Axonal transport of α-bungarotoxin binding sites in rat sciatic nerve

[¹²⁵I]α-Bungarotoxin (α-BuTX) binding sites accumulate both proximal and distal to a ligature positioned around the sciatic nerve of rats. [¹²⁵I]α-BuTX binding sites, localized using quantitative receptor autoradiography, were found to accumulate at nerve ligatures at a relatively constant rate which suggests that they undergo both anterograde and retrograde axonal transport. [¹²⁵I]α-BuTX binding to sections of ligated sciatic nerve was saturable with apparent dissociation constants of 0.97 nM proximal and 0.53 nM distal to the ligature.d-Tubocurarine, nicotine, decamethonium and atropine displaced [¹²⁵I]α-BuTX from sciatic nerve sections with affinities comparable to those previously reported for the toxin binding component of rat brain. These data indicate that [¹²⁵I]α-BuTX binding sites pharmacologically similar to those of rat brain are transported in sciatic nerve. Axonally transported toxin binding sites may correspond to those previously localized to the plasma membrane of peripheral nerve axons and on the terminals of motor neurons.     

α-Bungarotoxin binding to a high molecular weight component from lower vertebrate brain identified on dodecyl sulfate protein-blots

The binding of [¹²⁵I]iodo-α-bungarotoxin ([¹²⁵]α-BuTX) to the dissociated α-subunit of Torpedo acetylcholine receptor (AChR) can be readily demonstrated in a modified ‘protein-blot’ analysis utilizing electrophoretically transferred, dissociated subunits immobilized onto positively charged nylon membranes which are then incubated directly with [¹²⁵I]α-BuTX. We report here the use of the protein-blotting technique to detect the α-BuTX binding site present in the central nervous system of lower vertebrates and to characterize some of the physicochemical properties of the toxin binding site. High molecular weight (M200,000 and 120,000) α-BuTX-binding components can be readily demonstrated in avian and fish brain extracts upon protein-blotting with [¹²⁵I]α-BuTX following lithium dodecyl sulfate PAGE. Neither extensive reduction with dithiothreitol nor prior reduction followed by alkylation with iodoacetamide alter the mobility of the CNS-derived BuTX-binding sites. In contrast to our findings with Torpedo AChR or muscle AChR derived from a number of different species, no binding is observed in the molecular weight range of the α-subunit (M_r= 40,000) nor is any binding at any molecular weight observed in similar fractions prepared from adult, mammalian (rat, guinea pig) brain using this technique. These results demonstrated the existence in lower vertebrate brain of a BuTX binding site comparable in size to the AChR oligomeric complex of electric organ and muscle. They also suggest, however, striking structural differences between muscle AChR and the central neuronal BuTX-binding complex as well as a considerable difference between the neuronal BuTX-binding sites derived from lower and higher vertebrate brain.     

Cholinergic transmission regulates extrajunctional acetylcholine receptors

To determine the role of ACh transmission in the regulation of extrajunctional ACh receptors, we compared the effect of postsynaptic cholinergic blockade with that of surgical denervation. Blockade of ACh transmission was produced in the soleus muscles of rats by continuous local infusion of α-bungarotoxin, delivered by implantable osmotic pumps. Extrajunctional ACh receptors were measured by an ¹²⁵I-α-BuTx binding method. Our results showed an increase of extrajunctional ACh receptors quantitatively equivalent to that resulting from surgical denervation. This denervation-like effect is attributed to elimination of (i) impulse-dependent ACh transmission (which normally triggers muscle usage), and (ii) spontaneous quantal and nonquantal ACh transmission. The influence of the nerve in regulating extrajunctional ACh receptors appears to be due to the sum of these forms of ACh transmission.     

