In vitro reinnervation of adult rat muscle fibres by foreign neurons and transformed chromaffin PC12 cells

Adult rat muscle fibres were dissociated by using collagenase and maintained in culture. One to nine days later, neurons obtained from stages 22-30 Xenopus laevis embryos, or neonatal spinal cord, or pheochromocytoma (PC12) cells treated with nerve growth factor were added. Subsequently, the co-cultures were maintained for up to eight days. Functional synapses were formed with variable efficiency: 12% in rat-Xenopus nerve-muscle co-cultures, 23% in rat-rat and 33% in PC12 co-cultures. Miniature endplate potentials (MEPPs) and currents (MEPCs) were recorded, at frequencies ranging from 0.01 to 0.9 Hz. Their mean amplitude was smaller than in normal mammalian muscles. The rise time and time-constant of decay of MEPCs was about seven to ten times longer than that found in the original muscle, resembling immature synapses. (+)-Tubocurarine abolished the MEPPs in the rat-PC12 neuromuscular junctions. It is concluded that dissociated adult rat muscle fibres retain their ability of being reinnervated, and can form functional synapses with foreign neurons and transformed chromaffin cells.     

Estimates of quantal content during 'chemical potentiation' of transmitter release.

The number of quantal transmitter packets (m), released from motor nerve terminals in response to a single stimulus, has been estimated from the ratio of the amplitudes of endplate currents (e.p.c.) to spontaneous miniature endplate currents (m.e.p.c.), in voltage-clamped endplates of the frog. At 6 degrees C, the average value of m at normal nerve-muscle junctions was about 300. If allowance is made for the temporal dispersion of quantal transmitter release during the e.p.c., this value is increased by about 30%. After treatment with diaminopyridine or tetraethylammonium, transmitter release in response to a nerve stimulus is greatly enhanced and values of m exceeding 10(4) are frequently found. Moreover, the duration of the e.p.c. becomes much longer than that of the m.e.p.cs. The number of packets then liberated during the e.p.c. is much larger than the number of 'active zones' of the endplate and may even exceed the total number of vesicles lined up in twin-files adjacent to the presynaptic membrane.     

Effects of vinblastine and colchicine on the postsynaptic membrane at the frog neuromuscular junction

The possible effects of the alkaloids vinblastine and colchicine on the postsynaptic membrane of the frog neuromuscular junction were investigated using voltage-clamp techniques. Concentrations of vinblastine and colchicine which had been shown to exert no effect on the amplitude and duration of miniature endplate currents (MEPC) and the current-voltage relationship of low-quantal endplate currents (EPC) together with the coefficient of voltage-dependent EPC decay did produce a considerable rise in the amplitude of response to iontophoretically applied acetylcholine (ACh). In addition, vinblastine and colchicine accelerate MEPC and EPC during acetylcholine esterase inhibition while further depressing the amplitude of multi-quantal EPC succeeding at the rate of 10 Hz as well as response to regular (5–10 Hz) application of ACh from a micropipet. The dosage-frequency effects of vinblastine and colchicine on the postsynaptic membrane (as described) are presumed to be unconnected with the action of these agents on muscle fiber cytoskeleton but the results of accelerated desensitization of cholinoreceptors.S. V. Kurashov Medical Institute, Kazan. Translated from Neirofiziologiya, Vol. 20, No. 1, pp. 75–81, January–February, 1988.     

Butyrylcholinesterase and the control of synaptic responses in acetylcholinesterase knockout mice

