The effect of acetylcholine on the function and structure of the developing mammalian neuromuscular junction

Isolated soleus muscles from rats aged 9–12 days were exposed to acetylcholine for 2 h in normal Krebs solution. This treatment caused changes in the ultrastructural appearance of the neuro-muscular junction and a significant reduction of axon profiles per endplate. Nevertheless, most neuro-muscular junctions remained functional, since the ratio of the indirectly to directly elicited contraction was not reduced.If muscles were exposed to acetylcholine in Krebs solutions containing 12m M Ca²⁺ instead of the normal 1.9 m M, the ultrastructural changes produced by acetylcholine were more severe, and the number of axon terminals per endplate was further reduced so that many endplates became completely denervated. This was also reflected in the impaired function of the nerve-muscle preparation; the ratio of the indirectly to directly elicited contraction decreased and about 40% of the muscles fibres became functionally denervated. Addition of curare to the incubating medium prevented the functional deterioration of the preparation.Addition of the protease inhibitors leupeptin and pepstatin protected the nerve terminals from the damaging effects of acetylcholine in Krebs solution containing 12m M Ca²⁺ and the number of axon profiles per endplate remained normal. The functional deterioration was also much reduced when protease inhibitors were included in the incubation medium.These results suggest that acetylcholine causes the activation and release of proteolytic enzymes in developing muscles. The response is mediated by calcium and may have a role in the removal of superfluous nerve-muscle contacts during development.     

The release of acetylcholine and of catecholamine from the cat's adrenal gland

The cat's adrenal gland was perfused in situ with Krebs solution containing eserine; the amount of acetylcholine and of catecholamine released was measured. Splanchnic nerve stimulation (5 Hz for 2 min) increased the release of acetylcholine and catecholamine; the molar ratio of evoked release of catecholamine to acetylcholine was 122 +/- 8. It is suggested that this amplification is achieved because a chromaffin cell granule contains more mediator than does the acetylcholine quantum that releases it. The release per impulse of catecholamine during splanchnic nerve stimulation at 30 Hz was less than that released by stimulation at 1 or 5 Hz. This depression is attributed to a presynaptic failure, because the release of acetylcholine was similarly frequency dependent. The release of catecholamine was linearly related to the release of acetylcholine over the range tested, indicating that the input-output relationship at the splanchnic-adrenal medullary junction is linear. During continuous stimulation of the splanchnic nerve (5 Hz), catecholamine release declined to a level that was 32 +/- 2% of the initial output. This fatigue is attributed primarily to a postsynaptic depression, because the release of acetylcholine was maintained at 71 +/- 6% of its initial level. The presence of eserine in the perfusate was necessary for the release of acetylcholine to be detected, but in the presence of eserine catecholamine release was 90 +/- 10% that in the drug's absence. It is concluded that released acetylcholine is hydrolysed at some distance from its site of release and action. Glands perfused with raised K+ released acetylcholine and catecholamine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ontogenetic development of acetylcholine and atropine epileptogenic cortical foci in rats.

In acute experiments on male albino rats aged from 9 days to adulthood, cortical epileptogenic foci were elicited by the local application of acetylcholine and/or atropine. Focal spike discharges after the administration of acetylcholine were registered for the first time in 12-day old rats; during the third week of life, groups of spikes and ictal activity appeared, so that 20-day-old rats did not differ qualitatively from adult animals. Subsequent application of atropine blocked the actions of acetylcholine in all age groups. The isolated local application of atropine provoked focal discharges already in 9-day-old rats and the shape of the discharges did not change during the whole course of development. The transmission of discharges of acetylcholine- as well as atropine-foci to the other cortical areas was slight in all age groups.The results indicate that different mechanisms are involved in the epileptogenic actions of the two drugs.     

