Reduced Acetylcholine Release Research Articles

Neuromuscular recovery from botulism involves multiple forms of compensatory plasticity.

Botulinum neurotoxin (BoNT) causes neuroparalytic disease and death by blocking neuromuscular transmission. There are no specific therapies for clinical botulism and the only treatment option is supportive care until neuromuscular function spontaneously recovers, which can take weeks or months after exposure. The highly specialized neuromuscular junction (NMJ) between phrenic motor neurons and diaphragm muscle fibers is the main clinical target of BoNT. Due to the difficulty in eliciting respiratory paralysis without a high mortality rate, few studies have characterized the neurophysiological mechanisms involved in diaphragm recovery from intoxication. Here, we develop a mouse model of botulism that involves partial paralysis of respiratory muscles with low mortality rates, allowing for longitudinal analysis of recovery. Mice challenged by systemic administration of 0.7 LD50 BoNT/A developed physiological signs of botulism, such as respiratory depression and reduced voluntary running activity, that persisted for an average of 8-12 d. Studies in isolated hemidiaphragm preparations from intoxicated mice revealed profound reductions in nerve-elicited, tetanic and twitch muscle contraction strengths that recovered to baseline 21 d after intoxication. Despite apparent functional recovery, neurophysiological parameters remained depressed for 28 d, including end plate potential (EPP) amplitude, EPP success rate, quantal content (QC), and miniature EPP (mEPP) frequency. However, QC recovered more quickly than mEPP frequency, which could explain the discrepancy between muscle function studies and neurophysiological recordings. Hypothesizing that differential modulation of voltage-gated calcium channels (VGCC) contributed to the uncoupling of QC from mEPP frequency, pharmacological inhibition studies were used to study the contributions of different VGCCs to neurophysiological function. We found that N-type VGCC and P/Q-type VGCC partially restored QC but not mEPP frequency during recovery from paralysis, potentially explaining the accelerated recovery of evoked release versus spontaneous release. We identified additional changes that presumably compensate for reduced acetylcholine release during recovery, including increased depolarization of muscle fiber resting membrane potential and increased quantal size. In addition to identifying multiple forms of compensatory plasticity that occur in response to reduced NMJ function, it is expected that insights into the molecular mechanisms involved in recovery from neuromuscular paralysis will support new host-targeted treatments for multiple neuromuscular diseases.

Abstract 11494: Electrophysiological Effects of Sympathoexcitation are Mitigated by Blockade of Neuropeptide Y Y2 Receptors on Cardiac Parasympathetic Nerve Terminals

Introduction: Sympathetic activation releases both norepinephrine and co-transmitter neuropeptide Y (NPY). NPY can predispose to ventricular arrhythmias via receptors on cardiomyocytes. Ex vivo studies suggest that NPY may concomitantly reduce vagal tone via activation of NPY Y2 receptors (Y2R) on parasympathetic nerves, reducing acetylcholine release and further augmenting pro-arrhythmic sympathetic effects. We evaluated if parasympathetic Y2R blockade can prevent NPY-induced vagal inhibition, restore autonomic balance and stabilize cardiac electrophysiology. Methods: Yorkshire pigs (n = 12) underwent bilateral stellate ganglia stimulation (BSGS; 10 Hz, 4 ms) for 45 secs, before and after administration of selective Y2R antagonist BIIE0246 (BIIE; 32 nmol/kg bolus + 2 nmol/kg/min infusion). Hemodynamic and electrophysiological recordings were obtained with a left ventricular pressure (LVP) catheter, surface electrogram and a 56-electrode sock placed over the ventricles to measure activation recovery intervals (ARI), a surrogate of local action potential duration, from unipolar electrograms. Heart rate variability parameters, including low to high frequency ratio (LF/HF), were analyzed pre- and post-BIIE. Results: BSGS significantly increased heart rate (HR) and LVP, and shortened ventricular ARIs. BIIE significantly reduced both the rate of change and the maximal changes in HR and ARIs during BSGS. As anticipated, BSGS significantly increased LF/HF ratio, but this effect was abolished after BIIE. BIIE did not affect BSGS related increases in LVP. Conclusions: Cardiac effects of sympathetic activation are mediated, at least in part, by suppression of vagal tone through binding of NPY to parasympathetic Y2Rs. Blockade of this receptor partially counters pro-arrhythmic electrophysiological effects of BSGS and suggests a possible novel adjuvant role for Y2R blockade as a therapy for reducing consequences of sympatho-excitation.