At the neuromuscular junction (NMJ) acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) can hydrolyze acetylcholine (ACh). Released ACh quanta are known to diffuse rapidly across the narrow synaptic cleft and pairs of ACh molecules cooperate to open endplate channels. During their diffusion through the cleft, or after being released from muscle nicotinic ACh receptors (nAChRs), most ACh molecules are hydrolyzed by AChE highly concentrated at the NMJ. Advances in mouse genomics offered new approaches to assess the role of specific cholinesterases involved in synaptic transmission. AChE knockout mice (AChE-KO) provide a valuable tool for examining the complete abolition of AChE activity and the role of BChE. AChE-KO mice live to adulthood, and exhibit an increased sensitivity to BChE inhibitors, suggesting that BChE activity facilitated their survival and compensated for AChE function. Our results show that BChE is present at the endplate region of wild-type and AChE-KO mature muscles. The decay time constant of focally recorded miniature endplate currents was 1.04 +/- 0.06 ms in wild-type junctions and 5.4 ms +/- 0.3 ms in AChE-KO junctions, and remained unaffected by BChE-specific inhibitors, indicating that BChE is not limiting ACh duration on endplate nAChRs. Inhibition of BChE decreased evoked quantal ACh release in AChE-KO NMJs. This reduction in ACh release can explain the greatest sensitivity of AChE-KO mice to BChE inhibitors. BChE is known to be localized in perisynaptic Schwann cells, and our results strongly suggest that BChE's role at the NMJ is to protect nerve terminals from an excess of ACh.     

Cell-cell interaction during synaptogenesis

1. Neuromuscular synapse formation was studied using nerve and muscle cells dissociated from Xenopus embryos and kept in culture for 1 to 3 days. Within a few minutes of manipulated contact with isolated cholinergic neurons, miniature endplate potential-like depolarizations (MEPPs) due to spontaneous release of acetylcholine (ACh) from the neurons were detected in the muscle cells. 2. Addition of an antibody to a frog neural cell adhesion molecule (anti-NCAM) into the culture medium of nerve-muscle co-cultured for 1-3 days decreased the percentage of functional nerve-muscle contacts. 3. Acute exposure to anti-NCAM (1 hour) inhibited significantly muscle cell contact-triggered ACh release from initially identified cholinergic neurons. 4. Lysed muscle cells manipulated into contact with neurons induced ACh release, whereas lysed neurons did not, suggesting the presence of specific molecules on the muscle cell membrane capable of triggering ACh release from the cholinergic neuron. 5. Transient appearance of electrical coupling was detected between neuronal soma and muscle cell, suggesting the possibility of exchange of modulators for the formation and maintenance of neuromuscular synapses. 6. Neuromuscular synaptogenesis constitutes a complex process where at least two different types of direct cell-cell interaction seem to occur: a) cell surface molecule contact (and binding) for cell recognition and triggering of ACh release, and b) transient intercytoplasmic communication between the cells for possible passage of modulatory molecules.     

Discrepancies between spontaneous and evoked synaptic potentials at normal, regenerating and botulinum toxin poisoned mammalian neuromuscular junctions

Amplitudes and times to peak of spontaneous miniature endplate potentials (m.e.p.ps) and evoked quantal endplate potentials (e.p.ps) were compared at normal, regenerating and botulinum toxin poisoned neuromuscular junctions of the extensor digitorum longus muscle of the rat. At normal junctions the mean time to peak of m.e.p.ps was longer and more variable than that of similar-sized e.p.ps. At endplates where nerve regeneration was induced by mechanical crushing of the motor nerve the frequency of m.e.p.ps was reduced and their amplitude distribution was broader than normal. The distribution of times to peak of m.e.p.ps was considerably broader than that of quantal e.p.ps recorded at the same endplates. At neuromuscular junctions poisoned with botulinum toxin type A, spontaneous and evoked transmitter release were greatly reduced. The amplitude distribution of m.e.p.ps was wider than that of e.p.ps and the time to peak of e.p.ps was about twice as fast as and less variable than that of m.e.p.ps. To explain the observed differences in time to peak among m.e.p.ps and between m.e.p.ps and quantal e.p.ps we suggest that some m.e.p.ps, but not e.p.ps, originate from transmitter quanta released from sites at a greater distance from postsynaptic receptors or that the release or diffusion process for acetylcholine is more prolonged when producing some of the m.e.p.ps. Such mechanisms produce at normal junctions a small population of m.e.p.ps with prolonged times to peak, at regenerating junctions a greater proportion of such m.e.p.ps and in botulinum toxin poisoning a majority.     

