A comparison of nerve transection and chronic application of β-bungarotoxin on acetylcholine receptor distribution and other nerve-muscle properties

β-Bungarotoxin (β-BuTX), a snake venom neurotoxin which acts presynaptically to inhibit acetylcholine (ACh) release at the neuromuscular junction, was applied to the rat phrenic nerve-diaphragm muscle preparation to determine its effectiveness to mimic denervation. The distribution of junctional and extrajunctional ACh receptors on the muscle were assayed biochemically by [¹²⁵I]α-bungarotoxin ([¹²⁵I]α-BuTX) binding and electrophysiologically by iontophoretic application of ACh. Spontaneous transmitter release and muscle membrane potential were measured under conditions of denervation, β-BuTX treatment, and bee venom phospholipase A₂ exposure. Within 7 days after treatment with a single dose (5μg/kg) of enzymatically active β-BuTX, extrajunctional [¹²⁵I]α-BuTX binding increased fivefold, and there was a decrease in miniature end-plate potential (MEPP) frequency and in resting membrane potential (RMP) to values less than those of control muscles but greater than those of denervated muscles. The same dose of enzymatically inactive β-BuTX or snake venom phospholipase A₂ was without effect, but a fivefold greater dose of enzymatically inactive β-BuTX resulted in changes in extrajunctional binding and RMP similar to those of muscles exposed to the enzymatically active toxin. However, unlike muscles treated with active toxin, those treated with inactive toxin had MEPP frequencies similar to control muscles and exhibited contraction elicited by phrenic nerve stimulation. These results taken together indicate that the nerve exerts a trophic influence on muscle independent of ACh and muscle activity.     

On the appearance of acetylcholine receptors in denervated rat diaphragm,and its dependence on nerve stump length

Acetylcholine (ACh) sensitivity and extrajunctional receptor distribution of the rat diaphragm were closely monitored during the early period following denervation. Both contracture in response to 10 μg/ml of ACh and extrajunctional binding of [¹²⁵I]alpha-bungarotoxin ([¹²⁵I]α-BTX) were first detectable 30 h after cutting the phrenic nerve in the thorax. If the nerve were cut more proximally, leaving a 3.5 cm distal nerve stump, the same level of ACh contracture and [¹²⁵I]α-BTX binding did not appear until 40 h after operation. This 10-h delay was far longer than the 3-h delay in transmission failure reportedly dependent on stump length. The earliest detectable extrajunctional [¹²⁵I]α-BTX binding appeared throughout the entire muscle fiber, and was not localized to the endplate region as would be expected if degeneration in the nerve terminal induced new receptors. However, later significant increases in [¹²⁵I]α-BTX binding at the endplate region could have resulted from such degeneration. All these results are consistent with neurotrophic regulation of muscle ACh receptors, working via a mechanism involving axonal transport.     

Uptake of [H]colchicine from silastic implants by mammalian nerves and muscles

The uptake of [³H]colchicine, which had diffused from silastic cuffs placed around the right sciatic nerve of adult rats, by extensor and soleus muscles was compared with amounts taken up into these tissues in animals treated with intraperitoneal injections of the drug. Each cuff contained 120 μg of [³H]colchicine, and animals injected with the drug received either one or two doses of 0.3 mg/kg body weight. About 39% of the [³H]colchicine diffused from the silastic cuff during the first 5 days and became widely distributed in the body; however, extrajunctional sensitivity to ACh, tetrodotoxin-resistant action potentials and membrane depolarization were recorded only in the ipsilateral (drug-cuffed) leg. The electrical and chemosensitive properties of the muscles in the contralateral (sham-cuffed) legs were unaltered. The [³H]colchicine content in that portion of the nerve enclosed by the cuff was 3.67 pmoles/mg wet tissue. This amount was about sixfold greater than that observed in sciatic nerves of animals treated with two intraperitoneal injections of the drug. The concentration of [³H]colchicine in extensor and soleus muscles of animals treated with the intraperitoneal injections was twofold higher than that observed in muscles whose nerves were exposed to silastic cuffs containing the drug. There were no signs of denervation (e.g., appearence of tetrodotoxin-resistant action potentials, membrane depolarization or appearance of extrajunctional sensitivity to ACh) in the muscles of animals injected with colchicine even though levels of the drug were higher in these tissues than that found in animals exposed to the silastic cuffs. Furthermore, simultaneous injection of colchicine intramuscularly had no effect on the level of extrajunctional ACh sensitivity of denervated muscles which were under constant direct stimulation. It is concluded that colchicine cannot produce signs of denervation by a direct action on the muscle membrane.     