Effect of acetylcholine deficiency on neural oscillation in a brainstem-thalamus-cortex neurocomputational model related with Alzheimer’s disease

Previous works imply that involving brainstem in neuropathological studies of Alzheimer’s disease (AD) is of clinically significant. This work constructs a comprehensive neural mass model for cholinergic neuropathogenesis that involves brainstem, thalamus and cortex, wherein how acetylcholine deficiency in AD affects neural oscillation of the model output is systematically explored from the perspective of neurocomputation. By decreasing synapse connectivity parameters in direct cholinergic pathway from brainstem to thalamus or in indirect glutamatergic synapse pathway from cortex to brainstem to mimic the pathological condition of reduced acetylcholine release in patients with AD, the property of neural oscillation in this model is numerically investigated by means of power spectrum in frequency domain and amplitude distribution in time domain. Simulated results demonstrate that decreasing synapse connectivity whether in the direct cholinergic pathway or in the indirect glutamatergic synapse pathway can alter the neural oscillation significantly in three aspects: it induces an obvious decrease of dominant frequency; it leads to a degraded rhythmic activity in the alpha frequency band as well as an enhanced rhythmic activity in the theta frequency band; it results in reduced oscillation amplitude of the model output. These results are agreement with the characteristic of electrophysiological EEG measurement recorded in AD, especially support the hypothesis that cholinergic deficiency is a promising pathophysiological origin of EEG slowing in AD. Our analysis indicates that targeting the cholinergic system may have potential prospects in early diagnosis and treatment of AD.

Understanding Why Muscarinic Receptor Agonists Have Antipsychotic Properties

All current antipsychotics have direct dopamine (DA) D2 receptor activity, which is associated with problems such as dysphoria, EPS, or prolactin elevation. Muscarinic receptor agonists have shown antipsychotic-like activity across preclinical models and clinical trials. This poster reviews preclinical evidence as to how muscarinic receptor agonists, such as the M1/M4 preferring agonist xanomeline, might have clinically relevant antipsychotic effects. Highlight the novel mechanisms through which muscarinic receptor agonists are associated with antipsychotic effects without having any direct dopaminergic D2 receptor activity. The muscarinic receptor family is composed of 5 G protein-coupled receptors (GPCRs). One of the leading hypotheses explaining the antipsychotic activity of muscarinic receptor agonists is preclinical studies of muscarinic receptor modulation of those DA circuits associated with psychosis. Both M1 and M4 receptors are expressed in DA neural circuits implicated in psychosis, and provide unique regulation of these circuits. Xanomeline has both functional M1 and M4 receptor agonist activity, and shows robust antipsychotic-like effects in several animal models that require the presence of functional M1 and M4 receptors. M4 receptors are autoreceptors on cholinergic neurons that regulate DA circuits in two locations: ventral tegmental area (VTA) and nucleus accumbens (NAc). Cholinergic-rich neurons from the hindbrain at the VTA, where they release acetylcholine (ACh) into DA-rich synaptic spaces. M4 autoreceptors are present on these neurons, and their activation reduces ACh release and lowers ambient synaptic ACh concentrations, leading to reduced DA cell firing. Cholinergic interneurons residing in the NAc also express M4 autoreceptors. These ACh interneurons regulate ACh release with M4 activation also turning off ACh release. Therefore, M4 receptors serve as DA regulators at VTA and NAc, both key sites for dopamine's role in psychotic processes. M1 receptors regulate DA circuits in a different, "top down" manner. M1 receptors are found on cortical inhibitory interneurons. When activated, inhibitory drive onto excitatory output neurons is enhanced, which leads to reduced excitatory tone to VTA DA neurons. Over the last 25years, a growing body of evidence has shown potential for muscarinic receptor agonists to become a new class of medicines with potent antipsychotic activity. Preclinical data at micro-and macro-circuit levels suggest that the M1 and M4 receptor subtypes are most relevant in the regulation of DA circuits. The antipsychotic effects of muscarinic agonists effects may arise from influencing these key muscarinic receptor subtypes that are integral to the regulation of DA neural circuits. In summary, there has been great progress in understanding the potential for muscarinic receptor agonists for the treatment of psychosis. Karuna Therapeutics.