Catecholamine uptake into isolated adrenal chromaffin cells: inhibition of uptake by acetylcholine

We have investigated the process of catecholamine uptake in guinea-pig chromaffin cells. Isolated guinea-pig chromaffin cells accumulate [3H]norepinephrine and [3H]epinephrine by a saturable transport system. Catecholamine uptake is dependent upon temperature, energy, and extracellular Na+. The apparent KmS for norepinephrine and epinephrine transport are approximately 1 and 3.5 microM, respectively; the transport maximum (Vmax) for both compounds is about 100 pmol/min/mg protein. The uptake of norepinephrine into chromaffin cells is inhibited by imipramine (Ki = 50 nM) and by desmethylimipramine (IC50 = 20 nM). In both its substrate specificity and its sensitivity to pharmacological inhibition, the catecholamine uptake system in chromaffin cells is similar to the catecholamine transport system previously described in sympathetic neurons. Decreasing external Na+ from 130 to 19 mM increases the apparent Km for norepinephrine to 2.8 microM. Decreasing external norepinephrine increases the Na+ concentration required for half-maximal transport. Agents that depolarize chromaffin cells, such as acetylcholine and veratridine, significantly inhibit [3H]norepinephrine uptake. This decrease in uptake is due to an increase in the apparent Km for norepinephrine. The inhibition of [3H]norepinephrine uptake by depolarizing agents cannot be accounted for by the preferential release of newly-accumulated [3H]norepinephrine, or by the competitive inhibition of [3H]norepinephrine uptake by secreted catecholamines. The inhibition of catecholamine uptake by depolarizing agents suggests that the transport system may be regulated by the membrane potential. Norepinephrine and epinephrine that are spontaneously released from the adrenal medulla may be recaptured in vivo. The inhibition of transport by acetylcholine may prevent the re-uptake of catecholamine released during the physiological stimulation of secretion.     

Biosynthesis and degradation of acetylcholine receptors in rat skeletal muscles. Effects of electrical stimulation.

Synthesis and degradation of acetylcholine receptors in rat skeletal muscles were measured in organ culture. The rate of de novo biosynthesis and incorporation of acetylcholine receptors into extrajunctional membranes of denervated muscles was measured by determining the rate of appearance of [1] [²H, ¹³C, ¹⁵N]-acetylcholine receptors when muscles were cultured in medium containing [1] [²H, ¹³C, ¹⁵N]-amino acids. Denervated extensor digitorum longus and soleus muscles were found to synthesize new receptors for several days in organ culture at an average rate of 1.4%/h. The degradation rates for extrajunctional and junctional acetylcholine receptors were estimated by irreversibly labeling acétylcholine receptors on muscles with radioactive iodinated α-bungarotoxin and measuring the rate of release into the culture medium of mono- and di-iodotyrosine, breakdown products of the radioactive α-bungarotoxin. The rates of this proteolytic process yielded average lifetimes of 22 h and 13 days for [¹²⁵I]α-bungarotoxin bound to extrajunctional and junctional receptors, respectively, probably reflecting the average lifetimes of the acetylcholine receptors.Electrical stimulation at 100Hz for 1 s every 80s, producing visible contraction, but not maximal tetanic tension, barely altered the rate of incorporation of new acetylcholine receptors into the extrajunctional plasma membrane of extensor digitorum longus and soleus muscles, even when the stimulation continued for 5 days. Supra-maximal stimulation, resulting in maximal tetanic tension, with the same stimulation pattern produced a rapid decline of 10–20% in the rate of new receptor production and a corresponding decline in overall protein synthesis. Stimulation beyond 18–24 h (up to 68 h) resulted in a further decrease in new receptor production to about 30% of the control rate, but not more. Stimulation for longer than 16 h produced less than a 5–10% decrease in overall protein synthesis, compared with control muscles. The same pattern of electrical stimulation, producing maximal tetanic tension, had no effect on the apparent degradation rate of extrajunctional receptors in denervated muscles.Our results show that denervated adult muscle can be maintained in organ culture for at least 1 week, and that the muscles in culture will continue to degrade acetylcholine receptors and to synthesize new receptors, even when electrically stimulated for 5 days. The significance of this study is that electrical stimulation, producing frequent tetanic contractions, can affect extrajunctional acetylcholine receptor metabolism by selectively decreasing de novo synthesis, the receptor degradation remaining unchanged. The precise control point in the biosynthetic process has yet to be determined. However, our results indicate that electrically induced activity does exert a regulating influence, relatively rapidly, on extrajunctional acetylcholine receptor metabolism on muscles maintained in vitro.     

