Nerve-muscle Contact Research Articles

Time course of the regeneration of the endplate zone after autologous muscle transplantation.

Reinnervation of transplanted muscles occurs in 2 steps. During the first 5 months the motor axon terminals innervate primarily the border of the original endplate zone, re-establishing its previous outline. In the next 4 months, by further ramifying of the axons, new nerve-muscle contacts are formed exclusively within the boundaries of this zone.

Localization of acetylcholine receptors and cholinesterase on nerve- contacted and noncontacted muscle cells grown in the presence of agents that block action potentials

The possible role of nerve activity in triggering changes in the localization of acetylcholine receptors (AChRs) and cholinesterase (ChE) on nerve-contacted Xenopus muscle cells has been assessed. The localization of these molecules was examined on nerve-contacted and noncontacted muscle cells in cultures of spinal cord and myotomal muscle derived from Xenopus embryos. Sites of high AChR density were revealed by staining with fluorescent alpha-bungarotoxin and sites of ChE localization were revealed histochemically. Localization of AChRs and ChE at sites of nerve-muscle contact occurred when the culture medium contained 1.2 micron tetrodotoxin (TTX), 1.2 micron TTX, 10 mM magnesium, and no calcium salts, 1.2 micron TTX and 2 mM manganese, or 106 mM potassium methyl sulfate instead of sodium chloride. The nerve-contacted muscle cells in each of these modified culture media also exhibited a reduced incidence of AChR and ChE patches away from the site of contact. It is concluded that the neural factor(s) that triggers the local and remote changes in AChR and ChE distribution can be supplied to the neurites and externalized in the absence of nerve impulses, and that the nerve and muscle cells can interact even when they are largely depolarized.

Spontaneous release of transmitter from the growth cones of Xenopus neurons in vitro: The influence of Ca 2+ and Mg 2+ ions

To determine whether spontaneous release of transmitter from the growth cones of neurons exhibits properties similar to the spontaneous release which occurs from the neurons at the neuromuscular junction, release of transmitter from the growth cones of Xenopus neurons in culture was monitored in salines containing varying calcium and magnesium concentrations. Release was monitored by use of an outside-out piece of muscle membrane attached to a patch clamp electrode. Spontaneous release of transmitter from the growth cones in standard saline (2 m M CaCl 2, 1 m M MgCl 2) produces clusters of single-channel openings in the muscle membrane. Clusters are seen to consist of two types: a series of high-frequency channel openings, called “bursts,” and clusters of low-frequency channel openings called “singles.” The bursts were identified and examined for their possible relationship to MEPP-producing release, and the singles were identified and examined for their possible relationship to “leak” release of the neuromuscular junction. When the external saline contains high calcium (10 m M CaCl 2, 1 m M MgCl 2) or high magnesium (2 m M CaCl 2, 9 m M MgCl 2), the frequencies of both “bursts” and “singles” was greatly reduced. This reduction in release persists if the neurons are grown in the high-calcium or high-magnesium solutions. When the saline is a low-calcium solution (0 m M CaCl 2, 3 m M MgCl 2) the growth cones release transmitter at rates similar to those from standard saline. These results indicate that although the spontaneous release from the growth cone shares one characteristic with the leak release, neither the burst nor the singles release from the growth cones share exact relationship with either the MEPP producing release or the leak release. This suggests that further development of the mechanisms for spontaneous release of neurotransmitter occurs after nerve-muscle contact.