Long-term endogenous acetylcholine deficiency potentiates pulmonary inflammation in a murine model of elastase-induced emphysema

Acetylcholine (ACh), the neurotransmitter of the cholinergic system, regulates inflammation in several diseases including pulmonary diseases. ACh is also involved in a non-neuronal mechanism that modulates the innate immune response. Because inflammation and release of pro-inflammatory cytokines are involved in pulmonary emphysema, we hypothesized that vesicular acetylcholine transport protein (VAChT) deficiency, which leads to reduction in ACh release, can modulate lung inflammation in an experimental model of emphysema. Mice with genetical reduced expression of VAChT (VAChT KDHOM 70%) and wild-type mice (WT) received nasal instillation of 50 uL of porcine pancreatic elastase (PPE) or saline on day 0. Twenty-eight days after, animals were evaluated. Elastase instilled VAChT KDHOM mice presented an increase in macrophages, lymphocytes, and neutrophils in bronchoalveolar lavage fluid and MAC2-positive macrophages in lung tissue and peribronchovascular area that was comparable to that observed in WT mice. Conversely, elastase instilled VAChT KDHOM mice showed significantly larger number of NF-κB-positive cells and isoprostane staining in the peribronchovascular area when compared to elastase-instilled WT-mice. Moreover, elastase-instilled VAChT-deficient mice showed increased MCP-1 levels in the lungs. Other cytokines, extracellular matrix remodeling, alveolar enlargement, and lung function were not worse in elastase-instilled VAChT deficiency than in elastase-instilled WT-controls. These data suggest that decreased VAChT expression may contribute to the pathogenesis of emphysema, at least in part, through NF-κB activation, MCP-1, and oxidative stress pathways. This study highlights novel pathways involved in lung inflammation that may contribute to the development of chronic obstrutive lung disease (COPD) in cholinergic deficient individuals such as Alzheimer’s disease patients.

Botulinum Toxin A Ameliorates Neuroinflammation in the MPTP and 6-OHDA-Induced Parkinson's Disease Models.

Recently, increasing evidence suggests that neuroinflammation may be a critical factor in the development of Parkinson’s disease (PD) in addition to the ratio of acetylcholine/dopamine because dopaminergic neurons are particularly vulnerable to inflammatory attack. In this study, we investigated whether botulinum neurotoxin A (BoNT-A) was effective for the treatment of PD through its anti-neuroinflammatory effects and the modulation of acetylcholine and dopamine release. We found that BoNT-A ameliorated MPTP and 6-OHDA-induced PD progression, reduced acetylcholine release, levels of IL-1β, IL-6 and TNF-α as well as GFAP expression, but enhanced dopamine release and tyrosine hydroxylase expression. These results indicated that BoNT-A had beneficial effects on MPTP or 6-OHDA-induced PD-like behavior impairments via its anti-neuroinflammation properties, recovering dopamine, and reducing acetylcholine release.

Topical application of Zanthoxylum piperitum extract improves lateral canthal rhytides by inhibiting muscle contractions

Facial wrinkles are the predominant phenotypes of skin aging. To date, one of the most effective ways to improve wrinkles is botulinum toxin type A (BoNT/A) injection, which inhibits muscle contractions by reducing acetylcholine release from neurons. However, since BoNT/A is a hazardous neurotoxin, the injection can only be performed by medical doctors and the procedure is only possible through invasive injection, causing inconveniences such as pain. To overcome these inconveniences, we tried to find a way to reduce wrinkles non-invasively via mechanisms similar to BoNT/A. We first designed in vitro assays to test BoNT/A-like muscle contraction inhibition in two different model systems. By using the assays, we identified Zanthoxylum piperitum (Z. piperitum) fruit extract as a BoNT-like reagent (27.7% decrease of muscle contraction rates by 1000 ppm of Z. piperitum extract treatment). Next, we determined mechanisms of how Z. piperitum extract decreases muscle contraction rates and found that the extract treatment inhibits electrical signal transduction in neurons. We also showed that among known components of Z. piperitum extract, quercitrin is responsible for muscle contraction inhibition. We further identified that Z. piperitum extract has synergistic effects with acetyl hexapeptide-8 and BoNT/A light chain, which are well-known BoNT-like peptides. Finally, we showed that topical treatment of the Z. piperitum extract indeed decreases facial wrinkles and treatment of Z. piperitum extract with acetyl hexapeptide-8 has a tendency to improve wrinkles synergistically (14.5% improvement on average). The synergistic effect of the combination is expected to improve wrinkles effectively by implementing the BoNT/A mechanisms in a non-invasive way.

Evaluation of the neuromuscular junction in a middle-aged mouse model of congenital myasthenic syndrome.