Facilitation by acetylcholine of tetrodotoxin-resistant spikes in rat hippocampal pyramidal cells

The electrical activity of hippocampal pyramidal cells was studied in slice cultures during blockade of the regenerative Na currents. In the presence of tetrodotoxin, these neurones had a mean resting potential of -68 mV, a membrane input resistance of 87 M omega and displayed marked non-linearities in their current voltage relationship. In response to depolarizing stimuli, pyramidal cells generated action potentials of small amplitude, slow rise and long duration. These tetrodotoxin-resistant spikes were abolished by calcium conductance blockers such as cobalt and cadmium ions. Acetylcholine applied to the bath or by iontophoresis depolarized pyramidal cells, elicited spontaneous tetrodotoxin-resistant spikes and facilitated spiking evoked by depolarizing rectangular current pulses or a current ramp. The effects of acetylcholine were not only slow in onset, but also prolonged; they were completely reversible and sensitive to atropine and calcium-antagonists such as cadmium and cobalt ions which, respectively, reduced and abolished these effects. After hyperpolarizations following injection of depolarizing current pulses were suppressed by acetylcholine and often transformed into depolarizing afterpotentials. Acetylcholine had no effect on voltage-independent conductances as determined by application of hyperpolarizing current pulses. These results could be explained by inhibition of the voltage-dependent K+-current, i.e. the M current (blockade of the calcium current could remove any depolarizing influence resulting from M current inhibition) or by a direct activation of a voltage-dependent calcium current by muscarinic agonists.     

Cholinergic sensory inputs to command neurons in edible snail

We studied cholinergic component of visceral sensory input to defensive behavior command neurons in edible snail. Nicotinic receptor antagonist tubocurarine and muscarinic receptor antagonist atropine reversibly decreased the amplitude of the total excitatory postsynaptic potential induced by electrostimulation of the peripheral region in the mechanosensory receptor field of command neurons on the surface of internal organs. Our results indicate that acetylcholine is involved in sensory signal transduction from the visceral sac to command neurons of snail parietal ganglia. The subsynaptic membrane of visceral synaptic input contains nicotinic and muscarinic receptors. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 142, No. 9, pp. 244–247, September, 2006     

Disinhibitory action of acetylcholine in the rat's hippocampus: Extracellular observations

In rats under urethane anaesthesia, fimbrial-commissural stimulation at a frequency of 1 Hz or less evoked only a positive field in the pyramidal layer of CA1 (and the uppermost region of CA3). Stimulation at intensities 3–5 times threshold and frequencies of 2 or more Hz led to the appearance of large negative population spikes. When acetylcholine was released in the pyramidal layer from a micropipette (10–100 nA), the positive field was reduced and population spikes appeared, even at the lowest frequencies of stimulation (below 1 Hz).Known antagonists of γ-aminobutyrate (bicuculline, picrotoxin and penicillin) had a comparable effect, but with a slower time course—the action of acetylcholine consistently had a very rapid onset and offset (within 5 s). Population spikes were not evoked by acetylcholine when the accompanying fimbrial stimulation was not strong enough to generate spikes at higher frequencies (2 or more Hz), or when acetylcholine was released outside the narrow pyramidal zone where large positive fields are recorded; nor were they evoked by the release of glutamate instead of acetylcholine.On the basis of these observations, it was concluded that acetylcholine probably diminishes the efficiency of synaptic inhibition of pyramidal cells.     