Reinnervation of normal and dystrophic skeletal muscle

A comparative study was conducted of resting membrane potential (RMP), extrajunctional acetylcholine (ACh) sensitivity, spontaneous and neurally evoked transmitter release, and directly and indirectly elicited action potentials in posterior latissimus dorsi (PLD) muscles of normal (line 412) and dystrophic (line 413) chickens during reinnervation after nerve crush. Control (nondenervated) dystrophic muscle fibers had a significantly greater RMP (-77.2 vs. -74.1 mV), extrajunctional ACh sensitivity (0.34 vs. 0.04 mV/nC), and incidence of repetitive firing of directly elicited action potentials than did normals. Miniature end-plate potential (MEPP) amplitude in dystrophic fibers was significantly reduced (0.26 vs. 0.38 mV). Six to 7 days after nerve crush, muscle fibers from both lines of chickens showed a significant reduction in RMP and an increase in extrajunctional ACh sensitivity. During this time spontaneous MEPPs were absent and the incidence of repetitive firing was decreased. No significant difference was noted between chicken lines in any of the properties studied. The return of normal properties associated with reinnervation occurred primarily between days 9 and 40. Repolarization of the RMP was clearly evident by day 9 in both lines, but dystrophic fibers showed a slightly earlier and greater degree of repolarization. Similarly, initial decreases in extrajunctional ACh sensitivity and the reappearance of MEPPs were observed on day 9 with dystrophic and day 12 with normal fibers. Neurally elicited action potentials were first recorded on day 11 for dystrophic and day 12 for normal fibers. Finally, multiple firing of directly elicited action potentials associated with reinnervation attained the same incidence (20 to 21% of fibers) on day 12 in dystrophic and day 14 in normal fibers. The results suggest that dystrophic chicken muscle has an enhanced capacity for reinnervation following nerve crush.     

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.     

Block of locust muscle glutamate receptors by δ-philathotoxin occurs after receptor activations

One component (δ-philathottoxin (δ-PTX)) of the venom from the wasp (Philanthus triangulum blocks transmission postsynaptically at excitatory synapses on locust muscle. δ-PTX depresses both the iontophoretic glutamate potential and the excitatory junctional current (e.j.c.) in a glutamate receptor activation-dependent manner. The rate of recovery from the effects of the toxin is reduced following either prolonged application ofl-glutamate or repetitive iontophoretic application of this amino acid or high frequency neural stimulation of the muscle in the presence of δ-PTX. The decay phase of the e.j.c. is shortened by δ-PTX. The effects of δ-PTX on the e.j.c. are not voltage dependent. The open-close kinetics of glutamate channels in extrajunctional muscle membrane are modified by δ-PTX as shown by patch clamp analysis. The mean life time of the glutamate channel is reduced, whilst the mean interval between single opening events is increased with the events often occurring in bursts. These data are consistent with glutamate channel blocking by this toxin. It is proposed that the toxin blocks open channels gated by both junctional and extrajunctional glutamate receptors on locust muscle. It is further proposed that δ-PTX enters a compartment of the muscle through the glutamate open channels and that it can also block the open channels from this site.     

Characterization of neuronal nicotinic receptors by snake venom neurotoxins

Neuronal nicotinic ACh receptors differ pharmacologically from nicotinic receptors on skeletal muscle. The use of certain snake venom neurotoxins has now led to a more complete determination of the pharmacological properties of these neuronal receptors, as well as to their ultrastructural localization. This review highlights results found using one such neurotoxin, toxin F, (also called bungarotoxin 3.1 and κ-bungarotoxin. Toxin F blocks nicotinic receptors in several neuronal preparations while having little affinity for nicotinic receptors in skeletal muscle. Autoradiographic studies using [¹²⁵I] toxin F indicate that nicotinic receptors in autonomic ganglia are clustered at synaptic sites, though their density is 3–30 times lower than that of nicotinic receptors at the neuromuscular junction.     

α-Bungarotoxin-acetylcholine receptors in the chick ciliary ganglion: Effects of deafferentation and axotomy

α-Bungarotoxin (α-BuTX) binds in a saturable and practically irreversible fashion to membrane-associated receptors in the ciliary ganglion of the adult chick. The binding of toxin to receptors is competitively inhibited by nicotinic cholinergic ligands, and for these properties the receptors are regarded as acetylcholine receptors of the nicotinic type (α-BuTX-AChRs). The rate constant of association (K₁) and dissociation (K₋₁) of the toxin-receptor reaction has been estimated to be K₁ = 7.4 × 10⁴ M⁻¹sec⁻¹ and K₋₁ = 9.6 × 10⁻⁶sec⁻¹, respectively. Light autoradiography shows that most, if not all, the receptors are related to surface membrane, probably to synaptic areas of both choroid and ciliary neurons. The choroid neurons contain more receptors that the ciliary ones. A single chick ciliary ganglion binds specically 47 fmole of α-BuTX in situ corresponding to 2.83 × 10¹⁰ α-BuTX-AChRs/ganglion.No changes in number and distribution of the toxin receptors occur following preganglionic denervation. Conversely, postganglionic axotomy causes a rapid disappearance of the receptors in situ. Since binding experiments in vitro revealed a partial, instead of a total, loss of the receptors, it is suggested that the disappearance of the receptors in situ includes both a partial loss of the original receptors and the masking of the residual ones.     

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.     