Reduced expression of the vesicular acetylcholine transporter (VAChT) leads to changes in the distribution and shape of synaptic vesicles (SVs) at neuromuscular junctions (NMJs), suggesting vesicular acetylcholine (ACh) as a key component of synaptic structure and function. It is poorly understood how long-term changes in cholinergic transmission contribute to age- and disease-related degeneration in the motor system. In this study we performed confocal imaging, electrophysiology, electron microscopy, and analyses of respiratory mechanics of the diaphragm NMJ components in 12-month-old wild-type (WT) and VAChTKDHOM mice. Diaphragms of NMJs of the VAChTKDHOM mice were similar to those in WT mice in number, colocalization, and fragmentation of pre-/postsynaptic components. However, they had increased spontaneous SV exocytosis, miniature endplate potential frequency, and diminished MEPP amplitude. No impairment in respiratory mechanics at rest was observed, probably due to the large neurotransmission safety factor of the diaphragm. The present findings help us to understand the consequences of reduced ACh release at the NMJs during aging.

Effects of Three Anti-Seizure Drugs on Cholinergic and Metabolic Activity in Experimental Status Epilepticus.

Status epilepticus (SE) is characterized by recurrent seizure activity and can be drug- resistant. Knowledge of neuronal and metabolic activity of the brain during SE may be helpful to improve medical care. We here report the effects of three anti-seizure drugs on changes of acetylcholine energy metabolites and oxidative stress during SE. We used the lithium-pilocarpine model in rats to induce SE and in vivo- microdialysis to monitor cholinergic and metabolic activity in the hippocampus. We measured extracellular concentrations of acetylcholine, glucose, lactate, pyruvate, glycerol and isoprostanes before and during SE, and after acute treatment with pregabalin, valproic acid, and levetiracteam. Upon onset of SE, acetylcholine (ACh) release increased six- to eightfold. Glucose was increased only transiently by 30% but lactate levels rose four-fold, and extracellular concentrations of glycerol ten-fold. Isoprostanes are markers of oxidative stress and increased more than 20-fold. Two hours after pilocarpine adminstration, rats were treated with pregabalin (100 mg/kg), levetiracetam (200 mg/kg) or valproic acid (400 mg/kg) by i.p. injection. All three drugs stopped seizure activity in a delayed fashion, but at the doses indicated, only animals that received levetiracetam reached consciousness. All drugs reduced ACh release within 60-120 minutes. Lactate/pyruvate ratios, glycerol and isoprostanne levels were also reduced significantly after drug administration. Hippocampal ACh release closely follows seizure activity in SE and is attenuated when SE subsides. Pregabalin, valproic acid and levetiracetam all terminate seizures in the rat SE model and attenuate cholinergic and metabolic changes within two hours.

Toxina botulínica na Odontologia

Clostridium botulinum is an anaerobic bacter, which produces untileight types of toxins. The botulinum toxin A (BTX-A) is the most potentamong then, largely used in clinical procedures and approved forseveral therapeutic instances. The Allergan Inc. company was the first toproduce BTX-A commercially called as “Botox®”. The therapeutic useof BOTOX® is approved by ANVISA in Brazil since 2000, followed byFood and Drug Administrartion in 2002. In Brazil, the Federal Councilof Dentistry recognizes BOTOX® by 198/2019 law and its utilizationin procedures such as facial reconcilement; or in therapeutics suchas bruxism, hypertrophy of chew muscles, temporomandibular jointdysfunction, sialorrhea, asymmetry smile, and gingival profusion. Despiteits temporal use, the results are very interesting. BTX-A side effects areconsidered for uses in miastenia gravis and Lambert-Eaton Syndrome,which has, in common, a reduced acetylcholine release in synapses. Thus,the aim of this work was to review the main BTX-A uses in dentistry area,once its use has growing largely in aesthetics and in dentistry disorders.Herein, we report the low risk in the use of BTX-A, with several interestingbenefits on dentistry areas, improving the quality of life and the selfesteem

Altered gastrointestinal motility in an animal model of Lesch-Nyhan disease.

