Sharad S. Deshpande

A possible involvement of cyclic AMP in the expression of desensitization of the nicotinic acetylcholine receptor: A study with forskolin and its analogs

Forskolin, an activator of adenylate cyclase, and its analogs were studied on the nicotinic acetylcholine receptor‐ion channel complex (AChR) of rat and frog skeletal muscles. At nanomolar concentrations, forskolin caused desensitization of the AChR located at the junctional region of innervated and the extrajunctional region of chronically denervated rat soleus muscles. The desensitization of the AChR occurred without alteration of the conducting state (channel lifetime, conductance or bursting) as shown by single channel currents. Accordingly, forskolin decreased the peak amplitude of the repetitive evoked endplate currents in frog sartorius muscles. These findings taken together with the good correlation found between the effects of forskolin and its analogs on the desensitization of the nicotinic AChR and their ability to activate adenylate cyclase suggested a possible involvement of phosphorylation of AChR via cyclic AMP on the desensitization process.

Persistence of botulinum neurotoxin A demonstrated by sequential administration of serotypes A and E in rat EDL muscle.

Botulinum neurotoxin serotypes A (BoNT/A) and E (BoNT/E) inhibit neurotransmitter release from peripheral cholinergic nerve terminals by cleaving different sites on SNAP-25, a protein involved in synaptic vesicle docking and exocytosis. Since recovery from BoNT/A is protracted, but reversal of BoNT/E intoxication is relatively rapid, it was of interest to determine whether sequential exposure to BoNT/A and BoNT/E could provide insight into the factors responsible for persistence of BoNT action. Extensor digitorum longus (EDL) muscles from rats were injected locally with 5 mouse LD(50) units of BoNT/A or 20 mouse LD(50) units of BoNT/E; these doses were selected to produce total paralysis of EDL muscles within 48 hr. Additional groups of rats were injected sequentially with either BoNT/A followed 48 h later by BoNT/E or with BoNT/E followed 48 h later by BoNT/A. Muscle tensions were elicited in situ in response to supramaximal stimulation of the peroneal nerve to monitor recovery from BoNT intoxication. Tensions returned to 53% and 94% of control, respectively, 7 and 15 days after injection of BoNT/E. In contrast, tensions in muscles injected with BoNT/A returned to only 2% and 12% of control at these time points. Preparations injected sequentially with BoNT/A followed by BoNT/E or with BoNT/E followed by BoNT/A exhibited slow recovery times resembling those recorded in the presence of BoNT/A alone. Pronounced atrophy of the EDL muscle was observed in rats injected with BoNT/A or in those receiving serotype combinations in either sequence, whereas no loss of muscle mass was observed in animals treated with BoNT/E alone. Data suggesting that BoNT/E can enter BoNT/A-treated preparations was obtained by findings that 3,4-diaminopyridine, which readily reversed muscle paralysis after BoNT/A exposure, lost this ability within 1 h of BoNT/E addition. Evidence that BoNT/E was able to cleave SNAP-25 at its characteristic site during sequential neurotoxin exposure was demonstrated by western blot analysis of cultured primary cortical neurons. Since the sequential exposure studies indicate that recovery from BoNT intoxication is lengthened by exposure to serotype A, but not shortened by exposure to serotype E, the duration of BoNT/A intoxication appears to be determined predominantly by the intracellular stability of catalytically active BoNT/A light chain.

Nicotinic acetylcholine receptors in cultured neurons from the hippocampus and brain stem of the rat characterized by single channel recording

Single channel recording techniques have been applied to neurons cultured from the hippocampus and the respiratory area of the brain stem of fetal rats in order to search for nicotinic acetylcholine receptors (nAChR) in the central nervous system. In addition to acetylcholine (ACh), the potent and specific agonist (+)‐anatoxin‐a was also used to characterize nicotinic channels. nAChRs were concentrated on the somal surface near the base of the apical dendrite, and in some patches their density was sufficient to record 2 or more channel openings simultaneously. Although a multiplicity of conductance states was also evident, the predominant population showed a single channel conductance of 20 pS at 10°C. Thus, these neuronal nAChRs resembled the embryonic or denervated‐type nAChRs in muscle. However, channel opening and closing kinetics were faster than reported for similar conductance channels in muscle. Therefore the nicotinic channels described here are similar but not identical to those of the well‐characterized muscle nAChR, in agreement with biochemical, pharmacological, and molecular genetic studies on brain AChR.