EFFECTS OF NEUROMUSCULAR BLOCKING AGENTS ON THE DIFFERENTIATION OF NERVE-MUSCLE CONNECTIONS IN SLOW AND FAST CHICK MUSCLES

The effects of neuromuscular blocking drugs on the development of slow and fast muscle fibres and their neuromuscular junctions was studied in chick embryos.
Treatment of embryos with the depolarizing neuromuscular blocking agent suxamethonium affected the development of muscle fibres of the slow anterior latissimus dorsi (ALD) muscle more than that of muscle fibres of the posterior latissimus dorsi (PLD). The differentiation of the presynaptic elements of the neuromuscular junction was delayed and this was particularly obvious in PLD. Normally the number of axon profiles at individual endplates is reduced by 18 days of incubation, but in suxamethonium treated embryos this reduction took place only at 21 days. During earlier stages of development the axon profiles from treated embryos were small with sparse synaptic vesicles. Nevertheless the subsynaptic site of endplates on ALD and PLD muscle fibres became specialized earlier than normal and to a greater extent. Treatment with hemicholinium (HC-3), a drug that reduces the synthesis of acetylcholine (ACh) in nerve terminals affected the development of PLD muscle fibres more than ALD muscle fibres. Although in HC-3 treated embryos nerve-muscle contacts were formed, the axon terminals look immature and remain small even in 18-day old embryos at both ALD and PLD muscle fibres. The reduction of the number of axon profiles normally seen at 18 days failed to take place in treated embryos. At 18 days of incubation many endplates on PLD muscle fibres showed little sign of postsynaptic specilization and resembled endplates usually seen at this stage on ALD muscle fibres.
It is concluded that while neuromuscular activity may be important for the reduction of the number of axon profiles at individual endplates, the specialization of the subsynaptic membrane is brought about by depolarizing effect of ACh.     

In vitro regulation of the innervation pattern of quail muscle fibres by quail and mouse neurons

Myoblasts from rudiments of slow and fast muscle, anterior latissimus dorsi (ALD) and posterior latissimus dorsi (PLD) respectively, of 9-day-old quail embryos were cultured in vitro for a period of up to 60 days in order to give rise to well-differentiated muscle fibres. These fibres were innervated by neurons from either quail or mouse embryo spinal cord and their innervation pattern was examined by the visualization of acetylcholine receptors (ACh-R) and of acetylcholinesterase (ACh-E) activity at the neuromuscular contacts. In the culture system used, quail neurons always innervated muscle fibres at several sites and only when a fast-type activity was imposed on these neurons did a reduction in the number of the previously established neuromuscular contacts take place. In contrast, in the muscle fibres innervated by mouse neurons, a spontaneous reduction in the number of the previously established neuromuscular contacts occurred but this spontaneous reduction depended upon the level of differentiation reached by the muscle fibres in vitro. In the cultures of muscle fibres previously innervated by mouse neurons, the addition of quail neurons did not provoke any modification in the initial innervation pattern, and no quail ACh-R cluster was observed. In contrast, in the muscle fibres previously innervated by quail neurons, the mouse neurons contacted these fibres, resulting in a decrease in the number of quail ACh-R clusters. These results emphasize the part played by neurons in the establishment of the innervation pattern when muscle fibres have reached a high level of differentiation. In vitro, the slow and fast characteristics of the muscle fibres do not influence this pattern.     

Endplate channel block by guanidine derivatives

The effects of the n-alkyl derivatives of guanidine on the frog neuromuscular junction were studied using the two-microelectrode voltage clamp and other electrophysiological techniques. Methyl-, ethyl- , and propylguanidine stimulated the nerve-evoked release of transmitter. However, amyl-and octylguanidine had no apparent presynaptic action. All of the derivatives blocked the postsynaptic response to acetylcholine, the potency sequence being octyl-greater than amyl-greater than propyl-, methyl-greater than ethylguanidine. Methyl- and octylguanidine did not protect the receptor from alpha- bungarotoxin block, suggesting that these compounds do not bind to the receptor but probably block the ionic channel. Methyl-, ethyl-, and propylguanidine shortened inward endplate currents but prolonged outward currents. Amylguanidine prolonged both inward and outward endplate currents, and the currents became biphasic at negative membrane potentials. Octylguanidine increased the rate of decay of endplate currents at all potentials. All of the derivatives blocked inward endplate currents more markedly than outward currents, resulting in a highly nonlinear current-voltage relation. Methyl-, ethyl-, and propylguanidine reversed the voltage dependence of endplate current decay, while amyl-and octylguanidine reduced the voltage dependence of endplate current decay. Octylguanidine appears to block the ionic channel in both the open and the closed state. The block of the open channel follows pseudo-first-order kinetics with a forward rate constant of 4-6 X 10(7) M-1 s-1.     