The expression and localization of synaptic vesicle antigens at neuromuscular junctions in vitro

To examine the biochemical differentiation of presynaptic nerve terminals in vitro, we studied the expression and localization at synapses of two synaptic vesicle-specific antigens, synapsin I and protein p65. We purified these proteins from brain and raised a rabbit antiserum against each of them. Chick synapsin I had a slightly smaller molecular weight than its mammalian homologue, although the two share several properties. With the anti-p65 serum, the protein p65 could be detected consistently in presynaptic terminals at the neuromuscular junction in vivo, as had been previously shown for synapsin I. These antisera proved sufficiently sensitive to allow a study of the developmental expression of the antigens in embryonic chick brain, using protein blots. The two antigens are not coordinately regulated; protein p65 was detected substantially sooner in development that was synapsin I. Both synapsin I and protein p65 are expressed by ciliary ganglion neurons in vitro, as assessed by immunofluorescence using the affinity-purified antisera. The two antigens co-localized at all times in culture. Neurons grown alone were reliably stained only after 4 to 5 days in culture, but comparable levels of staining were found after 1 day when neurons were co-cultured with embryonic myotubes. In the co-cultures, staining was initially high in growth cones and neurites, but the brightest staining became confined to sites of nerve-muscle contact over 4 to 5 days in culture. In mature cultures, patches of bright staining for the vesicle antigens coincided with patches of acetylcholine receptors, suggesting that the antigens had become localized at synapses. The time course of this localization process suggests that it corresponds to the morphological maturation of synapses. It should be possible to exploit this system to obtain information about the molecules and processes involved in the induction of presynaptic differentiation.

On the mechanism of acetylcholine receptor accumulation at newly formed synapses on chick myotubes

We have examined the specificity and the mechanism of acetylcholine receptor (AChR) accumulation at embryonic chick nerve-muscle contacts that form in culture. Spinal cord motoneurons were identified in vitro after labeling them in vivo with Lucifer Yellow-wheat germ agglutinin conjugates. All of their processes induced receptor clusters on contacted myotubes; after 24 to 48 hr of co-culture, the incidence of neurite-associated receptor patches (NARPs) was approximately 1.2/100 microns of contact. In contrast, NARPs were rarely associated with spinal cord interneurons (approximately 0.1/100 microns of contact). Neurons dissociated from ciliary ganglia induce NARPS to the same extent as motoneurons. The relative contribution to NARPs of AChRs present in the membrane prior to plating ciliary ganglion neurons and of "new" AChRs inserted 8, 11, or 17 hr after addition of neurons was assessed with two fluorescent receptor probes. Rhodamine-conjugated alpha-bungarotoxin was used to label either old or new receptors; a monoclonal, anti-receptor antibody visualized with fluorescein-second antibody was used to label all (new and old) receptors. Analysis of digitized fluorescence images showed that NARPs contained both new and old receptors but that within the first 24 hr of co-culture the majority (60 to 80%) were new. We estimate that cholinergic neurites increase the rate of receptor insertion 4- to 5-fold during the first 8 hr of NARP formation. The contribution of new receptors to NARPs declines with time. After 3 days of co-culture, receptors inserted over an 8-hr interval comprised only 20% of the total NARP complement.(ABSTRACT TRUNCATED AT 250 WORDS)

N-CAM at the vertebrate neuromuscular junction.

We have detected the neural cell adhesion molecule, N-CAM, at nerve-muscle contacts in the developing and adult mouse diaphragm. Whereas we found N-CAM staining with fluorescent antibodies consistently to overlap with the pattern of alpha-bungarotoxin staining at nerve-muscle contacts both during development and in the adult, we observed N-CAM staining on the surfaces of developing myofibers and at much lower levels on adult myofibers. Consistent with its function, N-CAM was also detected on axons and axon terminals. Immunoblotting experiments with anti-N-CAM antibodies on detergent extracts of embryonic (E) diaphragm muscle revealed a polydisperse polysialylated N-CAM polypeptide, which in the adult (A) was converted to a discrete form of Mr 140,000; this change, called E-to-A conversion, was previously found to occur in different neural tissues at different rates. The Mr 140,000 component was not recognized by monoclonal antibody anti-N-CAM No. 5, which specifically recognizes antigenic determinants associated with N-linked oligosaccharide determinants on N-CAM from neural tissue. The relative concentration of the Mr 140,000 component prepared from diaphragm muscle increased during fetal development and then decreased sharply to reach adult values. Nevertheless, expression of N-CAM in muscle could be induced after denervation: one week after the sciatic nerve was severed, the relative amount of N-CAM increased dramatically as detected by immunoblots of extracts of whole muscle. Immunofluorescent staining confirmed that there was an increase in N-CAM, both in the cell and at the cell surface; at the same time, however, staining at the motor endplate was diminished. Our findings indicate that, in muscle, in addition to chemical modulation, cell-surface modulation of N-CAM occurs both in amount and distribution during embryogenesis and in response to denervation.