Electrophysiologic denervation changes of human muscle fibers in motoneuron diseases

Muscle denervation and reinnervation by sprouting of the surviving motoneurons characterizes motoneuron diseases (MND). In mammalian muscles, experimental denervation induces the appearance of extrajunctional acetylcholine (ACh) receptors and tetrodotoxin (TTX) resistant action potentials (AP). These denervation changes have been investigated in muscle biopsies from 10 MND patients and in 2 traumatically denervated normal human muscles. Extrajunctional ACh sensitivity was present in 113 of the 140 (73%) fibers from MND muscles studied. In 50 of 84 (70%) ACh-sensitive fibers, no TTX-resistant AP were present. The remaining fibers (30%) showed small regenerative responses. In contrast, all the traumatically denervated muscle fibers showed extrajunctional ACh sensitivity and TTX-resistant AP. Histochemical analysis of the biopsies showed no direct correlation between the frequency of ACh-sensitive fibers and that of the atrophic or normal-appearing fibers. The absence of TTX-resistant AP in ACh-sensitive fibers and its lack of correlation with the histochemical criteria of denervation suggest the presence of a state of innervation in MMD, where the motoneuron is not able to maintain its fully trophic influence on the muscle fiber membrane.     

The roles of disuse and loss of neurotrophic function in denervation atrophy of skeletal muscle

Denervated muscle fibers are characterized by a lowered resting membrane potential (RMP), increased extrajunctional acetylcholine (ACh) sensitivity, and decreased junctional acetylcholinesterase (AChE) activity. Whether these changes in denervated muscle result from cessation of contractile activity, from interruption of axonal transport, or from both is not known. Experiments were therefore designed to analyze whether or not the denervation changes could be ascribed solely to the loss of contractile activity. In one experiment, the hemidiaphragm of the rat was rendered quiescent for 1 to 3 weeks either by spinal hemisection at C2 (disuse) or by unilateral phrenicotomy (denervation). After denervation there was a spread of ACh sensitivity to extrajunctional regions, a decline in RMP, and a reduction in 16 S AChE (a measure of junctional AChE activity). Comparable changes did not occur after spinal hemisection, and we therefore conclude that inactivity alone does not induce these changes in denervated muscle. In another experiment, rats were chronically paralyzed by repeated administration of d-tubocurarine. During this time the extensor digitorum longus muscle of one hind limb was denervated. After 6 h of immobilization by d-tubocurarine, the RMP of denervated muscle fibers was significantly reduced whereas that of the contralateral innervated muscle fibers was unchanged. This result supports the previous interpretation, viz., that the change in RMP of denervated muscle fibers cannot be attributed solely to muscle inactivity. Experiments by others have shown that chronic disuse causes changes that are qualitatively but not quantitatively equivalent to those of denervation. Those observations, together with the present results, enable us to conclude that inactivity does not initiate the changes in extrajunctional ACh sensitivity, RMP, and junctional AChE activity seen in denervated muscle and that these properties of muscle are normally regulated by axonally transported neurotrophic influences.     

Neurotransmission regulates stability of acetylcholine receptors at the neuromuscular junction

The majority of acetylcholine receptors (AChRs) at normally innervated neuromuscular junctions are stable, with a half-life averaging about 12 d in most rodent muscles. Following denervation, the AChRs turn over much more rapidly after a lag period. The mechanism by which motor nerves normally maintain stabilization of junctional AChRs is not yet known. In order to determine whether synaptic transmission plays a role in this process, we have compared the effects of pre-and postsynaptic chloinergic blockade with those of surgical denervation. 125l-alpha-bungarotoxin was used to label junctional AChRs and follow their loss over time. Presynaptic blockade of quantal ACh transmission was produced in the soleus (SOL) and flexor digitorum brevis muscles of mice by repeated injections of type A botulinum toxin. Postsynaptic blockade of quantal and nonquantal ACh transmission was produced by continuous infusion of alpha-bungarotoxin in the SOL. Our findings show that treatment with botulinum toxin resulted in an accelerated loss of junctional AChRs that was similar to the effects of surgical denervation, though briefly delayed in its onset. Treatment with alpha-bungarotoxin produced an effect that was quantitatively equivalent to the accelerated loss of junctional AChRs following surgical denervation, with an identical time course. These results support the concept that cholinergic transmission is a mediator of the neural control of stability of junctional AChRs. The possibility that receptor stabilization may represent a mechanism of long-term postsynaptic "memory" dependent on neural transmission is discussed.     