A nerve stump dependent appearance of junctional and perijunctional acetylcholine receptors in organ culture

A rapid, nerve stump dependenl appearance of junctional and perijunctional acetylcholine receptors occurred after 42 h following the introduction of rat diaphragm into organ culture. Approximately2 × 10⁶ or more acetylcholine receptors (assayed by [¹²⁵I]alphabungarotoxin hinding) appeared at the endplate within a 2 h period, between 42 and 44 h, while only approximately2.5 × 10³ acetylcholine receptors appeared in an equivalent area of extrajunctional membrane during the same time. Autoradiographic studies confirmed that most of the new acetylcholine receptor sites appeared at the endplate, although a small perijunctional component was also detected. The presence of a long nerve stump prevented the increase in the number of receptors specific to the endplate region, but had no effect on the appearance of extrajunctional acetylcholine receptors. Pharmacological studies showed that the nerve stump effect did not involve nerve impulse transmission, or the interaction of nicotinic acetylcholine receptors with acetylcholine. The findings suggest that junctional and extrajunctional acetylcholine receptors are controlled by different neuronal mechanisms and that junctional receptors are subject to     

Intracellular injection of acetylcholine blocks various potassium conductances in vagal motoneurons

Injection of acetylcholine into cholinergic neurons of the dorsal motor nucleus of the vagus induced membrane depolarization, an increase in input resistance, a decrease of early and late afterhyperpolarizations and a prolongation of the action potential. These effects were reversible and within 10-20 min almost complete recovery was always observed. Externally applied acetylcholine, even with doses as high as 15 mM, was not effective. Acetylcholine appeared to block voltage- and Ca2+-dependent K+ conductances. This block was manifested by the reduction of both the early and late afterhyperpolarizations and a decrease of the delayed rectification. The reversal potential for the conductance decrease was 15-30 mV negative to the resting potential. As a result of this blockade an increased Ca2+ current ensues, which is responsible for most of the prolongation of the action potential. The same responses were obtained after the injection of carbamylcholine, neostigmine and choline. However, unlike acetylcholine no sign of recovery was observed. In fact injection of neostigmine, carbamylcholine or neostigmine, together with acetylcholine, produced a delayed response which may reflect the accumulation of endogenous acetylcholine.     

Evidence that transmitter can be released from regions of the nerve cell other than presynaptic axon terminal: axonal release of acetylcholine without modulation

Release of acetylcholine from isolated preganglionic axons of sympathetic nerve trunk (cervical preganglionic sympathetic branch) of the cat was studied. In response to depolarization (KCl, 48.4 mM) acetylcholine was released into the eserinized Krebs solution. This release was shown to be dependent on extracellular Ca2+. Electrical stimulation (1 Hz) enhanced the release of acetylcholine from the isolated axonal preparation. The release by stimulation proved to be tetrodotoxin-sensitive and Ca2+-dependent. Evidence has been obtained that the acetylcholine released from sympathetic nerve trunks originates from the axon and not from Schwann cells: 5 days after section of the nerve, there was no release in response to stimulation. The release of acetylcholine from the axon is unlike that from axon terminals in that the rate of release cannot be enhanced by the inhibition of Na, K-adenosine 5'-triphosphatase (ouabain 2 X 10(-5) M) and cannot be modulated by noradrenaline (10(-6) M) or by morphine. Furthermore, although isolated nerve trunks took up [3H]choline by a hemicholinium-sensitive process, no radioactivity could be released upon electrical stimulation. It is suggested that the release of acetylcholine is not confined to axon terminals, but that it can be non-synaptically released by depolarization from axons provided Ca2+ is present.     

Modulation of cortical release of acetylcholine by noradrenaline released from nerves arising from the rat locus coeruleus

The release of acetylcholine was studied in isolated cerebral cortex slices of the rat. There was no significant difference in the release between right and left sides of the cerebral cortex. Noradrenaline reduced the ouabain-stimulated release of acetylcholine and phentolamine prevented its action. The spontaneous and evoked release of acetylcholine was higher in those slices where noradrenergic input was somehow impaired: 6-hydroxydopamine pretreatment or locus coeruleus lesion ipsilaterally resulted in a higher release. Following a locus coeruleus lesion the spontaneous evoked release of acetylcholine from slices dissected from the ipsilateral side was higher in comparison to the contralateral side. Noradrenaline significantly reduced the resting release of acetylcholine only in those cases where the noradrenergic control has been previously removed.It is suggested that the release of acetylcholine is continously controlled by noradrenaline released from nerves arising from the locus coeruleus. The removal of this inhibitory system results in an increase of acetylcholine release.     