Acetylcholine receptors and acetylcholinesterase accumulate at the nerve-muscle contacts of de novo grown human monolayer muscle cocultured with fetal rat spinal cord

Adult human biopsied muscle grown de novo from myoblasts in monolayer was cocultured with spinal cord explants of 13- to 14-day-old rat embryos. Cocultures were established 15 days after myoblast fusion. Autoradiography of 125I-α-bungarotoxin and acetylcholinesterase staining were carried out on 27- to 56-day-old muscle-cord cocultures. Large clusters of 125I-α-bungarotoxin, indicating clusters of acetylcholine receptors, were present at nerve-muscle contacts but not elsewhere on the muscle fibers. Accumulation of acetylcholinesterase was also present at nerve-muscle contacts. This study demonstrates that monolayer cultured adult human muscle can be innervated by embryonic rat spinal cord neurons and thus there is no requirement for an original basal lamina tube as it is present when organ-cultured human muscle is innervated.

Influence of trophic substances in the regulation of resting membrane potential and ionic concentration in skeletal muscle

We studied the ionic and water content and the resting membrane potential of rat extensor digitorum longus EDL muscles at different times after unilateral nerve crush. Intracellular potassium concentration decreased progressively during the 1st week after nerve crush whereas intracellular sodium concentration increased significantly. At about day 10, when functional reinnervation (presence of end-plate potentials and miniature end-plate potentials) was detected, the above changes tended to return to control values. In addition, there was a significant difference between muscles with long and short nerve stumps. These results suggest a neurogenic dependency of muscle hydroelectrolytic composition. The decrease in resting membrane potential was greatest after 6.5 days of denervation when changes in the internal ionic concentration were maximum; however, these ionic changes contributed little to the decrease. The recovery of the resting membrane potential commenced at least 48 h before the first signs of functional reinnervation (10th day). This finding suggested an important contribution of some neurotrophic material in early stages of the reinnervation when nerve-muscle contacts were already established. Later, the contribution of mechanical activity to the restoration of the RMP became apparent (20th day); fibrillation potentials has disappeared by that time.

Response of nerve growth cone to focal electric currents.

Monopolar electric current pulses were focally applied through a micropipette to the growth cone of Xenopus embryonic neurons in culture. Application of the current directly in front of the growth cone modulated the rate of growth cone extension: Negative (sink) currents increased the growth rate, while positive (source) currents reduced the growth rate. When the currents were applied in a direction perpendicular to the direction of the neurite growth, both negative and positive currents produced inhibitory effects. Application of a negative focal current at a 45 degree angle with respect to the direction of neurite growth resulted in an oriented growth of the neurite toward the current sink. However, after the growth cone had been attracted to the vicinity of a current sink, further extension of the neurite was inhibited. These current effects occur rapidly after the onset of the current application, and are at least partially reversible within 1 hr after the termination of the current. The magnitude of current density required to induce a growth cone response was found to be in the order of a few pA per micron2. Such current density is close to that which may be generated at the muscle cell surface by the acetylcholine molecules released from the growth cone during the early phase of nerve-muscle contact.

Acetylcholine receptor aggregation parallels the deposition of a basal lamina proteoglycan during development of the neuromuscular junction.

