Search Results (72)

The gastrointestinal (GI) tract is under neural, endocrine and paracrine control. The release (basal and in the presence of either cholinergic and opioid antagonists) of Met-enkephalin, insulin-like growth factor 1 (IGF-1) and somatostatin (SRIF) was determined quantitatively in vitro using explants of the crop, proventriculus and duodenum from either day 0 or day 1 chicks. In addition, the effects of cholinergic and opioid antagonists on PENK gene expression were examined. Thus, the aim of this study was to determine the roles of cholinergic and opioid receptors in the GI tract in newly hatched chickens. Moreover, the effect of IGF-1 and Met-enkephalin on cell division in duodenal explants in vitro was determined. There was both the release of Met-enkephalin from, and PENK expression in, the explants of the crop, proventriculus and duodenum tissue. This is the first report of any neuropeptide(s) being synthesized in and/or released from the crop. In vitro release of Met-enkephalin, IGF-1 and SRIF from duodenal and proventriculus explants was influenced (p < 0.01) by either cholinergic or opioid antagonists; for instance, in the presence of atropine, decreases (p < 0.001) of 17.8% and 57.7% are seen, respectively, in Met-enkephalin release and PENK expression in crop explants from day 1 chicks. Moreover, in the presence of atropine, there were increases (p < 0.001) of 47.7% and 70.9% in IGF-1 release in proventriculus explants from, respectively, day 0 and day 1 chicks. Met-enkephalin and/or IGF-1 stimulated the cell division of duodenal explants in vitro. This is the first report of Met-enkephalin release and PENK expression in the avian crop and of the effects of cholinergic or opioid antagonists on these factors. It is also the first report of either cholinergic or opioid control of IGF-1 release in the periphery of any species. There were strong relationships (p < 0.05) between the release of Met-enkephalin and that of IGF-1 in the duodenum and between the release of SRIF and that of IGF-1 in the proventriculus. This is the first report of IGF-1 and Met-enkephalin stimulating (p < 0.001) the proliferation of duodenal cells and, together, exerting a synergist effect. It is concluded that the release of Met-enkephalin, IGF-1 and SRIF from foregut regions is under tonic cholinergic and opioid control. Full article

Background: This study investigates the effects of cadmium chloride (CdCl₂) on hippocampal peroxisome proliferator-activated receptor gamma (PPARγ) expression and examines whether PPARγ activation mediates the neuroprotective effects of quercetin (QUR). Methods: Sixty adult male rats were included in this study, separated into 12 rats per group as follows: control, CdCl₂ (0.5 mg/kg), CdCl₂ + PPARγ agonist (Pioglitazone, 10 mg/kg), CdCl₂ + QUR (25 mg/kg), and CdCl₂ + QUR + PPARγ antagonist (GW9662, 1 mg/kg). Treatments were administered orally for 30 days. At the end of the experiment, behavioral memory tests, hippocampal histology, markers of cholinergic function, neuroplasticity, oxidative stress, inflammation, and apoptosis, as well as transcription levels of some genes were carried out. Results: CdCl₂ exposure significantly reduced hippocampal PPARγ mRNA and DNA binding potential and nuclear levels. Additionally, CdCl₂ impaired spatial, short-term, and recognition memory, decreased granular cell density in the dentate gyrus (DG), and reduced levels of neuroprotective factors, including Nrf2, brain-derived neurotrophic factor (BDNF), acetylcholine (ACh), and several antioxidant enzymes including heme-oxygenase-1 (HO-1) and superoxide dismutase (SOD), as well as reduced glutathione (GSH). Conversely, CdCl₂ elevated levels of oxidative stress, inflammation, and apoptosis markers such as interleukin-6 (IL-6), malondialdehyde (MDA), Bax, tumor necrosis factor-α (TNF-α), and cleaved caspase-3. QUR and Pioglitazone reversed these effects, restoring expression and PPARγ activation, improving memory, and modulating antioxidant and anti-inflammatory pathways. In contrast, blocking PPARγ with GW9662 negated the neuroprotective effects of QUR, exacerbating oxidative stress and inflammation by reversing all their beneficial effects. Conclusions: Activation of PPARγ by QUR or Pioglitazone offers a promising therapeutic strategy for mitigating CdCl₂-induced neurotoxicity. Full article