Neurotrophic regulation of prejunctional and postjunctional membrane at the mammalian motor endplate

Abstract The trophic influence of nerve on muscle was investigated by studying the effect of transecting the sciatic nerve or its branches at various distances from the extensor digitorum longus and soleus muscles. If the rate at which the muscle responds to denervation were related to the level of nerve transection, a neurotrophic mechanism would have to be postulated, because the operation abolishes such non-neurotrophic factors as neuromuscular transmission and muscle activity. partial membrane depolarization of muscle fibers and spread of extrajunctional sensitivity to acetylcholine occurred much sooner when the nerve was transected near the muscle than when it was transected farther away. These changes preceded the disappearance of spontaneous miniature endplate potentials, thus indicating that quantal release of acetylcholine is not responsible for the maintenance of the resting potential or for the distribution of acetylcholine receptors in innervated muscle. An increase in spontaneous transmitter release was observed prior to the disappearance of miniature endplate potentials. This increase, which reflects a partial depolarization of the prejunctional nerve membrane, occurred sooner when the nerve was transected near the muscle than when it was cut farther away. We therefore conclude that both the prejunctional nerve membrane and the postjunctional muscle membrane are regulated by a neurohumoral, trophic mechanism.

Consequences of axonal transport blockade induced by batrachotoxin on mammalian neuromuscular junction I. early pre- and postsynaptic changes

Subperineural injections of batrachotoxin (BTX) (1.86 X 10-12 or 9.3 X 10-12 mol) were made into the peroneal nerve at 10-12 or 33-35 mm from the entrance of the nerve into the extensor muscle of the rats. Measurements of fast axonal transport in the nerve and the resting membrane potential (RMP) from the surface fibers of the extensor muscle were made at intervals up to 18 h after injection of the toxin. The transport of 3H-labeled proteins and nerve conduction were blocked almost instantaneously by either dose of toxin. At 18 h some radioactive material distal to the BTX injection site could be seen, indicating partial recovery in fast axonal transport. Membrane depolarization of about 4 mV was evident in the surface fibers of the extensor muscle 50 min after injecting BTX in the peroneal nerve at a distance of 10 mm from the muscle. If the toxin was injected into the nerve at a farther site (33-35 mm), the onset of muscle membrane depolarization occurred at 120 min. The muscle membrane depolarization seen after injection of BTX at these two sites in the nerve was not a result of the toxin acting directly on the muscle nor was the depolarization reversibly by bath applied tetrodotoxin (TTX). Similar subperineural injections of TTX (6.3 X 10-9 mol) into peroneal nerve failed to cause any membrane depolarization in the extensor muscle even up to 18 h although the leg on the injected side was paralyzed in the same fashion as was the one with BTX. Membrane potential consistently recovered at 18 h in all BTX-injected animals although spontaneous release of transmitter had completely ceased at this time. These results conclusively demonstrate the fact that blockade of axonal transport by BTX and not suppression of electrical activity in the nerve caused by this agent is responsible for the early membrane depolarization of surface fibers of the extensor muscle. Thus the notion that resting membrane potential is under neurotrophic control is further supported. Muscle inactivity produced by paralysis of the affected limb alone apparently plays very little role in the onset of muscle depolarization and cessation of transmitter release.