To determine the time course of synaptic differentiation, we made successive observations on identified, nerve-contacted muscle cells developing in culture. The cultures had either been stained with fluorescent alpha-bungarotoxin, or were maintained in the presence of a fluorescent monoclonal antibody. These probes are directed at acetylcholine receptors (AChR) and a basal lamina proteoglycan, substances that show nearly congruent surface organizations at the adult neuromuscular junction. In other experiments individual muscle cells developing in culture were selected at different stages of AChR accumulation and examined in the electron microscope after serial sectioning along the entire path of nerve-muscle contact. The results indicate that the nerve-induced formation of AChR aggregates and adjacent plaques of proteoglycan is closely coupled throughout early stages of synapse formation. Developing junctional accumulations of AChR and proteoglycan appeared and grew progressively, throughout a perineural zone that extended along the muscle surface for several micrometers on either side of the nerve process. Unlike junctional AChR accumulations, which disappeared within a day of denervation, both junctional and extrajunctional proteoglycan deposits were stable in size and morphology. Junctional proteoglycan deposits appeared to correspond to discrete ultrastructural plaques of basal lamina, which were initially separated by broad expanses of lamina-free muscle surface. The extent of this basal lamina, and a corresponding thickening of the postsynaptic membrane, also increased during the accumulation of AChR and proteoglycan along the path of nerve contact. Presynaptic differentiation of synaptic vesicle clusters became detectable at the developing neuromuscular junction only after the formation of postsynaptic plaques containing both AChR and proteoglycan. It is concluded that motor nerves induce a gradual formation and growth of AChR aggregates and stable basal lamina proteoglycan deposits on the muscle surface during development of the neuromuscular junction.

Myometrial ultrastructure and innervation in Myotis lucifugus, the little brown bat.

Uteri from hibernating bats, Myotis lucifugus, collected periodically from Renfrew County, Ontario, were fixed in 2% glutaraldehyde and processed for electron microscopy or incubated in glyoxylic acid to show adrenergic nerves by fluorescence. The bat uterus is structurally typical of mammalian species; although the right uterine horn is permanently enlarged in parous bats due to hypertrophy of both myometrium and endometrium. Nerves were abundant between both longitudinal and circular layers of muscle cells. Unmyelinated, and some myelinated, axons, ranging from few to many, coursed generally parallel to the uterine long axis. Numerous axonal varicosities containing small dense-cored (adrenergic) vesicles or, less often, small agranular (cholinergic) vesicles, were found forming close nerve-muscle contacts between myometrial cells and blood vessels. Fluorescent microscopy showed a dense network of adrenergic nerves in parous uteri, but a sparse network in nulliparous uteri. A specific adrenergic nerve marker, 5-hydroxydopamine, greatly increased the density and in some instances, the size of granular vesicles, while 6-hydroxydopamine, which depletes adrenergic neutrotransmitter, reduced the number of dense-cored vesicles. Nulliparous uteri appeared unchanged by six daily injections of 0.1 microgram estradiol-17 beta; 0.25 mg progesterone, or both; but parous uteri were greatly enlarged by all regimes. Nerve ultrastructure, however, appeared unaffected by steroid treatment; nor, despite the absolute dextral bias in implantation, were left-right differences observed. Gap junctions were not found between muscle cells in myometria of any bat uteri. Based on this study, we suggest that M. lucifugus may provide a most useful model for examination of neurogenic regulation of the uterus.

Coated vesicles are associated with acetylcholine receptors at nerve-muscle contacts.

Cholinergic synapses form between ciliary neurons and cultured myotubes. We have identified these synaptic contacts using alpha-bungarotoxin conjugated to horseradish peroxidase (alpha BTX-HRP). The enzymatic reaction product was limited to a small portion of the sarcolemma in direct apposition to the nerve terminal. Multiple neuronal processes contact the region of the myotube containing acetylcholine receptors (AChRs). At the early stages of nerve-muscle contacts neuronal processes protrude into the coated pits of the myoplasm. Numerous coated pits and coated vesicles were found beneath these early contacts. These vesicles may be involved in the transport of protein molecules at the newly formed cholinergic structures.