Mutations in the HGPRT1 gene, which encodes hypoxanthine-guanine phosphoribosyltransferase (HGprt), housekeeping enzyme responsible for recycling purines, lead to Lesch-Nyhan disease (LND). Clinical expression of LND indicates that HGprt deficiency has adverse effects on gastrointestinal motility. Therefore, we aimed to evaluate intestinal motility in HGprt knockout mice (HGprt¯). Spontaneous and neurally evoked mechanical activity was recorded in vitro as changes in isometric tension in circular muscle strips of distal colon. HGprt¯ tissues showed a lower in amplitude spontaneous activity and atropine-sensitivity neural contraction compared to control mice. The responses to carbachol and to high KCl were reduced, demonstrating a widespread impairment of contractility. L-NAME was not able in the HGprt¯ tissues to restore the large amplitude contractile activity typical of control. In HGprt¯ colon, a reduced expression of dopaminergic D1 receptor was observed together with the loss of its tonic inhibitory activity present in control-mice. The analysis of inflammatory and oxidative stress in colonic tissue of HGprt¯ mice revealed a significant increase of lipid peroxidation associated with over production of oxygen free radicals. In conclusion, HGprt deficiency in mice is associated with a decrease in colon contractility, not dependent upon reduction of acetylcholine release from the myenteric plexus or hyperactivity of inhibitory signalling. By contrast the increased levels of oxidative stress could partially explain the reduced colon motility in HGprt¯ mice. Colonic dysmotility observed in HGprt¯ mice may mimic the gastrointestinal dysfunctions symptoms of human syndrome, providing a useful animal model to elucidate the pathophysiology of this problem in the LND.

P2 The action of endothelial–derived acetylcholine is impaired in diabetes

The endothelium is highly sensitive to acetylcholine and responds to the neurotransmitter by releasing vasodilators that control smooth muscle tone. However, acetylcholine is not expected to reach the endothelium through the vessel wall or via blood. Here, by studying calcium signalling in large areas of the endothelium of intact arteries, we show: (1) the endothelium itself is a source of acetylcholine, (2) acetylcholine release from the endothelium underlies flow-mediated vascular responses; and (3) flow-mediated responses are all but abolished in a rat model of diabetes. Calcium signalling was studied in large fields of intact endothelia (~150 cells) of carotid and mesenteric arteries from healthy and diabetic rats (Sprague-Dawley, 150–250 g). In both carotid and mesenteric arteries, flow evoked repeatable endothelial calcium signalling. Importantly, flow-mediated calcium responses could be manipulated by modulating cholinergic signalling pathways: the muscarinic receptor blocker, atropine (100 nM), and the acetylcholine hydrolase, acetylcholinesterase (4 U/ml), each abolished flow-induced calcium activity, whilst the acetylcholinesterase inhibitor, neostigmine (10 µM), enhanced calcium activity, and the choline acetyltransferase inhibitor, bromoacetylcholine (50?µM), attenuated responses. Significantly, responses were reduced in diabetic, compared to control, endothelia. Indeed, the number of diabetic endothelial cells responding to flow was 45% of control, and the peak and time-averaged magnitude of calcium signals was reduced to 54% and 12% of control, respectively. These results identify the endothelium as a source of non-neuronal acetylcholine release and thus demonstrate the reason for the endothelium’s sensitivity to acetylcholine. Significantly, reduced acetylcholine release may underlie the pathophysiology and endothelial dysfunction that occurs in diabetes.

Beneficial effects of neuropeptide galanin on reinstatement of exercise-induced somatic and psychological trauma.

Galanin is a versatile neuropeptide that is distinctly upregulated by exercise in exercise-related tissues. Although benefits from exercise-induced upregulation of this peptide have been identified, many issues require additional exploration. This Review summarizes the information currently available on the relationship between galanin and exercise-induced physical and psychological damage. On the one hand, body movement, exercise damage, and exercise-induced stress and pain significantly increase local and circulatory galanin levels. On the other hand, galanin plays an exercise-protective role to inhibit the flexor reflex and prevent excessive movement of skeletal muscles through enhancing response threshold and reducing acetylcholine release. Additionally, elevated galanin levels can boost repair of the exercise-induced damage in exercise-related tissues, including peripheral nerve, skeletal muscle, blood vessel, skin, bone, articulation, and ligament. Moreover, elevated galanin levels may serve as effective signals to buffer sport-induced stress and pain via inhibiting nociceptive signal transmission and enhancing pain threshold. This Review deepens our understanding of the profitable roles of galanin in exercise protection, exercise injury repair, and exercise-induced stress and pain. Galanin and its agonists may be used to develop a novel preventive and therapeutic strategy to prevent and treat exercise-induced somatic and psychological trauma. © 2016 Wiley Periodicals, Inc.