Background/Objectives: Mebeverine hydrochloride (MBH) is an antispasmodic agent used to regulate bowel movements and relax intestinal smooth muscle, but its application is limited by specific side effects; therefore, this study investigates the effects of previously synthesized MBH-loaded silver nanoparticles (AgNPs) on smooth muscle contractile activity and their anti-inflammatory potential as an alternative delivery system. Methods: The interactions of AgNPs with cholinergic inhibitors, selective antagonists, Ca²⁺ blockers, and key neurotransmitters were analyzed. In vitro, albumin denaturation suppression and ex vivo assays evaluated the anti-inflammatory effects of AgNPs-MBH, validated using a DFT in silico approach. To comprehensively assess the systemic impact and IBS treatment potential of AgNPs-MBH, we also examined in vitro their antimicrobial activity and hepatic cell responses, as the liver is a key organ in evaluating the overall safety and efficacy of nanoparticles. Additionally, the drug-release capabilities of Ag NPs were established. Results: Our findings indicate that AgNPs with MBH do not affect blocked cholinergic receptors, but their effects are more pronounced and distinct in amplitude and character than MBH. MBH-loaded AgNPs showed a lower anti-inflammatory effect than MBH but were still better than diclofenac. They also affected hepatic cell morphology and proliferation, suggesting potential for enhanced therapeutic efficacy. Drug-loaded AgNPs are considered not bactericidal. Conclusions: Based on our results, drug-loaded AgNPs might be a promising medication delivery system for MBH and a useful treatment option for IBS. Future in vivo and preclinical experiments will contribute to the establishment of drug-loaded AgNPs in IBS treatment. Full article

The cholinergic pathways in the central nervous system (CNS) play a pivotal role in different cognitive functions of the brain, such as memory and learning. This review takes a dive into the pharmacological side of this important part of CNS function, taking into consideration muscarinic receptors and cholinesterase enzymes. Targeting a specific subtype of five primary muscarinic receptor subtypes (M1-M5) through agonism or antagonism may benefit patients; thus, there is a great pharmaceutical research interest. Inhibition of AChE and BChE, orthosteric or allosteric, or partial agonism of M1 mAChR are correlated with Alzheimer’s disease (AD) symptoms improvement. Agonism or antagonism on different muscarinic receptor subunits may lessen schizophrenia symptoms (especially positive allosteric modulation of M4 mAChR). Selective antagonism of M4 mAChR is a promising treatment for Parkinson’s disease and dystonia, and the adverse effects are limited compared to inhibition of all five mAChR. Additionally, selective M5 antagonism plays a role in drug independence behavior. M3 mAChR overexpression is associated with malignancies, and M3R antagonists seem to have a therapeutic potential in cancer, while M1R and M2R inhibition leads to reduction of neoangiogenesis. Depending on the type of cancer, agonism of mAChR may promote cancer cell proliferation (as M3R agonism does) or protection against further tumor development (M1R agonism). Thus, there is an intense need to discover new potent compounds with specific action on muscarinic receptor subtypes. Chemical structures, chemical modification of function groups aiming at action enhancement, reduction of adverse effects, and optimization of Drug Metabolism and Pharmacokinetics (DMPK) will be further discussed, as well as protein–ligand docking. Full article

Background/Objectives: Antipsychotic medicines are used to treat several psychological disorders and some symptoms caused by dementia and schizophrenia. Haloperidol (Hal) is a typical antipsychotic usually used to treat psychosis; however, its use causes motor or extrapyramidal symptoms (EPS) such as catalepsy. Hal blocks the function of presynaptic D2 receptors on cholinergic interneurons, leading to the release of acetylcholine (ACh), which is hydrolyzed by the enzyme acetylcholinesterase (AChE). Methods: This study was designed to investigate the Hal-inhibitory effects on AChE activity in regions representative of the cholinergic system of mice and potential associations between cataleptic effects generated by Hal using therapeutic doses and their inhibitory effects on AChE. Results: The distribution of the AChE activity in the different regions of the brain followed the order striatum > hippocampus > (prefrontal cortex/hypothalamus/ cerebellum) > brainstem > septo-hippocampal system. In ex vivo assays, Hal inhibited AChE activity obtained from homogenate tissue of the striatum, hippocampus, and septo-hippocampal system in a concentration-dependent manner. The inhibitory concentration of 50% of enzyme activity (IC₅₀) indicated that the septo-hippocampal system required a higher concentration of Hal (IC₅₀ = 202.5 µmol·L⁻¹) to inhibit AChE activity compared to the striatum (IC₅₀ = 162.5 µmol·L⁻¹) and hippocampus (IC₅₀ = 145 µmol·L⁻¹). In in vivo assays, male Swiss mice treated with concentrations of Hal higher than 0.1 mg·kg⁻¹ induced cataleptic effects. Positive correlations with Spearman’s correlation were observed only between the lack of cataleptic effect and the decreased AChE activity of the hippocampus in the mice treated with 0.01 mg·kg⁻¹ of Hal but not in the striatum and septo-hippocampal system. Conclusions: Our results suggest that Hal could increase cholinergic effects via AChE inhibition, in addition to its dopamine antagonist effect, as an alternative approach to the treatment of behavioral disturbances associated with dementia. Full article