Binding of D-tubocurarine and alpha-bungarotoxin in normal and denervated mouse muscles

The action of native and mono-³H-acetylated-α-bungarotoxin (³H-BuTX) was studied at the acetylcholine (ACh) receptors of innervated and of chronically denervated diaphragm and soleus muscles of the mouse. The ³H-BuTX was similar in potency to the native toxin, and both toxins produced complete blockade of endplate potentials of innervated muscles and of ACh sensitivity of chronically denervated muscles. When the innervated muscles were washed for 4–7 hr after exposure to either toxin (at 1–5 μg/ml), recovery to an endplate potential value of 0.5–1 mV was recorded in most of the endplate regions of the surface fibers. Parallel experiments on the chronically denervated muscle after a 4–7 hr wash showed a much larger fraction of reversibility; i.e., while in control denervated preparations the ACh sensitivity was 50–75 mV/nC, after the toxin treatment and washing the values were 5–10 mV/nC. When d-tubocurarine (d-TC, 28 × 10^?6m) was present to protect against the blockade of the toxin at the endplates, a complete recovery of neuromuscular transmission could always be obtained upon washing. In the chronically denervated muscles, however, much less protection from the ³H-BuTX blockade was observed. Uptake of ³H-BuTX at the endplates was measured by radioactivity analyses; these revealed that only about 60% of the endplate ACh receptors are protected at saturating d-TC levels. Kinetic analyses of the uptake confirmed this. The observations can be interpreted in terms of two types of sites at the endplate; the two are equally reactive with α-bungarotoxin, but only one of them binds d-TC firmly. Similar observations were made with d-TC protection in the denervated muscles; the results showed much lower affinity of d-TC in these muscles. The changes in d-TC effectiveness in the receptors after denervation are all parallel, when measured in several muscles by three approaches—blockade of ACh sensitivity, prevention of α-bungarotoxin blockade of ACh sensitivity, and inhibition of uptake of ³H-BuTX. We conclude that the ACh receptor molecules themselves, when induced by denervation, are different from normal receptors in that they interact much less strongly with d-TC.     

An electrophysiological study of the effects of D-tubocurarine, atropine, and alpha-bungarotoxin on the cholinergic receptor in innervated and chronically denervated mammalian skeletal muscles

An electrophysiological study of the action of d-tubocurarine (d-TC), atropine, and α-bungarotoxin (BuTX) was made on the innervated and chronically denervated diaphragm and soleus muscles of the rat and mouse. All three drugs were able to block endplate potentials of innervated muscles as well as acetylcholine (ACh) sensitivity of chronically denervated muscles. The effects of atropine and d-TC were fully reversed upon washing with Ringer's solution, white the effects of BuTX were only partially reversed. The reversibility of BuTX was more evident at extrajunctional areas in chronically denervated muscles than at the innervated endplate region, but in both cases only a fraction of the normal response could be detected after intensive washing for at least 2 hr. The blockade of ACh sensitivity in chronically denervated muscles required a concentration of d-TC 10-fold higher than that necessary to block the endplate potentials of innervated muscles. BuTX and d-TC did not affect the ionic permeabilities of the muscle fiber during an action potential while atropine decreased both Na⁺ and K⁺ conductances, the magnitude of the effect being dependent on the frequency of stimulation. At the endplate region, d-TC was much more effective than atropine in protecting against the irreversible effect of BuTX. In the chronically denervated preparation, however, neither of the two drugs effectively protected against BuTX. It is concluded that in terms of their reactivities to cholinolytic agents, the extrajunctional receptors induced by chronic denervation of skeletal muscles are qualitatively similar to those found at endplate regions of normal muscles, but that they exhibit differences in their quantitative interaction with different cholinolytic agents. The data further indicate that atropine interacts with the ionic conductance modulator unit associated with the cholinergic receptor, rather than with the ACh receptor itself.     

