beta-fodrin. Surprisingly, F-actin stain, which we localized to the nerve terminal itself, did not overlap a synaptic vesicle marker, suggesting that it was concentrated in nonrelease domains of nerve terminals between clusters of synaptic vesicles. These findings suggest that the majority of the presynaptic F-actin is not involved in tethering synaptic vesicles. Instead, the strategic presynaptic positioning of this cytoskeletal meshwork in nonrelease domains of the nerve terminal suggests alternate functions such as restricting synaptic vesicles to release domains, recycling synaptic vesicles, or stabilizing the nerve terminal.

Twitch and tonic muscle fibers of snake skeletal muscle differ in their synpatic as well as mechanical properties. These experiments were aimed at detemining the basis of the difference in vesicular release properties of nerve terminals at twitch and tonic endplates. Miniature endplate currents (MEPCs) were recorded from voltage-clamped garter snake muscle fibers depolarized by high K+ in either a control Ca2+ or high-Ca2+ solution. MEPC frequency increased at twitch and tonic endplates and remained elevated for 8 h during depolarization in control Ca2+. At twitch endplates depolarized in the presence of high Ca2+, an increase in MEPC frequency was followed by a progressive decline. In contrast, MEPC frequency remained elevated in high Ca2+ at tonic endplates. The observed decrease in MEPC frequency at depolarized twitch endplates in high Ca2+ was not a function of the level of depolarization or initial MEPC frequency, nor was it due to a reduction in MEPC amplitude and loss of MEPCs in baseline noise. An optical assay of presynaptic function in which the activity-dependent dye FM1-43 was used confirmed that quantal releases differs at twitch and tonic endplates. Most twitch nerve terminals were labeled by FM1-43 during prolonged depolarization with control Ca2+ or after brief depolarization with high Ca2+. In contrast, the number of twitch nerve terminals and the degree to which they were stained was greatly reduced after prolonged exposure to high K+ and high Ca2+, whereas depolarized tonic endplates were well stained by FM1-43 during brief and prolonged exposure to high Ca2+. FM1-43 staining also revealed variable levels of quantal release between individual boutons at twitch endplates after prolonged depolarization in high-Ca2+ solution. The observed reduction in presynaptic function at twitch nerve terminals after prolonged depolarization in high-Ca2+ solution was reversible and therefore not due to irreversible damage to terminal boutons. MEPC frequency increased at both twitch and tonic endplates when either Sr2+ or Ba2+ was substituted for high Ca2+ during K(+)-induced depolarization. Over time, in Sr2+ or Ba2+ solutions, MEPC frequency remained elevated at tonic endplates but declined at twitch endplates with a time course similar to that observed in high Ca2+. MEPC amplitudes at both endplates remained constant. We conclude that the regulation of quantal release differs in nerve terminals innervating twitch and tonic endplates and postulate that differential intraterminal accumulation of Ca2+ may underlie the observed difference in presynaptic function.