Non-quantal Acetylcholine Release Research Articles

Characterization of cellular sources for non-quantal ACh release in mouse airways

Acetylcholine (ACh) is the major transmitter to induce contraction of the airway smooth muscle (ASM). In addition to the quantal vesicular ACh release after stimulation of parasympathetic neurons, non-quantal ACh (nqACh) release is observed in tracheal preparations after eserine treatment, which is capable to induce ASM contraction. In order to identify which cell population in the airways contributes to nqACh synthesis, expression of the choline acetyltransferase (ChAT), high affinity choline transporter-1 (CHT1), and the low-affinity choline transporter-like family (CTL1 – 5) was evaluated by RT-PCR in primary isolated vagal and dorsal root ganglia, parasympathetic neurons, tracheal epithelial cells, lymphocytes, granulocytes, and in a smooth muscle and fibroblast cell line. All evaluated cell populations expressed ChAT-, CHT1- or CTL-mRNA, respectively. ACh release was functionally investigated by using M3WT4 cells, a reporter cell line overexpressing muscarinic M3 receptors. An increase in [Ca2+]i was induced in M3WT4 cells by supernatants of eserine-treated tracheal preparations and inhibited by atropine co-application. However, supernatants of eserine-treated isolated cell populations or cells co-cultured cells with M3WT4 cells, respectively, had no effect on [Ca2+]i levels. To address the question if mast cells might contribute to nqACh release, organ bath experiments with tracheas of mast cell-deficient B6.Cg-KitW-sh/HNihrJaeBsmGlliJ mice (kindly provided by Frank Petersen, Leibniz Forschungszentrum Borstel, ARCN) were performed. ASM contraction in response to eserine in mast cell-deficient mice was unaltered in comparison to wild-type mice. In conclusion, we were able to show that many different cell populations located in the trachea express transporters and/or enzymes associated with nqACh release. The fact that we were not able identify the major source of nqACh may be due to a low sensitivity of the M3WT4 cell assay. Alternatively, the possibility that the effect of nqACh-dependent ASM contraction is the sum of low-level ACh released from different cell populations has to be discussed.

Metabotropic GABAB receptors mediate GABA inhibition of acetylcholine release in the rat neuromuscular junction.

Gamma-aminobutyric acid (GABA) is an amino acid which acts as a neurotransmitter in the central nervous system. Here, we studied the effects of GABA on non-quantal, spontaneous, and evoked quantal acetylcholine (ACh) release from motor nerve endings. We found that while the application of 10 μM of GABA had no effect on spontaneous quantal ACh release, as detected by the frequency of miniature endplate potentials, GABA reduced the non-quantal ACh release by 57%, as determined by the H-effect value. Finally, the evoked quantal ACh release, estimated by calculating the quantal content of full-sized endplate potentials (EPPs), was reduced by 34%. GABA's inhibitory effect remained unchanged after pre-incubation with picrotoxin, an ionotropic GABAA receptor blocker, but was attenuated following application of the GABAB receptor blocker CGP 55845, which itself had no effect on ACh release. An inhibitor of phospholipase C, U73122, completely prevented the GABA-induced decrease in ACh release. Immunofluorescence demonstrated the presence of both subunits of the GABAB receptor (GABAB R1 and GABAB R2) in the neuromuscular junction. These findings suggest that metabotropic GABAB receptors are expressed in the mammalian neuromuscular synapse and their activation results in a phospholipase C-mediated reduction in the intensity of non-quantal and evoked quantal ACh release. We investigated the effect of gamma-aminobutyric acid (GABA) on neuromuscular transmission. GABA reduced the non-quantal and evoked quantal release of acetylcholine. These effects are mediated by GABAB receptors and are implemented via phospholipase C (PLC) activation. Our findings suggest that in the mammalian neuromuscular synapse, metabotropic GABAB receptors are expressed and their activation results in a reduction in the intensity of acetylcholine release.