A study of zinc-dependent metalloendopeptidase inhibitors as pharmacological antagonists in botulinum neurotoxin poisoning

Zinc-dependent metalloprotease inhibitors phosphoramidon, captopril and a peptide hydroxamate were studied as potential pretreatment compounds by examining their ability to delay the onset or to prolong the time to 50% block of nerve-elicited muscle twitch tension in the mouse phrenic-nerve diaphragm (in vitro at 36 degrees C) after botulinum neurotoxin serotypes A and B (BoNT-A, BoNT-B). Addition of BoNT-A or BoNT-B (1 x 10(-10) M) produced 50% block of the twitch response at 56 +/- 9 min and 76 +/- 4 min, respectively. Preincubation (45 min) of muscles with phosphoramidon (0.2 mM) prolonged the time to 50% block by 15 min in BoNT-B-poisoned muscles with no effect on the time-course of paralysis in BoNT-A exposed muscles. When the same quantities of BoNT-A or BoNT-B (equivalent to 1 x 10(-10) M bath concentration) were preincubated for 2 hr with phosphoramidon (equivalent to 0.2 mM final bath concentration), and the incubation mixture was added to the muscle chamber, the times to 50% block were prolonged by 38 min and 18 min for BoNT-B and BoNT-A, respectively. Preincubation of diaphragms with captopril (up to 10 mM) or peptide hydroxamate (75 microM) failed to antagonize BoNT-A or BoNT-B-induced neuromuscular block. Among the three metalloprotease inhibitors examined here, only phosphoramidon showed a significant protection against both serotypes of BoNT. A search for better inhibitor compounds specifically tailored to match the active site on BoNT molecule deserves attention.

Comparison of the ultrastructural myopathy induced by anticholinesterase agents at the end plates of rat soleus and extensor muscles

Rats were treated with single subcutaneous injections of the irreversible AChE inhibitors, sarin (90 to 100 micrograms/kg) or soman (55 micrograms/kg), and with chronic doses of the reversible carbamate inhibitor, pyridostigmine. In surviving animals with severe behavioral symptoms, we examined the end-plate regions of the slow-twitch soleus and the fast-twitch extensor digitorum longus muscles, using the electron microscope. Within 30 min, sarin administration caused a recognizable subjunctional myopathy. The progress of morphologic damage was followed for 7 days, during which time the occurrence of damage diminished. The initial swelling of subjunctional organelles and vacuole generation progressed to the point where nerve terminals and attached postjunctional folds were lifted away from the muscle surface. This appeared to be caused by a combination of enlarging vacuoles and insertion of Schwann and macrophage cells into the lesions, and was followed by degeneration of the postjunctional folds. A new component of anti-AChE myopathy was recognized: progressive swelling of chromatin in subjunctional muscle nuclei. The soleus muscle was considerably more sensitive to these effects than the extensor muscle. Soman had a much less prominent ultrastructural effect on the muscle end plates. Chronic pyridostigmine treatment had effects similar to those of a single sarin injection on the soleus as well as a pronounced effect on the extensor muscle.

Antagonism of botulinum toxin-induced muscle weakness by 3,4-diaminopyridine in rat phrenic nerve-hemidiaphragm preparations

The effects of the potassium channel inhibitor and putative botulinum toxin antagonist 3,4-diaminopyridine (3,4-DAP) were investigated in vitro on the contractile properties of rat diaphragm muscle. In the presence of 100 pM botulinum neurotoxin A (BoNT/A), twitches elicited by supramaximal nerve stimulation (0.1 Hz) were reduced to approximately 10% of control in 3 hr at 37 degrees C. Addition of 3,4-DAP led to a rapid reversal of the BoNT/A-induced depression of twitch tension. In the presence of 100 microM 3,4-DAP, antagonism of the BoNT/A-induced blockade began within 30-40 sec and reached 82% of control with a half-time of 6.7 min. The beneficial effect of 3,4-DAP was well maintained and underwent little or no decrement relative to control for at least 8 hr after addition. Application of 1 microM neostigmine 1 hr after 3,4-DAP led to a further potentiation of twitch tension, but this action lasted for < 20 min. Moreover, neostigmine caused tetanic fade during repetitive stimulation. In contrast to the efficacy of the parent compound, the quaternary derivative of 3,4-DAP, 3,4-diamino-1-methyl pyridinium produced little or no twitch potentiation up to a concentration of 1 mM. The potassium channel blocker, tetraethylammonium, generated a transient potentiation followed by a sustained depression of twitch tensions. It is concluded that 3,4-DAP is of benefit in antagonizing the muscle paralysis following exposure to BoNT/A. Co-application of neostigmine or tetraethylammonium with 3,4-DAP, however, appears to confer no additional benefit.