The rise times of miniature endplate currents suggest that acetylcholine may be released over a period of time.

Models of miniature endplate currents predict 20-80% rise times of 100 microseconds or less. These predictions are substantially less than most of the rise times recorded in the literature. New measurements were made of rise times at the frog neuromuscular junction using extracellular recording. The mean 20-80% rise time was 250 microseconds. Rise times were variable; at 20 degrees C, 95% of them fell in a range from 140 to 460 microseconds. The most questionable assumption in the models is that the acetylcholine (ACh) is released instantaneously. Modifying the model, so that ACh diffuses from the vesicle through a pore, lengthens the rise time to observed levels. It has been proposed that ACh is released from the vesicle in exchange for Na+. However, the rise times of miniature endplate currents recorded in solutions in which the Na+ is replaced by sucrose are in the normal range. The Q10 for the rise of miniature endplate currents is approximately 2, which is consistent with the models and with temperature effects on pore formation in mast cells.     

Effect of N-acetylaspartylglutamate (NAAG) on non-quantal and spontaneous quantal release of acetylcholine at the neuromuscular synapse of rat

N-Acetylaspartylglutamate (NAAG), known to be present in rat motor neurons, may participate in neuronal modulation of non-quantal secretion of acetylcholine (ACh) from motor nerve terminals. Non-quantal release of ACh was estimated by the amplitude of the endplate membrane hyperpolarization (H-effect) caused by inhibition of nicotinic receptors by (+)-tubocurarine and acetylcholinesterase by armin (diethoxy-p-nitrophenyl phosphate). Application of exogenous NAAG decreased the H-effect in a dose-dependent manner. The reduction of the H-effect by NAAG was completely removed when N-acetyl-beta-aspartylglutamate (betaNAAG) or 2-(phosphonomethyl)-pentanedioic acid (2-PMPA) was used to inhibit glutamate carboxypeptidase II (GCP II), a presynaptic Schwann cell membrane-associated ectoenzyme that hydrolyzes NAAG to glutamate and N-acetylaspartate. Bath application of glutamate decreased the H-effect similarly to the action of NAAG but N-acetylaspartate was without effect. Inhibition of NMDA receptors by dl-2-amino-5-phosphopentanoic acid, (+)-5-methyl-10,11-dihydro-5H-dibenzocyclohepten-5,10-imine (MK801), and 7-chlorokynurenic acid or inhibition of muscle nitric oxide synthase (NO synthase) by N(G)-nitro-l-arginine methyl ester and 3-bromo-7-nitroindazole completely prevented the decrease of the H-effect by NAAG. These results suggest that glutamate, produced by enzymatic hydrolysis of bath-applied NAAG, can modulate non-quantal secretion of ACh from the presynaptic terminal of the neuromuscular synapse via activation of postsynaptic NMDA receptors and synthesis of nitric oxide (NO) in muscle fibers. NAAG also increased the frequency of miniature endplate potentials (mEPPs) generated by spontaneous quantal secretion of ACh, whereas the mean amplitude and time constants for rise time and for decay of mEPPs did not change.     

Role of Acetylcholinesterase on the Structure and Function of Cholinergic Synapses: Insights Gained from Studies on Knockout Mice