Cholinergic activation of the murine trachealis muscle via non-vesicular acetylcholine release involving low-affinity choline transporters

In addition to quantal, vesicular release of acetylcholine (ACh), there is also non-quantal release at the motor endplate which is insufficient to evoke postsynaptic responses unless acetylcholinesterase (AChE) is inhibited. We here addressed potential non-quantal release in the mouse trachea by organ bath experiments and (immuno)histochemical methods. Electrical field stimulation (EFS) of nerve terminals elicited tracheal constriction that is largely due to ACh release. Classical enzyme histochemistry demonstrated acetylcholinesterase (AChE) activity in nerve fibers in the muscle and butyrylcholinesterase (BChE) activity in the smooth muscle cells. Acute inhibition of both esterases by eserine significantly raised tracheal tone which was fully sensitive to atropine. This effect was reduced, but not abolished, in AChE, but not in BChE gene-deficient mice. The eserine-induced increase in tracheal tone was unaffected by vesamicol (10−5M), an inhibitor of the vesicular acetylcholine transporter, and by corticosterone (10−4M), an inhibitor of organic cation transporters. Hemicholinium-3, in low concentrations an inhibitor of the high-affinity choline transporter-1 (CHT1), completely abrogated the eserine effects when applied in high concentrations (10−4M) pointing towards an involvement of low-affinity choline transporters. To evaluate the cellular sources of non-quantal ACh release in the trachea, expression of low-affinity choline transporter-like family (CTL1-5) was evaluated by RT-PCR analysis. Even though these transporters were largely abundant in the epithelium, denudation of airway epithelial cells had no effect on eserine-induced tracheal contraction, indicating a non-quantal release of ACh from non-epithelial sources in the airways. These data provide evidence for an epithelium-independent non-vesicular, non-quantal ACh release in the mouse trachea involving low-affinity choline transporters.

The effect of dynasore, a blocker of dynamin-dependent endocytosis, on spontaneous quantal and non-quantal release of acetylcholine in murine neuromuscular junctions.

The effect of dynasore, a blocker of dynamin-dependent endocytosis, on spontaneous quantal and non-quantal release of acetylcholine in murine neuromuscular junctions.

Noradrenergic regulation of non-quantal acetylcholine release from parasympathetic fibers in rat myocardium

Noradrenergic regulation of non-quantal acetylcholine release from parasympathetic fibers in rat myocardium

Different Effects of ATP on the Contractility and Nonquantal Acetylcholine Release of Rat Tonic and Phasic Muscles

Different Effects of ATP on the Contractility and Nonquantal Acetylcholine Release of Rat Tonic and Phasic Muscles

Modulatory role of purines in neuromuscular transmission

The data on purine modulation of myoneural transmission are reviewed. A particular attention is paid to adenosine-5′-triphosphoric acid (ATP), the co-transmitter of the principal mediator (acetylcholine), and adenosine, the final ATP metabolite in the synaptic cleft. The effects of these endogenous modulators on pre- and postsynaptic current are discussed. The contributions of purines to the process of quantal and non-quantal acetylcholine release into the synaptic cleft and the effects of ATP and adenosine on cholinoceptor function have been assessed. It is concluded that the role of endogenous purines is mainly confined to enhancement of the efficiency of neuromuscular transmission and synaptic adjustment of a motor unit at different modes of function.

Quantal and non-quantal acetylcholine release at neuromuscular junctions of muscles of different types in a model of hypogravity

Quantal and non-quantal acetylcholine release at neuromuscular junctions of muscles of different types in a model of hypogravity

Nitric oxide modulates intensity of non-quantal acetylcholine release in myocardium of the right atrium of rat

The main parasympathetic neurotransmitter acetylcholine (ACh) is released in the myocardium from the intramural postganglionic parasympathetic nerve endings. The mechanism of non-quantal ACh release has been recently demonstrated in these neurons. Non-quantal ACh release does not depend on exocytosis of ACh-containing vesicles in response to nerve impulse activity but is assumed to be mediated by the high-affinity choline uptake system. The intensity of non-quantal ACh release in the myocardium correlates with the degree of manifestation of the effects of acetylcholinesterase inhibitors inducing the accumulation of non-quantal ACh in the myocardium. The present study deals with the influence of putative modulators of non-quantal ACh release: nitric oxide (NO) and ATP, on the intensity of cholinergic effects induced by organophosphorous acetylcholinesterase inhibitor paraoxon. Intracellular registration of bioelectrical activity in isolated right atrium preparations from rats was used. Under normal conditions, paraoxon (10−7–10−5 M) induced a marked decrease in the action potential (AP) duration at a level of 50 and 90% repolarization in the working right atrial myocardium and slowed down the sinus rhythm. ATP, which is known to suppress nonquantal ACh release in the neuromuscular junction, did not induce significant reduction or augmentation of the effects of paraoxon (5 × 10−6 M). The NO donors, sodium nitroprusside (10−5 M) and SNAP (10−4 M), significantly reduced the paraoxon-induced AP shortening. Moreover, sodium nitroprusside decreased the negative chronotropic effect of paraoxon by 43.7%. On the contrary, NO synthase inhibitor L-NAME (10−4 M), which is known to suppress endogenous NO production, augmented the AP shortening caused by paraoxon. It may be deduced that NO is a universal regulator of non-quantal ACh release intensity both in the myocardium and in the neuromuscular junction.

