Embryonic Acetylcholine Receptor Research Articles

Altered active zones, vesicle pools, nerve terminal conductivity, and morphology during experimental MuSK myasthenia gravis.

Recent studies demonstrate reduced motor-nerve function during autoimmune muscle-specific tyrosine kinase (MuSK) myasthenia gravis (MG). To further understand the basis of motor-nerve dysfunction during MuSK-MG, we immunized female C57/B6 mice with purified rat MuSK ectodomain. Nerve-muscle preparations were dissected and neuromuscular junctions (NMJs) studied electrophysiologically, morphologically, and biochemically. While all mice produced antibodies to MuSK, only 40% developed respiratory muscle weakness. In vitro study of respiratory nerve-muscle preparations isolated from these affected mice revealed that 78% of NMJs produced endplate currents (EPCs) with significantly reduced quantal content, although potentiation and depression at 50 Hz remained qualitatively normal. EPC and mEPC amplitude variability indicated significantly reduced number of vesicle-release sites (active zones) and reduced probability of vesicle release. The readily releasable vesicle pool size and the frequency of large amplitude mEPCs also declined. The remaining NMJs had intermittent (4%) or complete (18%) failure of neurotransmitter release in response to 50 Hz nerve stimulation, presumably due to blocked action potential entry into the nerve terminal, which may arise from nerve terminal swelling and thinning. Since MuSK-MG-affected muscles do not express the AChR γ subunit, the observed prolongation of EPC decay time was not due to inactivity-induced expression of embryonic acetylcholine receptor, but rather to reduced catalytic activity of acetylcholinesterase. Muscle protein levels of MuSK did not change. These findings provide novel insight into the pathophysiology of autoimmune MuSK-MG.

Acetylcholinesterase deficiency contributes to neuromuscular junction dysfunction in type 1 diabetic neuropathy

Diabetic neuropathy is associated with functional and morphological changes of the neuromuscular junction (NMJ) associated with muscle weakness. This study examines the effect of type 1 diabetes on NMJ function. Swiss Webster mice were made diabetic with three interdaily ip injections of streptozotocin (STZ). Mice were severely hyperglycemic within 7 days after the STZ treatment began. Whereas performance of mice on a rotating rod remained normal, the twitch tension response of the isolated extensor digitorum longus to nerve stimulation was reduced significantly at 4 wk after the onset of STZ-induced hyperglycemia. This mechanical alteration was associated with increased amplitude and prolonged duration of miniature end-plate currents (mEPCs). Prolongation of mEPCs was not due to expression of the embryonic acetylcholine receptor but to reduced muscle expression of acetylcholine esterase (AChE). Greater sensitivity of mEPC decay time to the selective butyrylcholinesterase (BChE) inhibitor PEC suggests that muscle attempts to compensate for reduced AChE levels by increasing expression of BChE. These alterations of AChE are attributed to STZ-induced hyperglycemia since similar mEPC prolongation and reduced AChE expression were found for db/db mice. The reduction of muscle end-plate AChE activity early during the onset of STZ-induced hyperglycemia may contribute to endplate pathology and subsequent muscle weakness during diabetes.

Time Lapse in Vivo Visualization of Developmental Stabilization of Synaptic Receptors at Neuromuscular Junctions

The lifetime of nicotinic acetylcholine receptors (AChRs) in neuromuscular junctions (NMJs) is increased from <1 day to >1 week during early postnatal development. However, the exact timing of AChR stabilization is not known, and its correlation to the concurrent embryonic to adult AChR channel conversion, NMJ remodeling, and neuromuscular diseases is unclear. Using a novel time lapse in vivo imaging technology we show that replacement of the entire receptor population of an individual NMJ occurs end plate-specifically within hours. This makes it possible to follow directly in live animals changing stabilities of end plate receptors. In three different, genetically modified mouse models we demonstrate that the metabolic half-life values of synaptic AChRs increase from a few hours to several days after postnatal day 6. Developmental stabilization is independent of receptor subtype and apparently regulated by an intrinsic muscle-specific maturation program. Myosin Va, an F-actin-dependent motor protein, is also accumulated synaptically during postnatal development and thus could mediate the stabilization of end plate AChR.