Increased binding of alpha-bungarotoxin in dystrophic mouse muscle.

The content of ¹²⁵I-labeled α-bungarotoxin in muscles of normal and dystrophic mice was measured after in vivo administration of a lethal dose of the toxin. Both the uptake of toxin in vivo and the amount retained after washing in vitro were higher in most of the dystrophic hind limb muscles studied than in corresponding controls. In addition, there was an increase of toxin-binding sites which progressed with time after surgical denervation of normal muscles but the corresponding increase in dystrophic muscles was considerably less. However, autoradiography of dystrophic muscles indicates that the toxin is localized at end plate regions and that extrajunctional receptors still appear after surgical denervation.     

Neural regulation of mRNA for the alpha-subunit of acetylcholine receptors: role of neuromuscular transmission

Levels of mRNA for acetylcholine receptor (AChR) subunits are relatively low in innervated skeletal muscles. Following denervation they rise rapidly, leading to increased AChR synthesis. The mechanism by which motor nerves normally regulate these mRNA levels is not yet known. In order to determine the possible role of synaptic transmission in this process, we have compared the effect of blockade of cholinergic ACh transmission with that of surgical denervation. Blockade of quantal ACh transmission was produced by injection of type A botulinum toxin into the soleus muscles of rats. We measured mRNA for the alpha-subunit of the AChR (alpha-AChR mRNA) in RNA extracts of botulinum-treated, denervated, and normal control muscles by hybridization with a highly specific cDNA probe. Our findings show that treatment with botulinum toxin resulted in an increase in alpha-AChR mRNA which was similar to the effect of surgical denervation, although slower in its time course. Since botulinum toxin specifically inhibits quantal ACh release, these results support the concept that cholinergic synaptic transmission plays a key role in mediating the neural control of the alpha-AChR message. The difference between the effects of denervation and botulinum-treatment may be explained by the fact that botulinum toxin does not block the spontaneous non-quantal component of ACh transmission, which has previously been shown to have a partial influence in regulating certain properties of muscles. The present results suggest that synaptic transmission has an important influence in regulating gene expression in the target cell.     

Binding sites for 125I-labeled α-bungarotoxin in normal and denervated mouse muscle

The uptake of [¹²⁵I]α-bungarotoxin in vivo by hind-limb muscles of normal and denervated mice has been studied to determine the effect of denervation on the number and distribution of acetylcholine receptors in these muscles. Autoradiography and cholinesterase staining on sections of the gastrocnemius muscle show that the increased binding of toxin by denervated muscle, which reaches a peak at 16 days after nerve transection, is due mainly to the formation of extrajunctional receptors. At the same time, there is an increase in the number of endplate acetycholine receptors, which represent a small proportion of the total toxin binding sites, and the area of the endplate region covered by receptors also increases, although the acetylcholine esterase activity at endplates is markedly reduced.     

Acetylcholine receptors in extrajunctional regions of innervated muscle have a slow degradation rate.

Scanning EM autoradiography was used to determine the degradation rate of extrajunctional ACh receptors (AChRs) in innervated sternomastoid muscles of the mouse. We report that in innervated muscles, extrajunctional AChRs have a slow degradation rate (t1/2, approximately 8 d), similar to that seen at the neuromuscular junction. We conclude that slowly degrading AChRs (Rs) need not be localized at the specialized structure of the nerve-muscle junction. Degradation of extrajunctional as well as junctional AChRs may depend primarily on the state of innervation of the muscle.     

Neonatal denervation inhibits the normal postnatal decrease in endplate channel open time

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.     