Dopamine induces inhibitory effects on the circular muscle contractility of mouse distal colon via D1- and D2-like receptors.

Dopamine (DA) acts as gut motility modulator, via D1- and D2-like receptors, but its effective role is far from being clear. Since alterations of the dopaminergic system could lead to gastrointestinal dysfunctions, a characterization of the enteric dopaminergic system is mandatory. In this study, we investigated the role of DA and D1- and D2-like receptors in the contractility of the circular muscle of mouse distal colon by organ-bath technique. DA caused relaxation in carbachol-precontracted circular muscle strips, sensitive to domperidone, D2-like receptor antagonist, and mimicked by bromocriptine, D2-like receptor agonist. 7-Chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride (SCH-23390), D1-like receptor antagonist, neural toxins, L-NAME (nitric oxide (NO) synthase inhibitor), 2'-deoxy-N6-methyl adenosine 3',5'-diphosphate diammonium salt (MRS 2179), purinergic P2Y1 antagonist, or adrenergic antagonists were ineffective. DA also reduced the amplitude of neurally evoked cholinergic contractions. The effect was mimicked by (±)-1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol hydrobromide (SKF-38393), D1-like receptor agonist and antagonized by SCH-23390, MRS2179, or L-NAME. Western blotting analysis determined the expression of DA receptor proteins in mouse distal colon. Notably, SCH-23390 per se induced an increase in amplitude of spontaneous and neurally evoked cholinergic contractions, unaffected by neural blockers, L-NAME, MRS 2179, muscarinic, adrenergic, or D2-like receptor antagonists. Indeed, SCH-23390-induced effects were antagonized by an adenylyl cyclase blocker. In conclusion, DA inhibits colonic motility in mice via D2- and D1-like receptors, the latter reducing acetylcholine release from enteric neurons, involving nitrergic and purinergic systems. Whether constitutively active D1-like receptors, linked to adenylyl cyclase pathway, are involved in a tonic inhibitory control of colonic contractility is questioned.

Dysfunctional Presynaptic M2 Receptors in the Presence of Chronically High Acetylcholine Levels: Data from the PRiMA Knockout Mouse.

The muscarinic M2 receptor (M2R) acts as a negative feedback regulator in central cholinergic systems. Activation of the M2 receptor limits acetylcholine (ACh) release, especially when ACh levels are increased because acetylcholinesterase (AChE) activity is acutely inhibited. Chronically high ACh levels in the extracellular space, however, were reported to down-regulate M2R to various degrees. In the present study, we used the PRiMA knockout mouse which develops severely reduced AChE activity postnatally to investigate ACh release, and we used microdialysis to investigate whether the function of M2R to reduce ACh release in vivo was impaired in adult PRiMA knockout mice. We first show that striatal and hippocampal ACh levels, while strongly increased, still respond to AChE inhibitors. Infusion or injection of oxotremorine, a muscarinic M2 agonist, reduced ACh levels in wild-type mice but did not significantly affect ACh levels in PRiMA knockout mice or in wild-type mice in which ACh levels were artificially increased by infusion of neostigmine. Scopolamine, a muscarinic antagonist, increased ACh levels in wild-type mice receiving neostigmine, but not in wild-type mice or in PRiMA knockout mice. These results demonstrate that M2R are dysfunctional and do not affect ACh levels in PRiMA knockout mice, likely because of down-regulation and/or loss of receptor-effector coupling. Remarkably, this loss of function does not affect cognitive functions in PRiMA knockout mice. Our results are discussed in the context of AChE inhibitor therapy as used in dementia.

<i>α,β</i>-Methylene-ATP-Induced Inhibition of Acetylcholine Release on the Stellate Ganglion: Contribution of Nitric Oxide