Binding sites contribute unequally to the gating of mouse nicotinic alpha D200N acetylcholine receptors.

1. Single channel currents were recorded from HEK 293 cells expressing recombinant mouse adult (alpha 2 beta delta gamma) acetylcholine receptors (AChRs) containing a mutation at residue D200 of the alpha-subunit. Rate and equilibrium constants for AChR activation were estimated from open and closed time obtained over a range of ACh concentrations. 2. Mutation of alpha D200 to asparagine (alpha D200N) dramatically slows the rate constant of channel opening, with adult AChRs slowing 100-fold and embryonic AChRs slowing 400-fold. the rate constant of channel closing increased 3-fold, resulting in a decrease of the gating equilibrium constant of up to 1200-fold. In contrast to channel gating steps, ACh-binding steps are only modestly effected by alpha D200N. 3. Introduction of a potential glycosylation site in alpha D200N cannot account for the effect on channel gating because eliminating the consensus for glycosylation with the mutation alpha D200N + T202V fails to restore efficient gating. Gating is similarly impaired with the substitutions of E, K and Q at position alpha 200. 4. the agonists carbamylcholine and tetramethylammonium also activate the alpha D200N AChR, but with channel opening rates even slower than with ACh. The agonist dependence of the opening rate constant is similar in alpha D200N and wild type AChRs. 5. AChRs containing D200N at just one of the two alpha-subunits show either small or large changes in the gating equilibrium constant, presumably due to the presence of the mutation at either the alpha delta or alpha epsilon/alpha gamma sites. The changes in free energy of channel gating show that the contribution of each binding site is nearly independent. However, the sites do not contribute equally to gating, as an alpha D200N mutation at the alpha epsilon or alpha gamma binding site slows channel opening relatively more than at the alpha delta site.

Nicotinic acetylcholine receptor channels are influenced by the physical state of their membrane environment

We investigated the effect of the physical state of the cell membrane on the activity of the nicotinic acetylcholine receptor (AChR) in various clonal cell lines transfected with the cDNAs of embryonic or adult AChR by measuring single-channel properties and some membrane physicochemical properties as a function of temperature. Unitary conductance and channel closing rate, alpha, had Q(10) values of 1.2 and 2.2, respectively. Using Eyring's transition state theory, it was calculated that both embryonic and adult-type AChR had relatively low thermal sensitivity of ionic conductance and activation energy (E(a) of 3.0-5.0 kcal-mol(-1) at 20 degrees C), indicating that once the AChR channel opens, ion movement is dominated by diffusional processes. Channel closure exhibited higher energy requirements, with E(a) values of about 13 kcal-mol(-1). This process appears to be more endothermic (higher delta H(a) values) than ion permeation, and it is plausible that the energy acquired by the system can be used in the maintenance of its degree of order, as revealed by the delta S(a) 0 calculated for channel closure. The influence of the membrane environment on AChR function is reinforced by the observation that the conductance of the same, embryonic-type AChR protein, expressed in qualitatively different cellular lipid environments, appeared to have different energetic requirements. A correlation between the electrophysiological and thermodynamic parameters of the AChR and physicochemical properties of the membrane bilayer in which the protein is embedded could be established using measurements of the so-called generalized polarization (GP) of the lipophilic probe laurdan. Both embryonic and adult AChR exhibited a higher GP and a higher sensitivity to temperature-dependent changes in GP when heterologously expressed in stable form in Chinese hamster ovary (CHO)-derived cells than did the native embryonic AChR in BC3H-1 cells, indicating that these two properties are determined by the host membrane and are not inherent properties of the AChR type. In addition, the differences in the macroscopic physical states of the lipids and membrane-associated solvent (water) dipolar relaxation between BC3H-1 and CHO-derived cells indicated by the spectroscopic properties of laurdan suggest that both lipid and associated water may influence the microscopic activity of individual AChR molecules embedded in the lipid bilayer. Finally, the different dependence of AChR channel conductance and mean open time as a function of GP observed between the different AChR subtypes in clonal cell lines suggests the importance of specific lipid-protein interactions in addition to bulk membrane properties.