Both neuronal and non-neuronal acetylcholine take part in non-quantal acetylcholine release in the rat atrium

AimsIn mammalian myocardium acetylcholine (ACh), neurotransmitter which strikingly affects the cardiomyocytes, can be released from the neurons both via quantal (vesicular) and nonquantal (non-vesicular) mechanism of secretion. Non-quantal release is continuous, independent on vagus activity and provides accumulation of ACh in myocardium in the presence of acetylcholinesterase (AChE) inhibitors. The aim of the present study was to determine the source of non-quantal ACh in isolated atrial myocardium of adult and newborn rats. Main methodsStandard microelectrode technique was used to determine the cholinergic changes of electrical activity under the action of AChE inhibitor paraoxon, which correlates with the intensity of nonquantal ACh release. Key findingsIn adult rats selective inhibitor of neuronal choline uptake system hemicholinium III (10–5M) decreased all effects of paraoxon (5×10–6M) more than twofold. Inhibitor of polyspecific 3 organic cation transporters corticosterone (10–4M) also significantly decreased effects of paraoxon in adult rats, indicating that non-neuronal ACh, which is synthesized by cardiomyocytes, takes part in accumulation of ACh in the myocardium. When hemicholinium III and corticosterone were applied together, paraoxon effects in adult atrial myocardium were suppressed almost completely. In newborn rats cardiomyocytes do not excrete ACh. In accordance with this fact hemicholinium III completely abolished effects of paraoxon in newborn myocardium, while corticosterone was ineffective. Thus, non-quantal ACh is released both from cholinergic nerves and cardiomyocytes in adult rat myocardium, while it has exclusively neuronal nature in newborns. SignificanceThe study demonstrates dual neuronal and non-neuronal nature of non-quantal ACh in the heart.

Erratum to “Increased non-quantal release of acetylcholine after inhibition of endocytosis by methyl-β-cyclodextrin: the role of vesicular acetylcholine transporter”

The publisher regrets that the corrections requested by the authors at the proof stage were not incorporated. On page 2, thereference “Dawson et al.” should have been written as “Dason et al.”Additionally, in the References section, the reference “Dawson et al.” should be written as follows:Dason JS, Smith AJ, Marin L, Charlton MP (2010) Vesicular sterols are essential for synaptic vesicle cycling. J Neurosci30:15856–15865.

Purine P2Y receptors in ATP-mediated regulation of non-quantal acetylcholine release from motor nerve endings of rat diaphragm

We established the effect of ATP, which is released together with acetylcholine (ACh), on the non-quantal ACh release (NQR) in rat diaphragm endplates and checked what kind of purine receptors are involved. NQR was estimated by the amplitude of endplate hyperpolarization (the H-effect) following the blockade of postsynaptic nicotinic receptors and cholinesterase. 100 μM ATP reduced the H-effect to 66% of the control. The action of ATP remained unchanged after the inhibition of ionotropic P2X receptors by Evans blue and PPADS, but disappeared after the application of the broad spectrum P2 receptor antagonist suramin, metabotropic P2Y receptor blocker reactive blue 2 and U73122, an inhibitor of phospholipase C. P2Y-mediated regulation is not coupled to presynaptic voltage-dependent Ca 2+ channels. During the simultaneous application of ATP and glutamate (which is another ACh cotransmitter reducing non-quantal release), the additive depressant effect led to a disappearance of the H-effect. This can be explained by the independence of the action of ATP and glutamate. Unlike the effects of purines on the spontaneous quantal secretion of ACh, its non-quantal release is regulated via P2Y receptors coupled to G q/11 and PLC. ATP thus regulates the neuromuscular synapse by two different pathways.