Electrophysiological and ultrastructural studies were performed on phrenic nerve-hemidiaphragm preparations isolated from wild-type and acetylcholinesterase (AChE) knockout (KO) mice to determine the compensatory mechanisms manifested by the neuromuscular junction to excess acetylcholine (ACh). The diaphragm was selected since it is the primary muscle of respiration, and it must adapt to allow for survival of the organism in the absence of AChE. Nerve-elicited muscle contractions, miniature endplate potentials (MEPPs) and evoked endplate potentials (EPPs) were recorded by conventional electrophysiological techniques from phrenic nerve-hemidiaphragm preparations isolated from 1.5- to 2-month-old wild-type (AChE^+/+) or AChE KO (AChE^−/−) mice. These recordings were chosen to provide a comprehensive assessment of functional alterations of the diaphragm muscle resulting from the absence of AChE. Tension measurements from AChE^−/− mice revealed that the amplitude of twitch tensions was potentiated, but tetanic tensions underwent a use-dependent decline at frequencies below 70 Hz and above 100 Hz. MEPPs recorded from hemidiaphragms of AChE^−/− mice showed a reduction in frequency and a prolongation in decay (37%) but no change in amplitude compared to values observed in age-matched wild-type littermates. In contrast, MEPPs recorded from hemidiaphragms of wild-type mice that were exposed for 30 min to the selective AChE inhibitor 5-bis(4-allyldimethyl-ammoniumphenyl)pentane-3-one (BW284C51) exhibited a pronounced increase in amplitude (42%) and a more marked prolongation in decay (76%). The difference between MEPP amplitudes and decays in AChE^−/− hemidiaphragms and in wild-type hemidiaphragms treated with BW284C51 represents effective adaptation by the former to a high ACh environment. Electron microscopic examination revealed that diaphragm muscles of AChE^−/− mice had smaller nerve terminals and diminished pre- and post-synaptic surface contacts relative to neuromuscular junctions of AChE^+/+ mice. The morphological changes are suggested to account, in part, for the ability of muscle from AChE^−/− mice to function in the complete absence of AChE.     

The Role of Endogenous Calcitonin Gene-Related Peptide in the Neurotransmitter Quantal Size Increase in Mouse Neuromuscular Junctions

Changes in parameters of spontaneous acetylcholine (ACh) quantal secretion caused by prolonged high-frequency burst activity of neuromuscular junctions and possible involvement of endogenous calcitonin gene-related peptide (CGRP) and its receptors in these changes were studied. With this purpose, miniature endplate potentials (MEPPs) were recorded using standard microelectrode technique in isolated neuromuscular preparations of m. EDL–n. peroneus after a prolonged high-frequency nerve stimulation (30 Hz for 2 min). An increase in the MEPP amplitudes and time course was observed in the postactivation period that reached maximum 20–30 min after nerve stimulation and progressively faded in the following 30 min of recording. Inhibition of vesicular ACh transporter with vesamicol (1 μM) fully prevented this “wave” of the MEPP enhancement. This indicates the presynaptic origin of the MEPP amplitude increase, possibly mediated via intensification of synaptic vesicle loading with ACh and subsequent increase of the quantal size. Competitive antagonist of the CGRP receptor, truncated peptide isoform CGRP_8–37 (1 μM), had no effect on spontaneous secretion parameters by itself but was able to prevent the appearance of enhanced MEPPs in the postactivation period. This suggests the involvement of endogenous CGRP and its receptors in the observed MEPP enhancement after an intensive nerve stimulation. Ryanodine in high concentration (1 μM) that blocks ryanodine receptors and stored calcium release did not influence spontaneous ACh secretion but prevented the increase of the MEPP parameters in the postactivation period. Altogether, the data indicate that an intensive nerve stimulation, which activates neuromuscular junctions and muscle contractions, leads to a release of endogenous CGRP into synaptic cleft and this release strongly depends on the efflux of stored calcium. The released endogenous CGRP is able to exert an acute presynaptic effect on nerve terminals, which involves its specific receptor action and intracellular cascades leading to intensification of ACh loading into synaptic vesicles and an increase in the ACh quantal size.     

Postsynaptic potentiation and desensitization at the frog neuromuscular junction produced by repeated stimulation of the motor nerve

Investigations were performed on a split neuromuscular preparation of frog sartorial muscle during acetylcholinesterase inhibition. A study was made of the part played in postsynaptic potentiation (PSP) and desensitization (DS) in changes in the amplitude and time course of miniature endplate currents (MEPC) recorded directly after regular stimulation of the motor nerve at a frequency of 10 Hz for 5 or 60 sec, producing short and long series of multiquantal endplate currents (EPC) respectively. After the short train the amplitude of MEPC could hardly be distinguished from initial level, while the decay time constant (MEPC) increased by 32%, indicating PSP. Comparable but more pronounced biphasic changes occurred in the time course of endplate currents. These effects were not observed when acetylcholinesterase was uninhibited. Both PSP and DS were restored when 1×10^–6 M exogenous acetylcholine was added to the bath. The ratio between them could be changed by aprodifen — a substance which accelerates desensitization.S. V. Kurashov Medical Institute, Ministry of Public Health of the RSFSR, Kazan'. I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 18, No. 5, pp. 645–654, September–October, 1986.     