MHC Class II Presentation of Human Acetylcholine Receptor inMyasthenia gravis: Binding of Synthetic Gamma Subunit Sequences to DR Molecules

Embryonic muscle acetylcholine receptor (AChR) is composed of four subunits-alpha, beta, gamma, and delta. Upon innervation, the gamma subunit is substituted by a homologous epsilon subunit. Embryonic muscle AChR might be the original autoantigen in Myasthenia gravis (MG) because it is expressed in the thymus, where the anti-AChR response might start, as well as in adult extrinsic ocular muscles, which are a preferential target in MG. MG patients have antibodies specific for embryonic AChR and CD4+ T cells, which recognize epitopes on the gamma subunit, in association with DR molecules. In the present study, we investigated the binding to several purified DR molecules (DRB1*0101, DRB1*0201, DRB1*0401 and DRB1*0701) of overlapping synthetic peptides, screening the human gamma subunit sequence. Binding of the peptides to the DR molecules were determined from their ability to compete with radiolabelled peptide probes for binding to purified DR molecules. All peptides which were recognized by CD4+ cells bound to the relevant DR molecule. On the other hand, some AChR peptides not recognized by CD4+ cells of MG patients bound well to one or more DR molecules. In terms of relative ability to bind to the DR molecules tested, the gamma subunit behaved like an exogenous antigen: some AChR peptide sequences uniquely bound one DR molecule, others bound several DR alleles, while others did not bind any of the DR molecules tested.

Differential influence of electrical blocking agents on embryonic acetylcholine receptor mRNA levels in long-term cultures of aneural mammalian myotubes.

The influence of spontaneous muscle activity on acetylcholine receptor (AChR) expression was examined by exposing long-term cultures of mammalian myotubes to two pharmacological agents that have similar effects on the rate of spontaneous contractile activity but pharmacologically distinct actions on voltage gated Na+ channels. Previous studies by other investigators have shown that tetrodotoxin upregulates and that veratridine downregulates surface AChR expression in short-term mammalian muscle cultures. In order to determine whether these drugs have disparate actions on AChR mRNA levels, myotubes were exposed to either tetrodotoxin or veratridine for a period of 10 days, and measurements of the relative levels of embryonic AChR subunit mRNAs (alpha, beta, gamma, delta) were obtained during and following the period of drug exposure. Veratridine produced a substantial decrease (between 33% and 50% reduction), while tetrodotoxin produced a relatively small increase (between 17% and 23%), in each of the AChR subunit mRNAs after 6 days of drug exposure. At 23 days in culture, spontaneously active myotubes exhibited a decrease in the relative levels of each of the AChR subunit mRNAs. Myotubes previously exposed to either veratridine or tetrodotoxin exhibited elevated levels of beta, gamma, and delta AChR subunit mRNAs 6 days after cessation of drug treatment, thus suggesting that a period of muscle inactivity can induce sustained influences on some AChR mRNA levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Asynaptic expression of the adult nicotinic acetylcholine receptor in long-term cultures of mammalian myotubes

To determine whether synaptic contact is required to express adult-type nicotinic acetylcholine receptors (A-AChR) in developing mammalian muscle, we have examined single-channel AChR activity in primary muscle cultures maintained for up to 29 days. A-AChRs were first expressed after day 12 in culture (CD12), during a period characterized by the accumulation of embryonic acetylcholine receptors (E-AChR). The highest rate of A-AChR expression was observed between CD15 and 19, during a period of maximal E-AChR accumulation. Although the level of A-AChR expression between individual patches was quite variable during this period, A-AChRs accounted for up to 40% of the events produced by receptors expressed over a 3-day interval. Between CD19 and 29, the density of E-AChRs diminished while the expression of A-AChRs per patch continued to increase but at a lower rate than that observed between CD15 and 19. In 25–29-day cultures. 70.6% of patches exhibited both E-AChR and A-AChR activity, and the percentage of A-AChR events per patch ranged between 0 and 47% with a mean of 11.7 ± 3.2%. These results demonstrate that endogenous muscle mechanisms promote developmental increases in the expression of A-AChRs in myotubes that have no history of synaptic contact. This conclusion suggests that synaptic imprinting at developing junctions is mediated in part by endogenous muscle mechanisms, and does not require direct neurotrophic activation of ϵ mRNA transcription.