Effects of acetylcholinesterase inhibitor paraoxon denote the possibility of non-quantal acetylcholine release in myocardium of different vertebrates

Effects of organophosphorous acetylcholinesterase inhibitor paraoxon were studied in the isolated atrial and ventricular myocardium preparations of a fish (cod), an amphibian (frog) and a mammal (rat) using the microelectrode technique. Incubation of isolated atrium with paraoxon (5×10(-6)-5×10(-5)M) caused significant reduction of action potential duration and marked slowing of sinus rhythm. These effects were abolished by muscarinic blocker atropine and therefore are caused by acetylcholine, which accumulates in the myocardium due to acetylcholinesterase inhibition even in the absence of vagal input. Hemicholinium III is a blocker of high affinity choline-uptake transporters, which are believed to mediate non-quantal release of acetylcholine from cholinergic terminals in different tissues. In the atrial myocardium of all the three studied species, hemicholinium III (10(-5)M) significantly suppressed all the effects of paraoxon. Blocker of parasympathetic ganglionic transmission hexamethonium bromide (10(-4)M) and inhibitor of vesicular acetylcholine transporters vesamicol (10(-5)M) failed to attenuate paraoxon effects. Among ventricular myocardium preparations of three species paraoxon provoked marked cholinergic effects only in frog, hemicholinium III abolished these effects effectively. We conclude that paraoxon stops degradation of acetylcholine in the myocardium and helps to reveal the effects of acetylcholine, which is continuously secreted from the cholinergic nerves in non-quantal manner. Thus, non-quantal release of acetylcholine in the heart is not specific only for mammals, but is also present in the hearts of different vertebrates.

Increased non-quantal release of acetylcholine after inhibition of endocytosis by methyl-β-cyclodextrin: the role of vesicular acetylcholine transporter

We investigated the role of the vesicular acetylcholine transporter in the mechanism of non-quantal (non-vesicular) secretion of neurotransmitter in the neuromuscular synapse of the rat diaphragm muscle. Non-quantal secretion was estimated electrophysiologically by the amplitude of end-plate hyperpolarization after inhibition of cholinesterase and nicotinic receptors (H-effect) or measured by the optical detection of acetylcholine in the bathing solution. It was shown that 1 mM methyl-β-cyclodextrin (MCD) reduced both endocytosis and, to much lesser extent, exocytosis of synaptic vesicles (SV) thereby increasing non-quantal secretion of acetylcholine with a concurrent decrease in axoplasm pH. During high-frequency stimulation of the motor nerve, that substantially increases vesicles exocytosis, the non-quantal secretion was further enhanced if the endocytosis of SV was blocked by MCD. In contrast, non-quantal secretion of acetylcholine did not increase when the MCD-treated neuromuscular preparations were superfused with either vesamicol, an inhibitor of vesicular transporter of acetylcholine, or sodium propionate, which decreases intracellular pH. These results suggest that the proton-dependent, vesamicol-sensitive vesicular transporters of acetylcholine, which become inserted into the presynaptic membrane during SV exocytosis and removed during endocytotic recycling of SV, play the major role in the process of non-quantal secretion of neurotransmitter.

Non-quantal release of acetylcholine in guinea-pig airways: role of choline transporter

In the resting state, motor neurons continuously release ACh through quantal and non-quantal mechanisms, the latter through vesicular ACh transporter (VAChT) and choline transporter (ChT). Although in skeletal muscle these mechanisms have been extensively studied, the non-quantal release (NQR) from parasympathetic neurons of airway smooth muscle has not been described. Here we corroborated that the organophosphate paraoxon (acetylcholinesterase inhibitor) induced a contraction blocked by atropine (muscarinic antagonist) in guinea-pig tracheal rings. This contraction was not modified by two blockers of evoked quantal release, tetrodotoxin (voltage-dependent Na(+) channel blocker) and ω-conotoxin GVIA (N-type Ca(2+) channel blocker), nor by the nicotinic blocker hexamethonium, suggesting that acetylcholine NQR could be responsible of the paraoxon-induced contraction. We confirmed that tetrodotoxin, and to some extent -conotoxin, abolished the evoked quantal ACh release induced by electrical field stimulation. Hemicholinium-3 (ChT inhibitor), but not vesamicol (VAChT inhibitor), caused a concentration-dependent inhibition of the response to paraoxon. The highest concentration of hemicholinium-3 left ∼75% of the response to electrical field stimulation, implying that inhibition of paraoxon-induced contraction was not due to depletion of neuronal vesicles. Non-neuronal sources of ACh released through organic cation transporters were discarded because their inhibition by quinine or corticosterone did not modify the response to paraoxon. Calcium-free medium abolished the effect of paraoxon, and NiCl(2), 2-aminoethyl diphenyl-borate and SKF 96365 partly inhibited it, suggesting that non-specific cation channels were involved in the acetylcholine NQR. We concluded that a Ca(2+)-dependent NQR of ACh is present in cholinergic nerves from guinea-pig airways, and that ChT is involved in this phenomenon.