Effects of a Dendroaspis neurotoxin on synaptic transmission in the spinal cord and the neuromuscular junction of the frog

A neurotoxin from the venom of Dendroaspis jamesoni was tested at the neuromuscular junction and at a cholinergic pathway in the isolated spinal cord of the frog. The toxin reduced the amplitude and time constant of decay of miniature endplate currents in the presence of prostigmine, indicating a curare-like action. In the spinal cord it selectively blocked transmission in the cholinergic pathway and increased spontaneous activity. Partial protection against toxin action in the spinal cord was provided by atropine or carbachol. The results suggest that the toxin acts on cholinergic receptors at both sites and also provide further evidence that the pharmacology of the two sites is different.     

Effects of hyaluronidase on miniature endplate potentials and currents at the frog neuromuscular junction

The effects of 0.1% testicular hyaluronidase on miniature endplate potentials and currents (MEPP and MEPC) were investigated in frog pectorocutaneous muscle. The action of hyaluronidase on preparations with armine-induced blockade of acetylcholinesterase was associated with decreased amplitude and duration of MEPP and MEPC half-decay time and rising phase. The correlation between amplitude and half-decay time of MEPP and MEPC declined at the same time, while MEPC decay remained exponential. Treating preparations having intact acetylcholinesterase with hyaluronidase increased the length of MEPC halfdecay, with duration of the rising phase and amplitude remaining constant. It is suggested that enzymatic breakdown of a proportion of the glycocalix of cells forming the neuromuscular junction and a portion of the extracellular matrix at the synaptic cleft leads to attenuation of nonspectific acetylcholine binding, thus facilitating acetylcholine diffusion into the synaptic cleft.A. A. Zhdanov State University, Leningrad. Translated from Neirofiziologiya, Vol. 20, No. 1, pp. 113–119, January–February, 1988.     

Acetylcholine sensitivity of innervated and noninnervated Xenopus muscle cells in culture

The chemosensitivity of Xenopus muscle cells grown in culture to iontophoretically applied acetylcholine (ACh) in the presence or absence of neurons was examined. Muscle cells grown without nerve cells are sensitive to ACh over their entire surface (2.4 mV/pC) with occasional spots of high chemosensitivity (“hot spots”). In cultures containing neural tube cells, the ACh sensitivity of muscle cells increased by approximately 50% regardless of the presence of nerve contacts or functional synapses. A similar increase in the ACh sensitivity was observed in muscle cells cultured in medium conditioned by neural tube cells. The ACh sensitivity of the extrajunctional region in functionally innervated muscle cells was not different from that of noninnervated cells growing in the same cultures. However, the chemosensitivity at the junctional region was about fivefold higher than that of the extrajunctional area. This increase in junctional chemosensitivity may well account for the increase in miniature endplate potential amplitude which has previously been reported to occur during nerve-induced ACh receptor accumulation.     

Developmental changes in transmitter sensitivity and synaptic transmission in embryonic chicken sympathetic neurons innervated in vitro.

Dispersed neurons from embryonic chicken sympathetic ganglia were innervated in vitro by explants of spinal cord containing the autonomic preganglionic nucleus or somatic motor nucleus. The maturation of postsynaptic acetylcholine (ACh) sensitivity and synaptic activity was evaluated from ACh and synaptically evoked currents in voltage-clamped neurons at several stages of innervation. All innervated cells are more sensitive to ACh than uninnervated neurons regardless of the source of cholinergic input. Similarly, medium conditioned by either dorsal or ventral explants mimics innervation by enhancing neuronal ACh sensitivity. This increase is due to changes in the rate of appearance of ACh receptors on the cell surface. There are also several changes in the nature of synaptic transmission with development in vitro, including an increased frequency of synaptic events and the appearance of larger amplitude synaptic currents. In addition, the mean amplitude of the unit synaptic current mode increases, as predicted from the observed changes in postsynaptic sensitivity. Although spontaneous synaptic current amplitude histograms with multimodal distributions are seen at all stages of development, histograms from early synapses are typically unimodal. Changes in the synaptic currents and ACh sensitivity between 1 and 4 days of innervation were paralleled by an increase in the number of synaptic events that evoked suprathreshold activity in the postsynaptic neurons. The early pre- and postsynaptic differentiation described here for interneuronal synapses formed in vitro may be responsible for increased efficacy of synaptic transmission during development in vivo.     