Myasthenia gravis. CD4+ T epitopes on the embryonic gamma subunit of human muscle acetylcholine receptor.

In myasthenia gravis (MG) an autoimmune response against muscle acetylcholine receptor (AChR) occurs. Embryonic muscle AChR contains a gamma subunit, substituted in adult muscle by a homologous epsilon subunit. Antibodies and CD4+ cells specific for embryonic AChR have been demonstrated in MG patients. We identified sequence segments of the human gamma subunit forming epitopes recognized by four embryonic AChR-specific CD4+ T cell lines, propagated from MG patients' blood by stimulation with synthetic peptides corresponding to the human gamma subunit sequence. Each line had an individual epitope repertoire, but two 20-residue sequence regions were recognized by three lines of different HLA haplotype. Most T epitope sequences were highly diverged between the gamma and the other AChR subunits, confirming the specificity of the T cells for embryonic AChR. These T cells may have been sensitized against AChR expressed by a tissue other than innervated skeletal muscle, possibly the thymus, which expresses an embryonic muscle AChR-like protein, containing a gamma subunit. Several sequence segments forming T epitopes are similar to regions of microbial and/or mammalian proteins unrelated to the AChR. These findings are consistent with the possibility that T cell cross-reactivity between unrelated proteins ("molecular mimicry"), proposed as a cause of autoimmune responses, is not a rare event.

T cells in myasthenia gravis specific for embryonic acetylcholine receptor

In myasthenia gravis (MG) there is an autoimmune response against muscle acetylcholine receptor (AChR). Embryonic and adult muscles express different AChRs; embryonic AChR contains a gamma subunit, instead of the homologous epsilon subunit that contributes to form adult AChR. We report propagation from the blood of MG patients of T helper (TH) cell lines specific for human embryonic AChR, by cycles of stimulation with a pool of synthetic peptides corresponding to the complete sequence of the gamma subunit (gamma pool). The TH lines strongly recognized AChR from embryonic mammalian muscle, and reacted less or not at all with adult muscle AChR. The existence of TH cells specific for embryonic AChR strongly suggests that the primary anti-AChR sensitization in MG occurs in a tissue other than the innervated skeletal muscle. This may be within the thymus, which expresses an AChR similar or identical to embryonic muscle AChR.

Functional adult acetylcholine receptor develops independently of motor innervation in Sol 8 mouse muscle cell line.

We have defined culture conditions, using a feeder layer of cells from the embryonic mesenchymal cell line, 10T1/2 and a serum-free medium, which allow cells from the mouse myogenic cell line Sol 8 to form contracting myotubes for two weeks. Under these culture conditions, Sol 8 myotubes undergo a maturation process characterized by a sequential expression of two phenotypes. An early phenotype is typified by the expression of the nicotinic acetylcholine receptor (AChR) gamma-subunit transcripts and the presence of low conductance ACh-activated channels, typical of embryonic AChR. A late phenotype is characterized by the expression of AChR epsilon-subunit transcripts, the decreased accumulation of gamma-subunit transcripts and the appearance of high conductance ACh-activated channels, typical of adult AChR. These results indicate that the expression of functional adult type AChR does not require the presence of the motor nerve and therefore represents an intrinsic feature of the Sol 8 muscle cells. Chronic exposure of the cells to the voltage-sensitive Na+ channel blocking agent tetrodotoxin does not affect the appearance of the AChR epsilon-subunit transcripts but prevents the reduction of the steady-state level of the AChR gamma-subunit transcripts and yields a reduced proportion of the adult type channels. Thus, activity seems to facilitate the switch from the embryonic to the adult phenotype of the AChR protein. The Sol 8 cell system might be useful to analyse further the genetic and epigenetic regulation of muscle fibre maturation in mammals.

Differences between embryonic and adult Torpedo acetylcholine receptor γ subunit

Antibodies to a synthetic peptide corresponding to residues 346–359 of the Torpedo acetylcholine receptor (AChR) γ subunit, were employed to compare the adult and embryonic receptor. This peptide contains a consensus phosphorylation site for cAMP-dependent protein kinase (PKA). The anti-peptide antibodies discriminated between adult and embryonic AChRs, and reacted preferentially with the adult γ form. These observed immunological differences did not seem to stem from different phosphorylation states. Our results suggest that the embryonic Torpedo AChR may have a γ-like subunit that differs from the known adult form of this subunit, at least in the specific region that contains the phosphorylation site for PKA.