Immunocytochemical Demonstration of M1 Muscarinic Acetylcholine Receptors at the Presynaptic and Postsynaptic Membranes of Rat Diaphragm Endplates

M(1)-muscarinic acetylcholine (ACh) receptors (M(1)R) were directly demonstrated immunocytochemically in electronmicroscopic images of rat diaphragm neuromuscular junctions (NMJ). Specific electron-dense granules were located at presynaptic nerve ending membranes and in the sarcolemma in the depths of postsynaptic folds. This first visualization of M(1)R on both sides of the NMJ is in agreement with previous pharmacological data on the regulatory role of M(1)R in quantal and non-quantal ACh release.

Acetyl‐CoA and acetylcholine metabolism in nerve terminal compartment of thiamine deficient rat brain

The decrease of pyruvate and ketoglutarate dehydrogenase complex activities is the main cause of energy and acetyl-CoA deficits in thiamine deficiency-evoked cholinergic encephalopathies. However, disturbances in pathways of acetyl-CoA metabolism leading to appearance of cholinergic deficits remain unknown. Therefore, the aim of this work was to investigate alterations in concentration and distribution of acetyl-CoA and in acetylcholine metabolism in brain nerve terminals, caused by thiamine deficits. They were induced by the pyrithiamine, a potent inhibitor of thiamine pyrophosphokinase. The thiamine deficit reduced metabolic fluxes through pyruvate and ketoglutarate dehydrogenase steps, yielding deficits of acetyl-CoA in mitochondrial and cytoplasmic compartments of K-depolarized nerve terminals. It also inhibited indirect transport of acetyl-CoA though ATP-citrate lyase pathway being without effect on its direct Ca-dependent transport to synaptoplasm. Resulting suppression of synaptoplasmic acetyl-CoA correlated with inhibition of quantal acetylcholine release (r = 0.91, p = 0.012). On the other hand, thiamine deficiency activated non-quantal acetylcholine release that was independent of shifts in intraterminal distribution of acetyl-CoA. Choline acetyltransferase activity was not changed by these conditions. These data indicate that divergent alterations in the release of non-quantal and quantal acetylcholine pools from thiamine deficient nerve terminals could be caused by the inhibition of acetyl-CoA and citrate synthesis in their mitochondria. They in turn, caused inhibition of acetyl-CoA transport to the synaptoplasmic compartment through ATP-citrate lyase pathway yielding deficits of cholinergic functions.

Non-quantal acetylcholine release in the mammalian neuromuscular junction: Dependence on the extracellular concentration of magnesium and calcium ions

Non-quantal acetylcholine release in the mammalian neuromuscular junction: Dependence on the extracellular concentration of magnesium and calcium ions

Non‐quantal release of acetylcholine from parasympathetic nerve terminals in the right atrium of rats

Acetylcholinesterase (AChE) inhibitors provoke typical cholinergic effects in the isolated right atrium of the rat due to the accumulation of acetylcholine (ACh). Our study was designed to show that in the absence of vagal impulse activity, ACh is released from the parasympathetic nerve fibres by means of non-quantal secretion. The conventional microelectrode technique was used to study changes in action potential (AP) configuration in the right atrium preparation of rats during application of AChE inhibitors. Staining with the lipophilic fluorescent dye FM1-43 was used to demonstrate the presence of endocytosis in cholinergic endings. The AChE inhibitors armin (10(7)-10(5)m) and neostigmine (10(7) to 5 x 10(6)m) caused a reduction of AP duration and prolonged the cycle length. These effects were abolished by atropine and were therefore mediated by ACh accumulated in the myocardium during AChE inhibition. Putative block of impulse activity of the postganglionic neurons by tetrodotoxin (5 x 10(7)m) and blockade of ganglionic transmission by hexomethonium (2 x 10(4)m), as well as blockade of all forms of quantal release with Clostridium botulinum type A toxin (50 U ml(1)), did not alter the effects of armin. Experiments with FM1-43 dye confirmed the effective block of exocytosis by botulinum toxin. Selective inhibition of the choline uptake system using hemicholinium III (10(5)m), which blocks non-quantal release at the neuromuscular junction, suppressed the effects of AChE inhibitors. Thus, accumulation of ACh is likely to be caused by non-quantal release from cholinergic terminals. We propose that non-quantal release of ACh, shown previously at the neuromuscular junction, is present in cholinergic postganglionic fibres of the rat heart in addition to quantal release.