Autism spectrum disorder is a complex neurodevelopmental disorder. The available medical treatment options for autism spectrum disorder are very limited. While the etiology and pathophysiology of autism spectrum disorder are still not fully understood, recent studies have suggested that wide alterations in the GABAergic, glutamatergic, cholinergic, and serotonergic systems play a key role in its development and progression. Histamine neurotransmission is known to have complex interactions with other neurotransmitters that fit perfectly into the complex etiology of this disease. Multitarget-directed compounds with an affinity for the histamine H₃ receptor indicate an interesting profile of activity against autism spectrum disorder in animal models. Here, we present the results of our research on the properties of (4-piperazin-1-ylbutyl)guanidine derivatives acting on histamine H₃ receptors as potential multitarget ligands. Through the virtual screening approach, we identified promising ligands among 32 non-imidazole histamine H₃ receptor antagonists/inverse agonists with potential additional activity against the dopamine D₂ receptor and/or cholinesterases. The virtual screening protocol integrated predictions from SwissTargetPrediction, SEA, and PPB2 tools, along with molecular docking simulations conducted using GOLD 5.3 and Glide 7.5 software. Among the selected ligands, compounds 25 and 30 blocked radioligand binding to the D₂ receptor at over 50% at a screening concentration of 1 µM. Further experiments allowed us to determine the pK_i value at the D₂ receptor of 6.22 and 6.12 for compounds 25 and 30, respectively. Our findings suggest that some of the tested compounds could be promising multitarget-directed ligands for the further research and development of more effective treatments for autism spectrum disorder. Full article

Purpose: Anticholinergic drugs can cause adverse events (AEs) in older adults. Clinical decision support systems (CDSSs) can detect prescriptions with a high anticholinergic load. Our starting hypothesis was that the anticholinergic load could be reduced by combining a CDSS with a strategy for generating pharmacist interventions. The objective of the present study was to assess the impact of this combination on the change over time in the anticholinergic load in hospitalized older adults. Methods: This prospective, single-centre study was divided into two 6-week periods. During the interventional period, a pharmacist analyzed the alerts generated by the CDSS for 30 targeted anticholinergic drugs and decided whether to issue a pharmacist intervention. A control period corresponds to standard care. The primary endpoint of the study is the delta of the anticholinergic load between the alert and hospital discharge; the secondary endpoint is the incidence of anticholinergic adverse events (AEs). Results: Of the 144 alerts generated, 87 were considered to be relevant (36 in the interventional period and 51 in the control period). A significant difference was observed between the delta anticholinergic load between the experimental and control periods (1.61 vs. 0.67, p-value = 0.0115). For the targeted drugs (n = 94) over the 87 alerts, 46.8% were for antihistamines and 21.3% were for desloratadine. Of the 36 pharmacist interventions sent by the pharmacist, 19 (52.8%) were accepted. The most deprescribed drug class was the antihistamine class (n = 7), and the most deprescribed drug was amitriptyline (n = 5). Among these 87 patients with alerts, the correlation between the anticholinergic load and the number of AEs was not statistically significant (p = 0.887). The most common AE affecting the peripheral nervous system was constipation (28.6%), and the most common AE affecting the central nervous system was confusion (29.9%). Conclusions: Our results showed that the combination of specific CDSS rules with pharmacist-mediated risk management procedures could further reduce the anticholinergic load in hospitalized older adults, relative to routine care. It remains to be determined whether this reduction in the anticholinergic load has an impact on the incidence of peripheral and central anticholinergic AEs, and thus the health of these patients. Full article