Properties of embryonic and adult muscle acetylcholine receptors transiently expressed in COS cells

We used transient transfection in COS cells to compare the properties of mouse muscle acetylcholine receptors (AChRs) containing α, β, S, and either γ or ε subunits. γ- and ε-AChRs had identical association rates for binding 125I-a-bungarotoxin, and identical curves for inhibition of toxin binding by d-tubocurarine, but ε-AChRs had a significantly longer half-time of turnover in the membrane than γ-AChRs. A myasthenic serum specific for the embryonic form of the AChR reduced toxin binding to γ-, but not ε-AChRs. The γ-AChRs had channel characteristics of embryonic AChRs, whereas the major class of ε-AChR channels had the characteristics of adult AChRs. Two minor channel classes with smaller conductances were also seen with ε AChR. Thus, some, but not all, of the differences between AChRs at adult endplates and those in the extrasynaptic membrane can be explained by the difference in subunit composition of γ- and ε-AChRs.

Expression of functional mouse muscle acetylcholine receptors in Chinese hamster ovary cells

CHO cells were transfected with cDNAs for all 4 subunits of the mouse muscle acetylcholine receptor (AChR) to obtain a stable cell line (CHO-AR) expressing the AChR on its surface. Immunoprecipitation experiments established that the AChR contained alpha- and beta-subunits assembled with gamma- and/or delta-subunits. In addition, one of the toxin-binding sites of the AChR was blocked by a myasthenic serum that specifically recognizes AChR containing the gamma-subunit. AChR from the CHO-AR cells had the same sedimentation rate, association rate constant for the binding of alpha-bungarotoxin (alpha BTX), and the same metabolic half-life as the AChR in myotubes of the mouse muscle cell line C2C12. Electrophysiological assay of CHO-AR cells by single-channel recording showed the presence of ACh-responsive ion channels with the characteristics of the embryonic AChR (gamma = 40 pS, tau = 5.6 msec). In some patches a smaller conductance channel was also seen that may represent partially assembled receptor. Fluorescence microscopy of fixed, permeabilized cells stained with rhodamine-alpha-BTX demonstrated both perinuclear and diffuse surface staining. The expression of fully assembled, functional mammalian muscle AChR in nonmuscle cells will allow detailed investigation of its properties and interactions with other cellular components.

Embryonic and adult acetylcholine receptors: molecular basis of developmental changes in ion channel properties

Acetylcholine receptors (AChRs) undergo significant changes in their distribution and properties during nerve—muscle synapse formation. AChRs on newly formed skeletal myotubes are diffusely distributed and rapidly degraded, but AChRs at mature endplates are highly localized and metabolically stable. Embryonic and adult AChRs also differ in their ionic channels. When activated by ACh, embryonic AChR channels conduct less current but remain open several times longer than mature AChR channels. Although the mechanisms behind the changes in AChR distribution and degradation rate remain to be determined, a recent paper has elucidated the molecular basis for developmental changes in AChR channel properties in bovine muscle. Beginning in the last months of gestation, fetal calf muscle expresses a new type of AChR subunit (ϵ) that is used in place of its embryonic counterpart (γ). Functional tests using a Xenopus oocyte expression system and the appropriate mRNAs have shown that this subunit substitution can account for all the known differences between embryonic and adult AChR channel properties in bovine muscle. These data suggest that developmental changes in AChR channel properties are a result of nerve-induced changes in AChR gene expression.