Acetylcholinesterase is functional in embryonic rat muscle before its accumulation at the sites of nerve-muscle contact

We have examined the expression, the location, and the physiological activity of acetylcholinesterase (AChE) in developing intercostal muscles in the rat. Although focal accumulations of AChE at developing end plates do not appear until Embryonic Day (ED) 16–17, 16 S AChE is present at ED 14. Experiments with permeable and impermeable inhibitors established that prior to focal accumulation most of the 16 S enzyme is on the surface of muscle fibers, where it constitutes the major species. Intracellular recording from developing muscle fibers showed that as early as ED 14, AChE inhibitors prolonged evoked end-plate potentials. We conclude that prior to its focal accumulation, AChE is present on the surface of muscle fibers and is physiologically active. Histochemical staining of the focally accumulated enzyme demonstrated that the enzyme is concentrated both intracellularly and extracellularly at the sites of developing nerve-muscle contacts.

Nerve disperses preexisting acetylcholine receptor clusters prior to induction of receptor accumulation in Xenopus muscle cultures

We have investigated the sequential changes of acetylcholine receptor (AChR) distribution on identified Xenopus laevis muscle cells in culture before and after innervation. AChRs on muscle cells were stained with tetramethylrhodamine-conjugated α-bungarotoxin and the distribution of AChR clusters was examined on a fluorescence microscope using an image intensifier. Large receptor clusters were identified on muscle cells and their fate was followed afterward. In muscle cells cultured without neural tube cells, about one-half of the identified AChR clusters survived for 2 days. In nerve-muscle cocultures, preexisting AChR clusters survived longer on non-nerve-contacted muscle cells than on muscle cells cultured without nerve. However, in nerve-contacted muscle cells the great majority of preexisting AChR clusters dispersed within 2 days. The dispersal of preexisting AChR clusters preceded receptor accumulation along the path of nerve contact by about 12–16 hr. Therefore, an accelerated dispersal of receptor clusters in innervated muscle cells is not a consequence of receptor accumulation along the nerve. The preexisting AChR clusters located near and far from the nerve contact sites dispersed along a similar time course. Protease inhibitors, trasylol and leupeptin, reduced the nerve-induced dispersal of the preexisting AChR clusters in the period before AChR accumulation at the nerve contact sites but did not do so during the period when AChRs began to accumulate at nerve-muscle contact. The significance of the dispersal of preexisting receptor clusters is discussed with regard to neuromuscular junction formation.

Critical periods in rat motoneuron development

Reinnervation of rat internal intercostal muscles was examined 2 weeks after intramuscular axotomy in the period embryonic Day 17 (E17) to postnatal Day 2 (PN2). The efficiency of reinnervation depended on the day of denervation. Responses to nerve stimulation were common only in muscles denervated in the interval E19–E21. Functional reinnervation was seldom seen in muscles denervated earlier, and was absent in muscles denervated shortly after birth. We suggest that there are “critical periods” in the sequence of differentiation of motoneurons. At E17 these neurons are very susceptible to cell death and any that lose contact with their muscle will die. Motoneurons axotomized later, while motor unit size is still increasing, can regenerate some functional nerve-muscle junctions. Motor unit size reaches its maximum by E21 and if motoneurons are exotomized after that time, in neonates, they cannot form more synaptic terminals although they can still extend axons. During this early postnatal period motoneurons normally reduce their number of nerve-muscle contacts. Finally, at 3 weeks of age, motoneurons reach their adult state when they are capable of regeneration relatively unhindered by restrictions on the number and location of their peripheral connections.