Assessment of primary neuronal culture as a model for soman-induced neurotoxicity and effectiveness of memantine as a neuroprotective drug.

An in vitro mammalian model neuronal system to evaluate the intrinsic toxicity of soman and other neurotoxicants as well as the efficacy of potential countermeasures was investigated. The link between soman toxicity, glutamate hyperactivity and neuronal death in the central nervous system was investigated in primary dissociated cell cultures from rat hippocampus and cerebral neocortex. Exposure of cortical or hippocampal neurons to glutamate for 30 min produced neuronal death in almost 80% of the cells examined at 24 h. Hippocampal neurons exposed to soman for 15–120 min at 0.1 μM concentration caused almost complete inhibition (⋝90%) of acetylcholinesterase but failed to show any evidence of effects on cell viability, indicating a lack of direct cytotoxicity by this agent. Acetylcholine (ACh, 0.1 mM), alone or in combination with soman, did not potentiate glutamate toxicity in hippocampal neurons. Memantine, a drug used for the therapy of Parkinson’s disease, spasticity and other brain disorders, significantly protected hippocampal and cortical neurons in culture against glutamate and N-methyl-D-aspartate (NMDA) excitotoxicity. In rats a single dose of memantine (18 mg/kg) administered 1 h prior to a s.c. injection of a 0.9 LD50 dose of soman reduced the severity of convulsions and increased survival. Survival, however, was accompanied by neuronal loss in the frontal cortex, piriform cortex and hippocampus.

Physiological properties of the innervated and denervated neuromuscular junction of hibernating and nonhibernating ground squirrels

Abstract The physiological status of the neuromuscular junction of hibernating and nonhibernating 13-lined ground squirrels was studied to determine whether or not the metabolic changes during hibernation would alter the muscles response to denervation. It was anticipated that the observations might clarify some aspects of the trophic interrelationship between nerve and muscle. The properties of innervated muscles were not significantly altered after the animals entered hibernation. The strength of contraction, speed of contraction, and resting membrane potential remained unchanged. In addition, extrajunctional sensitivity to acetylcholine did not develop. Because the muscles are inactive during hibernation, we conclude that muscle activity alone does not maintain the physiological properties of muscles. Denervation of muscles from nonhibernating animals resulted in loss of neuromuscular transmission, cessation of miniature end-plate potentials, partial muscle membrane depolarization, and appearance of extrajunctional sensitivity to acetylcholine. In contrast, muscles whose nerves were transected during the hibernating state showed unimpaired neuromuscular transmission and normal miniature end-plate potentials. However, the muscle became partially depolarized, indicating that the regulation of the resting membrane potential is under neurotrophic control and is not influenced solely by the release of acetylcholine (which had remained unchanged). The denervated muscles of hibernating animals did not develop extrajunctional sensitivity to acetylcholine; this probably reflects the low rate of protein turnover in tissues maintained at the low (7°C) body temperature of hibernating animals. Transection of the sciatic nerve of hibernating animals produced histologically demonstrable retrograde changes in the motor neurons of the lumbar spinal cord. It thus appears that hibernation does not adversely affect certain fundamental functions of the nervous system, such as transmission of nerve impulses, anterograde transmission of neurotrophic influences, and the retrograde transmission of signals which initiate the cell bodys reaction to injury.