Properties of junctional acetylcholine receptors that appear rapidly after denervation

It was previously found that the number of junctional acetylcholine receptors of rat diaphragm, as measured with [¹²⁵I]alphabungarotoxin binding, suddenly increased 2 days after denervation in vivo or in vitro. Organ culture was used here to characterize further this unusual class of junctional receptors. The ‘new’ acetylcholine receptors were physiologically functional and were functionally located only in the junctional region. The rate of degradation of new receptors was slower than that of extrajunctional receptors and similar (in the first 24 h) to that of typical junctional receptors. In addition, the appearance of new junctional receptors was inhibited by cycloheximide and actinomycin D given at critical periods, implicating a protein synthetic step. Finally, nerve stimulation in the presence of a post-synaptic blocker (pancuronium) advanced the time of appearance of new junctional receptors.This last finding coupled with our previous report of nerve stump length effects on junctional acetylcholine receptors²⁶ reinforces the suggestion that under certain conditions the level of junctional receptors can be regulated by the motor neuron.     

Changes in the distribution of the extrajunctional acetylcholine sensitivity along muscle fibers during development and following cordotomy in the rat

Acetylcholine sensitivity along the entire length of muscle fibers was studied during postnatal development and following transection of the spinal cord in the rat. During postnatal development, the acetylcholine sensitivity in the soleus and extensor digitorum longus muscles decreased faster at the juxtajunctional region than near the tendons. Thus, the adult pattern of low acetylcholine sensitivity at the extrajunctional membrane was achieved through the uneven change of acetylcholine sensitivity during normal development. This uneven pattern of the sensitivity was found to appear in both muscles in older rats after cordotomy, and is in striking contrast to the uniform pattern in denervated muscles. The uneven appearance of the sensitivity could not be explained by changes in input resistance or resting membrane potential. In the soleus muscle whose nerve was implanted at an ectopic site, the lowest sensitivity also appeared at the ectopic juxtajunctional region after cordotomy. These results indicate that the motor nerve exerts regionally different effects along a fiber with respect to the appearance of acetylcholine receptors.     

The potentiating action of acetylcholine on the chemosensitivity of denervated skeletal muscle

The acetylcholine sensitivity of soleus and extensor digitorum longus muscles of young rats was studied in vitro by measuring the isometric tension and membrane depolarisation developed in response to bath application of acetylcholine. Contractures of denervated muscles to acetylcholine gradually increased when the same concentration of acetylcholine was applied at 10 min intervals up to 60 min incubation whilst contractures evoked by potassium sulphate did not change significantly. The twitch tension of the muscles elicited in response to direct electrical stimulation did not increase and direct electrical stimulation failed to increase the acetylcholine sensitivity. Increased responses in vitro to acetylcholine were observed only in muscles denervated between 2 and 10 days previously with an optimum increase in sensitivity at 4–6 days. The increases in sensitivity were due to an action of acetylcholine since muscles left untreated exhibited no increases. Responses to carbamylcholine chloride, which is not hydrolysed by acetylcholinesterase, also increased.Membrane depolarisations in response to acetylcholine increased in a similar manner to the contracture responses in both soleus and extensor digitorum longus muscles, whilst the resting membrane potentials did not change. Denervated muscles which had been treated with acetylcholine bound more¹²⁵I-labelled α-bungarotoxin than muscles which had not been treated with acetylcholine. Denervated soleus muscles which had been previously blocked with unlabelled α-bungarotoxin responded to applications of acetylcholine with a gradually increasing depolarisation response and subsequently bound more [¹²⁵I]α-bungarotoxin than muscles left without acetylcholine treatment.The possibility is discussed that acetylcholine acts upon the surface membranes of denervated muscles to increase the number of active acetylcholine receptors.