Intracellular regulation of neuronal nicotinic cholinergic receptors

Effects of substances affecting intracellular secondary messengers on the membrane currents evoked by ionophoretic application of acetylcholine (ACh currents) and on the excitatory postsynaptic currents (EPSC) evoked by single stimuli applied to preganglionic nerve fibres, were studied in neurones of the rat isolated superior cervical ganglion. Forskolin, the protein kinase A activator, and isobutyl-methyxanthine, the phosphodiesterase inhibitor, decreased the ACh currents. Neither forskolin nor isobutyl-methylxanthine affected the EPSC amplitude or the EPSC decay time constant. Phorbol ester, the protein kinase C activator, decreased the ACh current but did not affect either EPSC amplitude or the EPSC decay time constant. Thapsigargin, the intracellular calcium releaser, decreased the ACh current and the EPSC amplitude but did not affect the EPSC decay time constant. The data obtained suggest that nicotinic acetylcholine receptors (nAChRs) of ganglion neurones are not modulated through the pathways involving protein kinase A or protein kinase C. The nAChRs sensitivity to both exogenous and nerve-released acetylcholine is reduced by intracellular calcium without affecting kinetics of their ionic channels.     

Reaction of tetraethylammonium with the open and closed conformations of the acetylcholine receptor ionic channel complex

The effect of tetraethylammonium (TEA) bromide on the neurally and iontophoretically evoked endplate current (EPC) of frog sartorius muscle was investigated using voltage-clamp and noise analysis techniques, and its binding to the acetylcholine (ACh) receptor ionic channel complex was determined on the electric organ of Torpedo ocellata. TEA (250-500 microM) produced an initial enhancement followed by a slow decline in the amplitude of the endplate potential and EPC, but caused only depression in the amplitude of the miniature endplate potential and current. In normal ringer's solution, the EPC current-voltage relationship was approximately linear, and the decay phase varied exponentially with membrane potential. Upon addition of 50-100 microM TEA, the current-voltage relationship became markedly nonlinear at hyperpolarized command potentials, and with 250-2000 microM TEA, there was an initial linear segment, an intermediate nonlinear segment, and a region of negative conductance. The onset of nonlinearity was dose-dependent, undergoing a 50 mV shift for a 10-fold increase in TEA concentration. The EPC decay phase was shortened by TEA at hyperpolarized but not depolarized potentials, and remained a single expotential function of time at all concentrations and membrane potentials examined. These actions of TEA were found to be independent of the sequence of polarizations, the length of the conditioning pulse, and the level of the initial holding potential. TEA shifted the power spectrum of ACh noise to higher frequencies and produced a significant depression of single channel conductance. The shortening in the mean channel lifetime agreed closely with the decrease in the EPC decay time constant. At the concentrations tested, TEA did not alter the EPC reversal potential, nor the resting membrane potential, and had little effect on the action potential duration. TEA inhibited the binding of both [3H] ACh (Ki = 200 microM) and [3H]perhydrohistrionicotoxin (Ki = 280 microM) to receptor-rich membranes from the electric organ of Torpedo ocellata, and inhibited the carbamylcholine-activated 22Na+ efflux from these microsacs. It is suggested that TEA reacts with the nicotinic ACh-receptor as well as its ion channel; the voltage-dependent actions are associated with blockade of the ion channel. The results are compatible with a kinetic model in which TEA first binds to the closed conformation of the receptor-ionicchannel complex to produce a voltage-depdndent depression of endplate conductance and sudsequently to its open conformation, giving rise to the shortening in the EPC decay and mean channel lifetime.