Regulation of NMDA-stimulated [C]GABA and [H]acetylcholine release by striatal glutamate and dopamine receptors

Striatal function is heavily influenced by glutamatergic and dopaminergic afferent input. To ultimately better understand how the N-methyl- -aspartate (NMDA) antagonist, phencyclidine (PCP), alters striatal function, we sought to determine how NMDA receptor function is influenced by activation of other glutamatergic receptors and by dopaminergic receptors. To this end, we used NMDA-stimulated efflux of [¹⁴C]GABA and [³H]acetylcholine (ACh) from striatal slices to assess the influence of these receptors on NMDA function. NMDA-stimulated [¹⁴C]GABA release was more sensitive to NMDA and glycine antagonists than was [³H]ACh release, suggesting that different NMDA receptors regulate the release of these neurotransmitters. Furthermore, NMDA-stimulated [³H]ACh release was inhibited by a D₂ receptor mechanism whereas NMDA-stimulated [¹⁴C]GABA release was enhanced by D₁ receptor activation. NMDA and (±)-α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid hydrobromide (AMPA) interact additively to evoke [³H]ACh release, and synergistically to evoke [¹⁴C]GABA release. An additive effect of NMDA and kainate (KA) was found on [¹⁴C]GABA release, but NMDA and KA acted in a less than additive manner in evoking [³H]ACh release. KA-stimulated [³H]ACh release was largely blocked by NMDA antagonists, suggesting mediation through activation of NMDA receptors, probably secondary to KA-induced glutamate release. A selective group II metabotropic receptor agonist inhibited NMDA-stimulated [¹⁴C]GABA and [³H]ACh release. On the other hand, NMDA-stimulated [¹⁴C]GABA release was potentiated by activation of group I metabotropic receptors. Thus, in addition to the differential modulation by D₁- and D₂-like receptors, the release of striatal neurotransmitters by NMDA receptor activation depends on the extent to which the other glutamate receptors, both ionotropic and metabotropic, are activated.     

5-HT2 receptor regulation of acetylcholine release induced by dopaminergic stimulation in rat striatal slices

The role of 5-hydroxytryptamine (5-HT) receptor subtypes in acetylcholine (ACh) release induced by dopamine or neurokinin receptor stimulation was studied in rat striatal slices. The dopamine D₁ receptor agonist SKF 38393 potentiated in a tetrodotoxin-sensitive manner the K⁺-evoked [³H]ACh release while SCH 23390, a dopamine D₁ receptor antagonist, had no effect. [³H]ACh release was decreased by the dopamine D₂ receptor agonist LY 171555 (quinpirole) and slightly potentiated by the dopamine D₂ receptor antagonist haloperidol. The selective neurokinin NK₁ receptor agonist [Sar⁹, met(O₂)¹¹]SP also potentiated K⁺-evoked release of [³H]ACh. GR 82334, a NK₁ receptor antagonist, blocked not only the effect of [Sar⁹, met(O₂)¹¹]SP but also the release of ACh induced by the D₁ receptor agonist SKF 38393. Among the 5-HT agents studied, only the 5-HT_2A receptor antagonists ketanserin and ritanserin were able to reduce the ACh release induced by dopamine D₁ receptor stimulation. Mesulergine, a more selective 5-HT_2C antagonist, showed an intrinsic releasing effect but did not affect K⁺-evoked ACh release induced by SKF 38393. Methysergide and methiothepin, mixed 5-HT_1/2 antagonists, as well as ondansetron, a 5-HT₃ receptor antagonist, showed an intrinsic effect on ACh release, their effects being additive to that of SKF 38393. 5-HT₂ receptor agonists were ineffective. However, the 5-HT₂ agonist DOI was able to prevent the antagonism by ketanserin of the increased [³H]ACh efflux elicited by SKF 38393, suggesting a permissive role of 5-HT_2A receptors. None of the above indicated 5-HT agents was able to reduce the ACh release induced by the selective NK₁ agonist. The results suggest that 5-HT₂ receptors, probably of the 5-HT_2A subtype, modulate the release of ACh observed in slices from the rat striatum after stimulation of dopamine D₁ receptors. It seems that this serotonergic control is exerted on the interposed collaterals of substance P-containing neurons which promote ACh efflux through activation of NK₁ receptors located on cholinergic interneurons.     