Motoneuron death and motor unit size during embryonic development of the rat

Chronic paralysis of rat embryos during the last 4 to 6 prenatal days causes a diminution in skeletal muscle fiber numbers but inhibits motoneuron death. Consequently, as paralyzed muscles develop, an increased number of motoneurons attempts to form synapses at a reduced number of synaptic sites. Paralyzed muscle fibers receive their synapses at a single endplate, as in control muscles, but these endplates are hyperinnervated, with about twice the normal number of inputs. Counts of axons, synaptic inputs, and muscle units showed that motoneurons normally contact a maximum number of muscle fibers shortly before birth, and this number remains stable for several days postnatal until it finally is reduced to the adult number. The average motor unit size in paralyzed embryos at the time of birth was the same as in controls. We suggest that it is not necessary to postulate the existence of competition between embryonic nerve terminals in order to explain regulation of the number of muscle fibers initially contacted by a motoneuron. Motoneuron death was not immediately affected by paralysis, but paralysis "rescued" all motoneurons whose death normally would have occurred 24 hr or more after the time when paralysis was initiated, regardless of when this was. This implies that the peak period for determination to die is during embryonic day 14, when myotube formation is just beginning and no recognizable endplate structures are present in muscles. When paralyzed, motoneurons normally destined to die are capable of forming a normal number of functional nerve-muscle contacts.

Spatial distribution of acetylcholine receptors at developing chick neuromuscular junctions.

The development of high-density clusters of acetylcholine receptors (AChRs) and the relationship of these clusters to nerve contacts on embryonic chick wing muscle fibres has been studied. Fluorescent labelling of AChRs with rhodamine-conjugated alpha-bungarotoxin (R-Bgt) revealed the presence of irregularly shaped AChR clusters in wing buds at 4 1/2-5 days of incubation. This is within a day of when myotubes first appear in the wing bud, and close to the time when functional innervation becomes established. At 10 days of incubation AChR clusters present on muscle cells in anterior and posterior latissimus dorsi appear as round or oval, uniformly labelled plaques. At about the time of hatching, however, these plaques break into numerous smaller clusters. Similar changes in the morphology of AChR clusters have been observed previously in mammalian skeletal muscle during development. Using horseradish peroxidase labelled alpha-bungarotoxin (HRP-Bgt), the relationship between AChR clusters and motor nerve terminals was studied at the ultrastructural level. At all stages of development nerve-muscle contacts were labelled with HRP-Bgt. In wing buds, however, the majority (90%) of labelled clusters observed were not in contact with a motor nerve terminal. The incidence of AChR clusters with axon contacts increased sharply during development such that by 10 days more than 50% and by hatching more than 90% of all sections through labelled AChR clusters contained nerve terminal profiles. At all times studied nerve-contacted receptor clusters were longer (about 5 micron) than non-contacted clusters (about 2 micron).

Neurotrophic control of channel properties at neuromuscular synapses of rat muscle.

1. Ectopic neuromuscular synapses formed when the fibular nerve was implanted into the proximal part of rat soleus muscle and the soleus nerve was cut. The gating properties of acetylcholine (ACh) receptor channels in the newly formed ectopic and in the denervated original end-plates were examined at various stages of ectopic synapse formation.2. At ectopic end-plates the apparent mean open time of ACh receptor channels changes during synaptic development. Channels in immature ectopic end-plates, examined 1 week after cutting the soleus nerve, have apparent mean open times of approximately 4 ms (at -70 mV, 22 degrees C), similar to those of the extrasynaptic ACh receptor channels of completely denervated fibres. The channel gating of mature ectopic end-plates, examined 3-7 weeks after nerve section, is characterized by a mean open time of approximately 1 ms and resembles that found in normal end-plates of adult fibres.3. The conversion of end-plate channel gating occurs during the second and third week of synapse formation. During this period two discrete classes of channels with different gating behaviour are present in the ectopic end-plate.4. Examination of ectopic end-plates in the electron microscope reveals that junctional folds begin to appear in the subsynaptic membrane during the period of channel conversion.5. At the denervated original end-plates of ectopically innervated fibres the apparent mean open time of ACh receptor channels remains similar to that of normally innervated end-plates. Original end-plates retain the normal synaptic class of channel for at least 42 days after denervation. At this time, most of the ACh receptors present originally in the membrane have been replaced by newly inserted receptors.6. At former end-plates of completely denervated fibres ACh activates two classes of channels, even when most of the ACh receptors originally present in the end-plate have been replaced by new receptors.7. The results show that during synapse formation a neurally controlled conversion of ACh receptor channels occurs about 2-3 weeks after establishment of the nerve muscle contact. Thereafter end-plate channel properties are independent of neural influences. These observations are consistent with a mechanism of channel conversion whereby the nerve modifies not the ACh receptor channel itself, but another constituent of the end-plate membrane which determines the gating properties of end-plate channels.