Cognitive impairment (CI) is a type of mental health disorder that mainly affects cognitive abilities, such as learning, memory, perception, and problem-solving. Currently, in clinical practice, the treatment of cognitive impairment mainly focuses on the application of cholinesterase inhibitors and NMDA receptor antagonists; however, there is no specific and effective drug yet. Procatechuic acid (PCA) possesses various functions, including antibacterial, antiasthmatic, and expectorant effects. In recent years, it has received growing attention in the cognitive domain. Therefore, by summarizing the mechanisms of action of procatechuic acid in the treatment of cognitive impairment in this paper, it is found that procatechuic acid has multiple effects, such as regulating the expression of neuroprotective factors, inhibiting cell apoptosis, promoting the autophagy-lysosome pathway, suppressing oxidative stress damage, inhibiting inflammatory responses, improving synaptic plasticity dysfunction, inhibiting Aβ deposition, reducing APP hydrolysis, enhancing the cholinergic system, and inhibiting the excitotoxicity of neuronal cells. The involved signaling pathways include activating Pi3K-akt-mTor and inhibiting JNK, P38 MAPK, P38-ERK-JNK, SIRT1, and NF-κB/p53, etc. This paper aims to present the latest progress in research on procatechuic acid, including aspects such as its chemical properties, sources, pharmacokinetics, mechanisms for treating neurodegenerative diseases. Full article

Memory is one of the most important abilities of our brain. The process of memory and learning is necessary for the proper existence of humans in the surrounding environment. However, sometimes there are unfavourable changes in the functioning of the brain and memory deficits occur, which may be associated with various diseases. Disturbances in the cholinergic system lead to abnormalities in memory functioning and are an essential part of clinical symptoms of many neurodegenerative diseases. However, their treatment is difficult and still unsatisfactory; thus, it is necessary to search for new drugs and their targets, being an alternative method of mono- or polypharmacotherapy. One of the possible strategies for the modulation of memory-related cognitive disorders is connected with the endocannabinoid system (ECS). The aim of the present study was to determine for the first time the effect of administration of natural cannabinoid compound (cannabidiol, CBD) and rivastigmine alone and in combination on the memory disorders connected with cholinergic dysfunctions in mice, provoked by using an antagonist of muscarinic cholinergic receptor—scopolamine. To assess and understand the memory-related effects in animals, we used the passive avoidance (PA) test, commonly used to examine the different stages of memory. An acute administration of CBD (1 mg/kg) or rivastigmine (0.5 mg/kg) significantly affected changes in scopolamine-induced disturbances in three different memory stages (acquisition, consolidation, and retrieval). Interestingly, co-administration of CBD (1 mg/kg) and rivastigmine (0.5 mg/kg) also attenuated memory impairment provoked by scopolamine (1 mg/kg) injection in the PA test in mice, but at a much greater extent than administered alone. The combination therapy of these two compounds, CBD and rivastigmine, appears to be more beneficial than substances administered alone in reducing scopolamine-induced cognitive impairment. This polytherapy seems to be favourable in the pharmacotherapy of various cognitive disorders, especially those in which cholinergic pathways are implicated. Full article

Gynecological diseases are triggered by aberrant molecular pathways that alter gene expression, hormonal balance, and cellular signaling pathways, which may lead to long-term physiological consequences. This study was able to identify highly preserved modules and key hub genes that are mainly associated with gynecological diseases, represented by endometriosis (EM), ovarian cancer (OC), cervical cancer (CC), and endometrial cancer (EC), through the weighted gene co-expression network analysis (WGCNA) of microarray datasets sourced from the Gene Expression Omnibus (GEO) database. Five highly preserved modules were observed across the EM (GSE51981), OC (GSE63885), CC (GSE63514), and EC (GSE17025) datasets. The functional annotation and pathway enrichment analysis revealed that the highly preserved modules were heavily involved in several inflammatory pathways that are associated with transcription dysregulation, such as NF-kB signaling, JAK-STAT signaling, MAPK-ERK signaling, and mTOR signaling pathways. Furthermore, the results also include pathways that are relevant in gynecological disease prognosis through viral infections. Mutations in the ESR1 gene that encodes for ERα, which were shown to also affect signaling pathways involved in inflammation, further indicate its importance in gynecological disease prognosis. Potential drugs were screened through the Drug Repurposing Encyclopedia (DRE) based on the up-and downregulated hub genes, wherein a bacterial ribosomal subunit inhibitor and a benzodiazepine receptor agonist were the top candidates. Other drug candidates include a dihydrofolate reductase inhibitor, glucocorticoid receptor agonists, cholinergic receptor agonists, selective serotonin reuptake inhibitors, sterol demethylase inhibitors, a bacterial antifolate, and serotonin receptor antagonist drugs which have known anti-inflammatory effects, demonstrating that the gene network highlights specific inflammatory pathways as a therapeutic avenue in designing drug candidates for gynecological diseases. Full article