Intraspinal distribution and reaction in the grey matter with tetanus toxin of intracisternally injected anti-tetanus toxoid F (ab′)2 fragments

Anti-tetanus toxoid F(ab′)₂ fragments were purified using immune-affinity chromatography on tetanus toxoid-Sepharose. Fragments were labeled with¹²⁵I. Labeled or non-labeled fragments were injected into the intrathecal space of rats. The labeled fragments were found in the spinal cord outside but not inside neurons.Tetanus toxin was injected into a muscle and 36 h later labeled fragments were injected intracisternally. After another 24 h the label was elevated in the spinal cord half segments giving neural supply to the injected muscle and in these half-segments the label was concentrated around some α-motoneurons.[¹²⁵I]Tetanus toxin was injected into a muscle and at different times thereafter non-labeled fragments were injected intracisternally. The development of hindlimb rigidity but not the accumulation of [¹²⁵I]tetanus toxin in α-motoneurons was prevented by early intracisternal injection of fragments. Injection of fragments after the appearance of hindlimb rigidity did not revert the rigidity but prevented the further development of symptoms.It is concluded that an action of tetanus toxin inside α-motoneurons is of no importance for the development of motor symptoms in clinical tetanus. The data suggest that in order to evoke spinal symptoms of toxicity tetanus toxin has to reach interneurons by transneuronal migration. In the very early stages of clinical tetanus the intrathecal injection of fragments may be useful.     

Effects of lead on neuromuscular transmission in the frog

The acute effects of Pb²⁺ on synaptic transmission at the frog neuromuscular junction were measured using conventional microelectrode techniques. Experiments were performed on preparations bathed in high magnesium/low calcium Ringer solution in order to record subthreshold endplate potentials (EPPs). The effects of Pb²⁺ on the muscle membrane and postsynaptic membrane were minimal since relatively high doses of Pb²⁺ caused no significant change in the input resistance of the muscle fiber and in the amplitude of the acetylcholine (ACh) iontophoteric potential when the ACh micropipette was highly localized. However, when the ACh micropipette was moved away from the receptors, the resulting ACh potential was reduced significantly by Pb²⁺. Pb²⁺ is a potent blocker of the EPP. Extracellular recordings from motor nerve terminals showed that endplate currents (EPCs) were reduced by Pb²⁺ while the nerve terminal potentials were unaffected. Therefore, Pb²⁺ blocks evoked transmitter release at a step following the depolarization of the nerve terminal. The blocking effect on the EPP was overcome when [Ca²⁺]_o was raised. The log-log relationship between [Ca²⁺]_o (abscissa) and EPP amplitude was shifted to the right in the presence of 1 μM Pb²⁺; the x¯±S.E. slopes were 4.16 ± 0.12 (control) and 4.05 ± 0.13 (Pb²⁺). Reciprocal plots relating [Ca²⁺]_o⁻¹ to (EPP)^−1/5 confirmed that Pb²⁺ competitively antagonized the action of Ca²⁺. The dissociation constant between Pb²⁺ and the Ca²⁺ receptor site was found to be 0.99 μM. Pb²⁺ is about 3 × 10³ times more potent than is Mg²⁺, about 150 times more potent than is either Mn²⁺ or Co²⁺, and about 3 times more potent than Cd²⁺ is in blocking evoked release of ACh. After Pb²⁺ decreased the EPP, the MEPP frequency began to increase; this was probably the result of intracellular Pb²⁺ disrupting the Ca²⁺ sequestering activity of mitochondria and/or other intraterminal organelles. [Ca²⁺]_i was thereby increased and an increase in MEPP frequency followed. Decreased MEPP amplitudes were observed when the MEPP frequency had been increased by Pb²⁺. Pb²⁺ may affect most chemical synapses in a manner which is similar to its effects on the neuromuscular junction and that this may be one of its important neurotoxic effects.     

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.     

Presence of an endogenous factor which inhibits binding of α-bungarotoxin 2.2 to its receptor