In this study, we investigated the possible involvement of nitric oxide pathways in the presynaptic inhibition of acetylcholine release induced by ATP analogs in dogs. We performed the study using HPLC with electrochemical detection and the nitric oxide detection-HPLC system. The amount of acetylcholine released in response to preganglionic stimulation at 5 Hz for 10 min was reduced in a concentration-dependent manner after exposure to 10-7 - 10-4 M α,β-methylene-ATP (α,β-meATP), but not by the P2Y receptor agonist, 2-methyl-thio-ATP (2MeSATP) or the P2X1 receptor agonist, β,γ-methylene-ATP (β,γ-meATP), at the same concentrations. The inhibition of acetylcholine release induced by α,β-meATP was antagonized by: the nonselective P2 receptor antagonist, pyridoxalphosphate-6-azophenyl-2’,4’-disulphonic acid (PPADS); the P2X1, P2X3 and P2X2/3 receptors antagonist, 2’-(or-3’)-O-trinitrophenyl-ATP (TNP-ATP); the neuronal nitric oxide synthase (nNOS) inhibitor, 3-bromo-7-nitroindazole; the soluble guanylyl cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ); the NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethy-limidazoline-1-oxyl-3-oxide (carboxy-PTIO). Exposure to 10-5 M, but not 10-4 M, α,β-meATP, for 30 min increased the levels of , and this increase was antagonized by TNP-ATP and 3-bromo-7-nitroindazole. These results show that P2X receptor activation inhibits stellate ganglionic transmission by reducing acetylcholine release from presynaptic nerve terminals and that this inhibition seems to involve, at least in part, the activation of endogenous NO production and cGMP pathways.

Dynamics of hippocampal acetylcholine release during lithium‐pilocarpine‐induced status epilepticus in rats

The lithium-pilocarpine model is a rat model of epilepsy that mimics status epilepticus in humans. Here, we report changes of acetylcholine (ACh) release in the hippocampus before, during and after status epilepticus as monitored by microdialysis in unanesthetized rats. Administration of pilocarpine (30 mg/kg s.c.) to rats pretreated with lithium chloride (127 mg/kg i.p.) caused a massive, six-fold increase of hippocampal ACh release, paralleling the development of tonic seizures. When seizures were stopped by administration of diazepam (10 mg/kg i.p.) or ketamine (75 mg/kg i.p.), ACh levels returned to normal. Extracellular concentrations of glutamate remained unchanged during this procedure. Administration of atropine (1 mg/kg i.p.) 2 h after pilocarpine caused a further increase of ACh but did not affect seizures, whereas injection of mecamylamine (5 mg/kg i.p.) reduced ACh levels and seizures in a delayed fashion. Local infusion of tetrodotoxin, 1 μM locally) or hemicholinium (10 μM locally) strongly reduced ACh release and had delayed effects on seizures. Administration of glucose or inositol (250 mg/kg each i.p.) had no visible consequences. In parallel experiments, lithium-pilocarpine-induced status epilepticus also enhanced striatal ACh release, and hippocampal ACh levels equally increased when status epilepticus was induced by kainate (30 mg/kg i.p.). Taken together, our results demonstrate that seizure development in status epilepticus models is accompanied by massive increases of extracellular ACh, but not glutamate, levels. Treatments that reduce seizure activity also reliably reduce extracellular ACh levels.

Histaminergic modulation of cholinergic release from the nucleus basalis magnocellularis into insular cortex during taste aversive memory formation.

The ability of acetylcholine (ACh) to alter specific functional properties of the cortex endows the cholinergic system with an important modulatory role in memory formation. For example, an increase in ACh release occurs during novel stimulus processing, indicating that ACh activity is critical during early stages of memory processing. During novel taste presentation, there is an increase in ACh release in the insular cortex (IC), a major structure for taste memory recognition. There is extensive evidence implicating the cholinergic efferents of the nucleus basalis magnocellularis (NBM) in cortical activity changes during learning processes, and new evidence suggests that the histaminergic system may interact with the cholinergic system in important ways. However, there is little information as to whether changes in cholinergic activity in the IC are modulated during taste memory formation. Therefore, in the present study, we evaluated the influence of two histamine receptor subtypes, H1 in the NBM and H3 in the IC, on ACh release in the IC during conditioned taste aversion (CTA). Injection of the H3 receptor agonist R-α-methylhistamine (RAMH) into the IC or of the H1 receptor antagonist pyrilamine into the NBM during CTA training impaired subsequent CTA memory, and simultaneously resulted in a reduction of ACh release in the IC. This study demonstrated that basal and cortical cholinergic pathways are finely tuned by histaminergic activity during CTA, since dual actions of histamine receptor subtypes on ACh modulation release each have a significant impact during taste memory formation.