Effect of nerve extract on number of acetylcholine receptors in denervated muscles of rats

We have shown elsewhere that injection of an extract of peripheral nerves reduces the atrophy of denervated muscle fibers in vivo. Denervated muscle fibers exhibit supersensitivity to acetylcholine owing to the production of extrajunctional acetylcholine receptors. We sought to determine whether or not injection of nerve extract can influence the numbers of acetylcholine receptors in normal, immobilized, or denervated extensor digitorum longus muscles of rats. The receptors were assayed by measuring the binding of ¹²⁵I-α-bungarotoxin. Normally innervated muscles injected with nerve extract exhibited slightly increased binding of the toxin, but this was due to the injections per se. Immobilization caused a small, transient increase in binding of α-bungarotoxin, whereas denervated muscles bound considerably more toxin than innervated controls. The nerve extract did not reduce or prevent the increase in acetylcholine receptors caused by denervation but instead caused an even greater increase. We concluded that the neurotrophic factor extracted from peripheral nerve that is responsible for the maintenance of the sizes of the fibers probably does not down-regulate extrajunctional acetylcholine receptors. The limitation of acetylcholine receptors to the end-plate regions is probably effected by a different mechanism which has yet to be elucidated.     

α1-Noradrenergic regulation of hypothalamic progestin receptors and guinea pig lordosis behavior

Experiments were conducted to determine whetherα₁- orα₂-receptors mediate noradrenergic (NA) regulation of guinea pig lordosis behavior and hypothalamic progestin receptors. When infused into a lateral cerebroventricle at a dose that inhibits lordosis and that decreases the concentration of estradiol-inducible hypothalamic progestin receptors, phenoxybenzamine decreased binding of theα₁-ligand [³H]WB4101 but not theα₂-ligand [³H]clonidine to brain membranes. Thus, under the conditions used, phenoxybenzamine appears to blockα₁-receptors with little or no effect onα₂-receptors.Experiments with the selectiveα₁-antagonist prazosin also indicatedα₁-receptor reguilation of lordosis and hypothalamic progestin receptors. Prazosin inhibited lordosis induced by estradiol benzoate (EB) plus progesterone and by EB + clonidine and decreased the concentration of cytoplamic progrestin receptors in hypothalamus (but not in area or frontal cortex) of EB-primed females. The inhibition of lordosis is apparently not due to some unknown side effect of prazosin because pretreatment with a high dose of clonidine attenuated the inhibition.The possibility that a causal relationship exists between effects ofα₁-NA transmission on hypothalamic progestin receptors and lordosis was discussed. Also, because effects of NA transmission on hypothalamic progestin receptors are dependent on prior treatment with EB, it was suggested that NA transmission might influence estradiol action in addition to progestin action in hypothalamic cells.     

Acetylcholine receptor availability and transmission efficacy

Recovery of cholinergic transmission after in vivo blockade with α-bungarotoxin (α-BTX), and the relationship of recovery to availability of unbound acetylcholine receptors (AChR) were studied in rat diaphragm. When 83% of endplate acetylcholine receptor binding sites were blocked, transmission was absent. A barely detectable recovery of the blocked receptors (25 h after exposure to α-bungarotoxin) restored transmission. In fact, 25% of the endplate receptor ACh binding sites were just sufficient for action potential generation. As discussed, slow turnover (t₁₂ = 11 days) of junctional receptors would be sufficient to provide the observed recovery of transmission. Good agreement was observed between the minimum fraction of total receptor sites required for transmission and the computed fraction of maximum quantal release of acetylcholine required to reach threshold. In addition, the data are consistent with the hypothesis that at the neuromuscular junction, AChR exists in considerable excess.     

Extrajunctional acetylcholine receptors. Alterations in human and experimental neuromuscular diseases.

Diffuse extrajunctional acethycholine receptors (AChR) of skeletal muscle fibers were readily visualized by light and electron microscopy in muscle biopsy specimens of experimental denervation and human denervating diseases by use of an alpha-bungarotoxin immunoperoxidase technique. In peripheral neuropathies and various motor neuron diseases, a significant number of muscle fibers appearing denervated by histochemical criteria have diffuse extrajunctional AChR like those experimentally denervated by cutting the motor nerve supply. In portions of muscle fibers experimentally deprived of neuronal influence by direct injury, diffuse extrajunctional AChR developed, demonstrating that a denervation-like diffuse appearance of extrajunctional AChR can develop other than with neuronal damage, ie, it can be myogenous. Similar extrajunctional AChR was seen in some regenerating fibers of human myopathies, especially inflammatory myopathies.