Neural control of embryonic acetylcholine receptor and skeletal muscle.

The manner by which motor neurons exert control over the distribution and number of acetylcholine receptors, and muscle development was investigated in the superior oblique muscle of white Peking duck embryos. Clusters of receptors in the normally developing muscle first appeared on day 10 of incubation as determined with I125 alpha-bungarotoxin autoradiography. The initial appearance of receptor clusters coincided with the arrival of motor nerve fibers in the muscle. Clusters of receptors also appeared in normal fashion in muscles made aneural by destruction of motor neurons on day 7. But after day 14 these clusters had disappeared and no new clusters were seen thereafter in the aneural muscle. Receptor clusters persisted throughout development in muscle in which neuromuscular transmission was blocked with either curare or botulinum toxin and in muscles denervated on day 10.5, i.e., shortly after the initial nerve-muscle contact but prior to the onset of muscle activity. A progressive increase in the total number of receptors and in the total amount of protein occurred during the course of normal development. However, the specific activity of the receptor protein declined sharply following innervation on day 10. The total number of receptors and the specific activity of the receptor was affected depending on whether the motor neurons were destroyed before or after innervation and following chronic blockade of neuromuscular transmission. The half-life of the receptor protein was similar in normal, aneural, and paralyzed muscles (26, 25, 26 h, respectively). Measurements of total protein indicated that essentially no muscle growth occurred in the complete absence of innervation. Paralyzed muscles continued to develop but at a slower pace.

Initial synaptic transmission at the growth cone in Xenopus nerve-muscle cultures.

The excellent visibility of cultured cells allows the early events during formation of the neuromuscular junction to be suitably studied. It has been shown in various culture systems that synaptic transmission occurs early after nerve-muscle contact. Early synaptic potentials are small in amplitude and slow in time course reflecting a low acetylcholine receptor density at the site of nerve contact. Acetylcholine receptors accumulate later at the contact region. We have examined initial synaptic transmission at the growth cone-muscle contact in Xenopus nerve-muscle cultures. The approaching growth cone was observed under a phase-contrast microscope while the membrane potential of its target muscle cell was continuously monitored by using an intracellular microelectrode. The innervating neuron was stimulated extracellularly at the cell body. No synaptic potential was evoked when the growth cone was contacting the muscle only at the tip of filopodia. However, as soon as the main portion of the growth cone contacted the muscle membrane, nerve-evoked synaptic potentials were detected after stimulation of the nerve. This immediate appearance of synaptic potentials raises the possibility that acetylcholine could be released at the growth cone even prior to contact with muscle cells. As the area of contact enlarged during the observation period the amplitude of end-plate potentials also increased. Spontaneous synaptic potentials (miniature end-plate potentials) were rarely observed in these early growth cone-muscle contacts. Although there were several inherent difficulties, quantal analysis of the end-plate potentials was attempted by using binomial statistics. This analysis suggests that nerve-evoked transmitter release at the growth cone occurs in a quantal fashion.