Letters to the Editor: Cardiomyocyte‐secreted acetylcholine

I read the paper, “Cardiomyocyte-secreted acetylcholine is required for maintenance of homeostasis in the heart” (1), on acetylcholine (ACh) originating in cardiac myocytes, with great interest. The significance of this phenomenon continues to fascinate since it was first described over 40 years ago. The authors have overlooked reports and experimental limits from the earlier papers that deserve careful consideration. Edouard Coraboeuf and colleagues (2) reported in 1970 that cardiac myocytes could synthesize and release ACh. Of note, they obtained data from embryonic hearts before innervation occurred. They also observed that electrical stimulation of the embryonic chick heart caused a transient slowing of heart rate and an increased release of ACh (2). The transient slowing was opposed by atropine. So far, more recent reports (1, 3) have not asked whether stimuli can regulate ACh secretion. Divergent results on the age of animals whose heart cells can secrete ACh are another unsettled issue. You report that myocytes from neonatal mice secrete ACh. This is opposite the report by Rana et al. (3), who state that “… adult, but not neonatal cardiomyocytes are able to synthesize, transport and excrete acetylcholine in the rat heart.” This discrepancy calls for an explanation, inasmuch as rats and mice develop similarly after nearly equal gestation periods. The immunofluorescent detection of choline acetyltransferase (ChAT) requires validation of specificity. Some years ago (4), it was reported that the method to detect ChAT must carefully distinguish ChAT from carnitine acetyltransferase, which is found in cardiac muscle. Thus, Roskoski et al. (4) showed that embryonic chick heart cells, per se, had no ChAT activity and no neurons. Neonatal rat heart cells in culture had no ChAT, but carnitine acetyltransferase was present. Thus, in the absence of cholinergic neurons, heart cells did not synthesize ACh (4). Does the immunofluorescent method you use distinguish between ChAT and carnitine acetyltransferase? The experiments with carbachol and pyridostigmine overlook important drug properties. It is reported that addition of carbachol caused ACh release from cardiac myocytes, as indicated by increased NO production, a downstream fluorescent signal (1). There are concerns with this observation. First, cholinergic neurons have muscarinic receptors whose occupancy by ACh reduces ACh release (5). This negative-feedback mechanism, a physiological hallmark of homeostasis, seems lacking in your system. Second, “… pyridostigmine also augmented the fluorescent signal (Supplemental Fig. S2F).” Pyridostigmine, like neostigmine (and carbachol), has a quaternary N that not only renders it impermeable to cell membranes but also makes it an agonist acting directly at cholinoceptive sites, just like carbachol. Thus, pyridostigmine and neostigmine can activate receptors, in addition to inhibiting acetylcholinesterase. Similarly, hemicholinium-3 (HC-3) is another quaternary N compound whose actions against pyridostigmine can be explained by HC-3 binding to the same site as pyridostigmine and occluding its effect. Indeed, one should test vesamicol and HC-3 against carbachol, as was done against pyridostigmine in Supplemental Fig. 2SF. Such testing should clarify some essential elements of the hypothesis. The current state of the problem seems not to have evolved significantly from that described by earlier investigators. This is a consequence, in part, of a failure to attend to precedent.

Sleep pattern and learning in knockdown mice with reduced cholinergic neurotransmission

Impaired cholinergic neurotransmission can affect memory formation and influence sleep-wake cycles (SWC). In the present study, we describe the SWC in mice with a deficient vesicular acetylcholine transporter (VAChT) system, previously characterized as presenting reduced acetylcholine release and cognitive and behavioral dysfunctions. Continuous, chronic ECoG and EMG recordings were used to evaluate the SWC pattern during light and dark phases in VAChT knockdown heterozygous (VAChT-KDHET, n=7) and wild-type (WT, n=7) mice. SWC were evaluated for sleep efficiency, total amount and mean duration of slow-wave, intermediate and paradoxical sleep, as well as the number of awakenings from sleep. After recording SWC, contextual fear-conditioning tests were used as an acetylcholine-dependent learning paradigm. The results showed that sleep efficiency in VAChT-KDHET animals was similar to that of WT mice, but that the SWC was more fragmented. Fragmentation was characterized by an increase in the number of awakenings, mainly during intermediate sleep. VAChT-KDHET animals performed poorly in the contextual fear-conditioning paradigm (mean freezing time: 34.4±3.1 and 44.5±3.3 s for WT and VAChT-KDHET animals, respectively), which was followed by a 45% reduction in the number of paradoxical sleep episodes after the training session. Taken together, the results show that reduced cholinergic transmission led to sleep fragmentation and learning impairment. We discuss the results on the basis of cholinergic plasticity and its relevance to sleep homeostasis. We suggest that VAChT-KDHET mice could be a useful model to test cholinergic drugs used to treat sleep dysfunction in neurodegenerative disorders.