Parasympathetic cholinergic transmission, minus the vesicles

Our understanding of quantal transmission developed initially through the skeletal neuromuscular junction (Fatt & Katz, 1952), and it took about 30 years for a similar phenomenon to be confirmed in autonomic nerves (Blakeley & Cunnane, 1979; Hirst & Neild, 1980). The first convincing demonstration of non-quantal release of acetylcholine at the neuromuscular junction occurred about 30 years ago (Katz & Miledi, 1977), and it is only now, in this issue of Experimental Physiology, that similar effects are shown for parasympathetic junctions (Abramochkin et al. 2009). One problem hampering the study of parasympathetic nerve terminals is their diffuse innervation, forming complex and irregular three-dimensional networks amidst other cells types. The electrical coupling between smooth and cardiac muscles in situ makes the interpretation of electrophysiological experiments more complex, while the presence of G-protein-coupled receptors (such as muscarinic receptors) on parasympathetic targets rather than ligand-gated ion channels (such as nicotinic receptors) makes the detection of neurotransmitter release inherently less sensitive. Abramochkin and colleagues (2009) show that when acetylcholinesterase is pharmacologically inhibited, several cardiac parameters (including heart rate) are sensitive to the muscarinic receptor antagonist, atropine. This suggests that there is tonic release of a cholinergic agonist, almost certainly acetylcholine, from within the atria that persists when vesicular release is prevented with botulinum toxin. Further evidence that acetylcholine itself is involved comes from experiments with hemicholinium III, which inhibits choline transport into the nerve terminals, hence preventing local acetylcholine production and reducing the atropine-sensitive effects. The effectiveness of the botulinum-toxin-induced inhibition of vesicular release was elegantly demonstrated using a fluorescent indicator of vesicle cycling, FM1-43. One of the anticholinesterases used, armin, has a contentious geopolitical history, but its use is not critical for the interpretation of the study because of the additional use of hexamethonium. The relative contributions of non-quantal and quantal transmitter release from parasympathetic nerves in normal conditions still need to be established, although such ratios do not necessarily reflect their relative importance. For example, at the skeletal neuromuscular junction the non-quantal leak of acetylcholine may be two orders of magnitude higher than the quantal release associated with the endplate potentials (reviewed by Katz & Miledi, 1977), yet the importance of quantal transmission for skeletal muscle contraction can be seen in the effectiveness of botulinum toxin for relaxing skeletal muscle, for which it is commonly used in general anaesthesia and therapeutically, to manage muscles that spasm. Botulinum toxin is also useful for treating some smooth muscles that spasm, such as in the oesophagus (achalasia) or in the overactive bladder; in both cases, botulinum toxin is an effective yet temporary treatment that causes the muscles to relax because they are no longer driven by the quantal (or packeted and vesicular) release of acetylcholine. Could non-quantal acetylcholine release be important outside the heart? Perhaps, but the pleomorphism and functional diversity of autonomic targets makes generalization difficult. It is worth considering whether separate targeting of non-vesicular acetylcholine release might have some therapeutic benefit. So far, functional effects of such release have only been shown in the presence of acetylcholinestase inhibition, so as yet there is no evidence for an important role in normal cardiac physiology. However, acetylcholinesterase inhibitors are not infrequently given, in the context of reversing non-depolarizing neuromuscular blocking drugs or for myasthenia gravis, so it may be that during such pharmacotherapy non-vesicular acetylcholine release has some acute cardiac effects. Abramochkin et al. (2009) suggest that a low concentration of acetylcholine released from the parasympathetic nerves may be trophic or anti-apoptotic, both reasonable and testable hypotheses. For all of the very elegant studies presented by Abramochkin et al. (2009), the non-vesicular release of acetylcholine still needs further investigation. For example, to be sure that acetycholine was the cholinergic agonist activating muscarinic receptors it would be helpful to assay acetylcholine more directly, but the required sensitivity (while maintaining specificity) is difficult to achieve. Non-neuronal sources of acetylcholine (for a review, see Kawashima & Fujii, 2008) might also play a role. While Abramochkin et al. (2009) wisely block ganglionic transmission in their model system, it would be interesting to explore the role of non-vesicular acetylcholine release in the cardiac intrinsic plexus, an understudied network that should be capable of significant information processing and signalling. Let us hope that research into autonomic junctions does not continue to lag 30 years behind its somatic cousin.