Basal forebrain cholinergic dysfunction, most likely linked with tau protein aggregation, is a characteristic feature of Alzheimer’s disease (AD). Recent evidence suggests that tau protein is a putative target for the treatment of dementia, and the tau aggregation inhibitor, hydromethylthionine mesylate (HMTM), has emerged as a potential disease-modifying treatment. However, its efficacy was diminished in patients already receiving approved acetylcholinesterase inhibitors. In this study, we ask whether this negative interaction can also be mimicked in experimental tau models of AD and whether the underlying mechanism can be understood. From a previous age profiling study, 6-month-old line 1 (L1) tau transgenic mice were characterized by a severe reduction in several cholinergic markers. We therefore assessed whether long-term pre-exposure with the acetylcholinesterase inhibitor rivastigmine alone and in conjunction with the tau aggregation inhibitor HMTM can reverse cholinergic deficits in L1. Rivastigmine and HMTM, and combinations of the two compounds were administered orally for 11 weeks to both L1 and wild-type mice. The brains were sectioned with a focus on the basal forebrain, motor cortex and hippocampus. Immunohistochemical staining and quantification of choline acetyltransferase (ChAT), tyrosine kinase A (TrkA)-positive neurons and relative optical intensity (ROI) for vesicular acetylcholine transporter (VAChT), and acetylcholinesterase (AChE) reactivity confirmed reversal of the diminished cholinergic phenotype of interneurons (nucleus accumbens, striatum) and projection neurons (medial septum, nucleus basalis magnocellularis) by HMTM, to a greater extent than by rivastigmine alone in L1 mice. Combined administration did not yield additivity but, in most proxies, led to antagonistic effects in which rivastigmine decreased the benefits shown with HMTM alone. Local markers (VAChT and AChE) in target structures of the basal forebrain, motor cortex and hippocampal CA3 seemed to be normalized by HMTM, but not by rivastigmine or the combination of both drugs. HMTM, which was developed as a tau aggregation inhibitor, strongly decreased the tau load in L1 mice, however, not in combination with rivastigmine. Taken together, these data confirm a cholinergic phenotype in L1 tau transgenic mice that resembles the deficits observed in AD patients. This phenotype is reversible by HMTM, but at the same time appears to be subject to a homeostatic regulation induced by chronic pre-treatment with an acetylcholinesterase inhibitor, which interferes with the efficacy of HMTM. The strongest phenotypic reversal coincided with a normalization of the tau load in the cortex and hippocampus of L1, suggesting that tau accumulation underpins the loss of cholinergic markers in the basal forebrain and its projection targets. Full article

Many people around the world suffer from neurodegenerative diseases associated with cognitive impairment. As life expectancy increases, this number is steadily rising. Therefore, it is extremely important to search for new treatment strategies and to discover new substances with potential neuroprotective and/or cognition-enhancing effects. This study focuses on investigating the potential of astragaloside IV (AIV), a triterpenoid saponin with proven acetylcholinesterase (AChE)-inhibiting activity naturally occurring in the root of Astragalus mongholicus, to attenuate memory impairment. Scopolamine (SCOP), an antagonist of muscarinic cholinergic receptors, and lipopolysaccharide (LPS), a trigger of neuroinflammation, were used to impair memory processes in the passive avoidance (PA) test in mice. This memory impairment in SCOP-treated mice was attenuated by prior intraperitoneal (ip) administration of AIV at a dose of 25 mg/kg. The attenuation of memory impairment by LPS was not observed. It can therefore be assumed that AIV does not reverse memory impairment by anti-inflammatory mechanisms, although this needs to be further verified. All doses of AIV tested did not affect baseline locomotor activity in mice. In the post mortem analysis by mass spectrometry of the body tissue of the mice, the highest content of AIV was found in the kidneys, then in the spleen and liver, and the lowest in the brain. Full article