Characterization and distribution of acetylcholine receptors and acetylcholinesterase during electric organ development in Torpedo marmorata

The changes that occur in the distribution and properties of the nicotinic acetylcholine receptor and acetylcholinesterase during the development of the electric organ of Torpedo marmorata have been investigated. At early stages of development, both proteins are distributed diffusely over the myotube surface and with differentiation of the myotubes into electrocytes, they become increasingly restricted to the ventral cell surface. This process occurs before axons contact the electrocytes. The concentrations of the acetylcholine receptor and acetylcholinesterase remain at rather low and stable levels during these developmental changes. The acetylcholine receptor concentration begins to increase rapidly as soon as electromotor axons begin to contact the electrocytes. No significant differences in the subunit composition, affinity for d-tubocurarine, isoelectric point or immunochemical properties of the embryonic acetylcholine receptors were detected when they were compared to the receptors of the adult electric organ. The onset of receptor accumulation occurs before the increase in the amount of 17 S acetylcholinesterase, suggesting that increases in acetylcholine receptor and in acetylcholinesterase are not regulated by the same mechanisms. The various molecular forms of acetylcholinesterase undergo characteristic changes during development. Sequential extraction of the esterase forms indicates that their interaction with cellular compartments changes during development. The solubility properties of the esterase forms suggest that most of the 17 S and 13 S acetylcholinesterase become strongly associated with cellular components via ionic interactions and that a hydrophobic 6 S form begins to accumulate at later embryonic stages when the number of mature presynaptic terminals is beginning to show its rapid phase of increase. The results show that events concerning the distribution and some properties of essential components of the electromotor synapse occur during early embryonic development before synaptogenesis, whereas the sequential accumulation of acetylcholine receptor and esterase begins after the axons start contacting the electrocytes. This suggests that the ingrowing nerves exert some regulative influence on the metabolic state of the electrocytes during development.

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.

Nonjunctional acetylcholine receptor channel open time decreases during development of Xenopus muscle.

The mean channel open time of junctional acetylcholine receptors (AChRs) decreases during development of mammalian skeletal neuromuscular junctions1–3. The junctional AChRs of newborn rats have long mean channel open times of about 4.5 ms and in this respect are similar to nonjunctional AChRs of adult denervated muscle3–5. Mature neuromuscular junctions have channels with shorter mean open times of about 1 ms, whereas junctions at intermediate stages seem to have a mixture of channels with short and long open times1–3. These observations, taken together with findings on receptor distribution during development6,14, suggest that the establishment of nerve–muscle contact induces an aggregation of embryonic AChRs with long mean channel open times at the point of contact and that these embryonic junctional receptors are then replaced, modified or subjected to local membrane changes leading to a discrete reduction in channel open time. However, the fate of those embryonic nonjunctional receptors which are not aggregated at the newly formed neuromuscular junction is unknown. Are they an unchanging population, or do the changes in kinetic behaviour which occur at the junctional regions also take place at other regions of the muscle membrane? The kinetics of nonjunctional receptors in mammalian skeletal muscle have not been studied during development. We have now, however, examined this question in amphibian myotomal muscle, where nonjunctional receptors are present in substantial numbers throughout development, making it possible to measure the mean open time of both classes of AChR channel. We observed a shortening of mean channel open time of junctional AChRs, from about 3 ms to 1 ms, and an identical change in mean channel open time of AChRs at nonjunctional regions of membrane.

Effects of deformation on inter-impurity recombination in Ge

To determine whether synaptic contact is required to express adult-type nicotinic acetylcholine receptors (A-AChR) in developing mammalian muscle, we have examined single-channel AChR activity in primary muscle cultures maintained for up to 29 days. A-AChRs were first expressed after day 12 in culture (CD12), during a period characterized by the accumulation of embryonic acetylcholine receptors (E-AChR). The highest rate of A-AChR expression was observed between CD15 and 19, during a period of maximal E-AChR accumulation. Although the level of A-AChR expression between individual patches was quite variable during this period, A-AChRs accounted for up to 40% of the events produced by receptors expressed over a 3-day interval. Between CD19 and 29, the density of E-AChRs diminished while the expression of A-AChRs per patch continued to increase but at a lower rate than that observed between CD15 and 19. In 25–29-day cultures. 70.6% of patches exhibited both E-AChR and A-AChR activity, and the percentage of A-AChR events per patch ranged between 0 and 47% with a mean of 11.7 ± 3.2%. These results demonstrate that endogenous muscle mechanisms promote developmental increases in the expression of A-AChRs in myotubes that have no history of synaptic contact. This conclusion suggests that synaptic imprinting at developing junctions is mediated in part by endogenous muscle mechanisms, and does not require direct neurotrophic activation of ϵ mRNA transcription.