Cerebral cortical membranes and supernatant from rat were prepared by centrifugation of tissue homogenates at 45,000 g for 10 min. The supernatant fraction thus obtained was found to significantly inhibit α-bungarotoxin binding to the membrane preparation. After a 3 min incubation period, the supernatant inhibited toxin binding by approximately 65%, while the inhibition declined to about 40% after 30 min of incubation, presumably due to the slow reversility of α-bungarotoxin binding. The choice of buffer was found to be an important determinant of the degree of inhibition observed, with 10 mM Tris pH 7.4 providing the most effective condition. This inhibition of toxin binding to cortical membranes by the 45,000 g supernatant was shown not to be due to adsorption of the radiolabeled compound to soluble or residual particulate material in the supernatant fraction. Specificity of the supernatant for the α-bungarotoxin site was demonstrated; a supernatant fr action could be prepared which inhibited α-bungarotoxin binding by 50% but had no effect on [³H]spiroperidol (DA₂ and 5-HT₂), [³H]prazosin, (α₁-adrenergic), [³H]5-hydroxytryptamine (5-HT₁) and [³H]quinuclidinylbenzilate (muscarinic cholinergic) binding. The inhibition of toxin binding also occurred in several other CNS regions including hippocampus, brainstem, spinal cord and cerebellum with an 80 to 90% inhibition of binding occuring in the latter two regions. In addition, the 45,000 g cortical supernatant completely prevented the binding of α-bungarotoxin to extrajunctional neuromuscular receptors and inhibited the binding to junctional receptors by 50%. Supernatants prepared from heart, liver and kidney or bovine serum albumin, at a concentration similar to the supernatant fraction, did not alter radiolabeled toxin binding to cortical membranes, while supernatant prepared from striated muscle tissue was effective. These results suggest there may be an endogenous ligand for the α-bungarotoxin 2.2 binding site in tissues which receive nicotinic cholinergic innervation.     

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.     

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.     

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.     

Consequences of axonal transport blockade by batrachotoxin on mammalian neuromuscular junction. II. Late pre- and postsynaptic changes

The present study describes the time course of recovery in the fast axonal transport of 3H-labeled proteins in the nerve and the electrophysiological parameters in the extensor muscle of rats after single subperineural injection of batrachotoxin (BTX) (9.3 x 10(-12) mol) into the peroneal nerve. The fast axonal transport of 3H-labeled proteins in the sciatic nerve showed significant accumulation of the labeled proteins proximal to the site of BTX injection at day 2. However, by day 7, when no locomotor deficit was visible, complete recovery of fast axonal transport had occurred. The recovery of membrane potential in this surface fibers of the extensor muscle, which showed atrophy even after 22 days of toxin administration, lagged far behind the recovery in the fast axonal transport. Between day 2 and 14 the extensor muscle showed partial membrane depolarization with almost complete recovery by day 22. From 18 h to 7 days after subperineural BTX injection, spontaneous (m.e.p.p.s.) or nerve evoked transmitter release were absent at the endplates of the affected extensor muscles. M.e.p.p.s. of very low frequency began to appear between day 7 and 10. Even at days 14 and 22, when most of the fibers tested showed m.e.p.p.s, the frequency of these mepps was significantly lower than that shown in corresponding control muscles. The profile of sensitivity of the extrajunctional region to microiontophoretically applied acetylcholine (ACh) showed extensive sensitivity (about 200 mV/nC) up to 14 days after BTX injection when the extensor muscle membrane was considerably depolarized. However, at day 22, when the RMP had returned to the control level, neither extrajunctional supersensitivity to ACh nor TTX-resistant action potentials could be detected. The persistence of membrane depolarization and other denervation signs in the extensor muscle, despite recovery in the fast axonal transport, suggests the existence of a blockade of other key particulates delivered to the synaptic region by slow axonal transport. The time course of recovery in the extensor muscle after BTX injection resembles in many respects that seen after nerve crush injury. The rate of recovery is fast due mostly to the ability of the toxin to produce disorganization and to depolarize the nerve without producing a break in continuity of the cytoarchitecture of axon.     

Effects of histrionicotoxin on the chemosensitive and electrical properties of skeletal muscle

Histrionicotoxin (HTX), a spiropiperidine alkaloid from the Colombian frog, Dendrobates histrionicus, has a variety of effects on the function of mammalian and amphibian nerve-muscle preparations. At a concentration of 70 × 10⁻⁶m, HTX blocks indirectly elicited muscle twitches in the mouse phrenic nerve-diaphragm preparation within 20 min, while potentiating directly elicited twitches. Resting membrane potential and passive electrical properties are little affected by the drug. HTX prolongs the falling phase of the action potential and blocks delayed rectification, indicating that the potassium conductance is suppressed. Endplate potentials are also blocked by the toxin, and the onset of this blockade is dependent on the frequency of stimulation. Similarly, HTX decreases the sensitivity of denervated mammalian skeletal muscles to repetitive applications of acetylcholine, the degree of blockade also being dependent on the rate and duration of iontophoretic application of acetylcholine. All effects of HTX are reversed by washing the muscle. HTX does not prevent the irreversible effects of an acetylcholine antagonist, α-bungarotoxin, in the mouse phrenic nerve-diaphragm preparation. It is suggested that HTX acts on the ionic conductance modulator of the acetylcholine receptor in a manner similar to its semisynthetic derivative perhydrohistrionicotoxin.     

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.