Alzheimer’s disease (AD), marked by cognitive impairment, predominantly affects the brain regions regulated by cholinergic innervation, such as the cerebral cortex and hippocampus. Cholinergic dysfunction, a key contributor to age-related cognitive decline, has spurred investigations into potential therapeutic interventions. We have previously shown that choline alphoscerate (α-GPC), a cholinergic neurotransmission-enhancing agent, protects from Aβ-mediated neurotoxicity. Herein, we investigated the effects of α-GPC on the microglial phenotype in response to Aβ via modulation of the nicotinic alpha-7 acetylcholine receptor (α7 nAChR). BV2 microglial cells were pre-treated for 1 h with α-GPC and were treated for 24, 48, and 72 h with Aβ_1–42 and/or α-BTX, a selective α7nAchR antagonist. Fluorescent immunocytochemistry and Western blot analysis showed that α-GPC was able to antagonize Aβ-induced inflammatory effects. Of note, α-GPC exerted its anti-inflammatory effect by directly activating the α7nAChR receptor, as suggested by the induction of an increase in [Ca²⁺]_i and Ach-like currents. Considering that cholinergic transmission appears crucial in regulating the inflammatory profiles of glial cells, its modulation emerges as a potential pharmaco-therapeutic target to improve outcomes in inflammatory neurodegenerative disorders, such as AD. Full article

The tunica muscularis of mammalian esophagi is composed of striated muscle and smooth muscle. Contraction of the esophageal striated muscle portion is mainly controlled by cholinergic neurons. On the other hand, smooth muscle contraction and relaxation are controlled not only by cholinergic components but also by non-cholinergic components in the esophagus. Adenosine triphosphate (ATP) is known to regulate smooth muscle contraction and relaxation in the gastrointestinal tract via purinergic receptors. However, the precise mechanism of purinergic regulation in the esophagus is still unclear. Therefore, the aim of the present study was to clarify the effects of ATP on the mechanical responses of the esophageal muscle in mice. An isolated segment of the mouse esophagus was placed in a Magnus’s tube and longitudinal mechanical responses were recorded. Exogenous application of ATP induced contractile responses in the esophageal preparations. Tetrodotoxin, a blocker of voltage-dependent sodium channels in neurons and striated muscle, did not affect the ATP-induced contraction. The ATP-evoked contraction was blocked by pretreatment with suramin, a purinergic receptor antagonist. RT-PCR revealed the expression of mRNA of purinergic receptor genes in the mouse esophageal tissue. The findings suggest that purinergic signaling might regulate the motor activity of mouse esophageal smooth muscle. Full article

Botulinum neurotoxin A (BoNT/A) is a potent neurotoxin that silences cholinergic neurotransmission through the cleavage of the synaptic protein SNAP-25. Previous studies have shown that, in addition to its paralytic effects, BoNT/A can inhibit sensory nerve activity. The aim of this study was to identify how BoNT/A inhibits afferent signalling from the bladder. To investigate the role of SNAP-25 cleavage in the previously reported BoNT/A-dependent inhibition of sensory signalling, we developed a recombinant form of BoNT/A with an inactive light chain, rBoNT/A (0), unable to paralyse muscle. We also developed recombinant light chain (LC)-domain-only proteins to better understand the entry mechanisms, as the heavy chain (HC) of the protein is responsible for the internalisation of the light chain. We found that, despite a lack of catalytic activity, rBoNT/A (0) potently inhibited the afferent responses to bladder distension to a greater degree than catalytically active rBoNT/A. This was also clear from the testing of the LC-only proteins, as the inactive rLC/A (0) protein inhibited afferent responses significantly more than the active rLC/A protein. Immunohistochemistry for cleaved SNAP-25 was negative, and purinergic and nitrergic antagonists partially and totally reversed the sensory inhibition, respectively. These data suggest that the BoNT/A inhibition of sensory nerve activity in this assay is not due to the classical well-characterised ‘double-receptor’ mechanism of BoNT/A, is independent of SNAP25 cleavage and involves nitrergic and purinergic signalling mechanisms. Full article