Increased expression of the dopamine transporter leads to loss of dopamine neurons, oxidative stress and l-DOPA reversible motor deficits.

The dopamine transporter is a key protein responsible for regulating dopamine homeostasis. Its function is to transport dopamine from the extracellular space into the presynaptic neuron. Studies have suggested that accumulation of dopamine in the cytosol can trigger oxidative stress and neurotoxicity. Previously, ectopic expression of the dopamine transporter was shown to cause damage in non-dopaminergic neurons due to their inability to handle cytosolic dopamine. However, it is unknown whether increasing dopamine transporter activity will be detrimental to dopamine neurons that are inherently capable of storing and degrading dopamine. To address this issue, we characterized transgenic mice that over-express the dopamine transporter selectively in dopamine neurons. We report that dopamine transporter over-expressing (DAT-tg) mice display spontaneous loss of midbrain dopamine neurons that is accompanied by increases in oxidative stress markers, 5-S-cysteinyl-dopamine and 5-S-cysteinyl-DOPAC. In addition, metabolite-to-dopamine ratios are increased and VMAT2 protein expression is decreased in the striatum of these animals. Furthermore, DAT-tg mice also show fine motor deficits on challenging beam traversal that are reversed with l-DOPA treatment. Collectively, our findings demonstrate that even in neurons that routinely handle dopamine, increased uptake of this neurotransmitter through the dopamine transporter results in oxidative damage, neuronal loss and l-DOPA reversible motor deficits. In addition, DAT over-expressing animals are highly sensitive to MPTP-induced neurotoxicity. The effects of increased dopamine uptake in these transgenic mice could shed light on the unique vulnerability of dopamine neurons in Parkinson's disease.

Buspirone anti-dyskinetic effect is correlated with temporal normalization of dysregulated striatal DRD1 signalling in L-DOPA-treated rats.

Dopamine replacement with l-DOPA is the most effective therapy in Parkinson's disease. However, with chronic treatment, half of the patients develop an abnormal motor response including dyskinesias. The specific molecular mechanisms underlying dyskinesias are not fully understood. In this study, we used a well-characterized animal model to first establish the molecular differences between rats that did and did not develop dyskinesias. We then investigated the molecular substrates implicated in the anti-dyskinetic effect of buspirone, a 5HT1A partial agonist. Striatal protein expression profile of dyskinetic animals revealed increased levels of the dopamine receptor (DR)D3, ΔFosB and phospho (p)CREB, as well as an over-activation of the DRD1 signalling pathway, reflected by elevated ratios of phosphorylated DARPP32 and ERK2. Buspirone reduced the abnormal involuntary motor response in dyskinetic rats in a dose-dependent fashion. Buspirone (4 mg/kg) dramatically reduced the presence and severity of dyskinesias (by 83%) and normalized DARPP32 and ERK2 phosphorylation ratios, while the increases in DRD3, ΔFosB and pCREB observed in dyskinetic rats were not modified. Pharmacological experiments combining buspirone with 5HT1A and DRD3 antagonists confirmed that normalization of both pDARPP32 and pERK2 is required, but not sufficient, for blocking dyskinesias. The correlation between pDARPP32 ratio and dyskinesias was significant but not strong, pointing to the involvement of convergent factors and signalling pathways. Our results suggest that in dyskinetic rats DRD3 striatal over-expression could be instrumental in the activation of DRD1-downstream signalling and demonstrate that the anti-dyskinetic effect of buspirone in this model is correlated with DRD1 pathway normalization.

Botulinum toxin for conditions of the female pelvis.

INTRODUCTION AND HYPOTHESIS: Botulinum toxin has recently been approved by the Food and Drug Administration (FDA) for the treatment of urinary incontinence associated with neurogenic detrusor overactivity. However, it has also been used off-label for a multitude of other conditions in the female pelvis, including urological, gynecological, and colorectal. This article reviews the most recent data regarding its efficacy and safety, and administration techniques for those conditions. METHODS: A literature review of the most relevant reports published between 1985 and 2012. RESULTS: Urinary incontinence related to neurogenic detrusor overactivity is currently the only approved indication in the female pelvis. Other supported off-label uses include: idiopathic detrusor overactivity, interstitial cystitis/bladder pain syndrome, detrusor sphincter dyssynergia, high-tone pelvic floor dysfunction, anal fissure, anismus, and functional anal pain. CONCLUSIONS: Botulinum toxin may effectively and safely be used in many conditions of the female pelvis. More high quality research is needed to better clarify its role in the therapeutic algorithm for those indications.

Single treatments that have lasting effects: some thoughts on the antidepressant effects of ketamine and botulinum toxin and the anxiolytic effect of psilocybin.

Recent clinical trials suggest that 3 single biological treatments have effects that persist. Based on research showing that the muscles involved in facial expressions can feed back to influence mood, a single trial diminishing glabella frown lines with botulinum toxin demonstrated a significant antidepressant effect for 16 weeks. Based primarily on research with animal models of depression suggesting that glutamate may be involved in depression, the N-methyl-D-aspartate antagonist ketamine has been tested in several trials. A single dose decreased depression for up to a week. The reported effects of the use of mushrooms containing psilocybin by a number of cultures around the world has stimulated several trials showing beneficial effects of a single dose of psilocybin for over a year in healthy people, and for up to 3 months in patients with anxiety disorders who have advanced cancer. This article discusses these studies, their rationale, their possible mechanisms of action, the future clinical research required to establish these therapies and the basic research required to optimize single treatments that have lasting effects.

Implications of Parkinson's disease pathophysiology for the development of cell replacement strategies and drug discovery in neurodegenerative diseases.

Parkinson's disease (PD) is a progressive neurodegenerative disorder traditionally characterized by the loss of dopaminergic neurons in the substantia nigra (SN) at the midbrain. The potential use of adult or embryonic stem cells, induced pluriputent stem (iPS) cells and endogenous neurogenesis in cell replacement strategies has lead to numerous studies and clinical trials in this direction. It is now possible to differentiate stem cells into dopaminergic neurons in vitro and clinical trials have shown an improvement in PD-related symptoms after intra-striatal embryonic transplants and acceptable cell survival rates on the mid term. However, clinical improvement is transitory and associated with a strong placebo effect. Interestingly, recent pathological studies in PD patients who received embryonic stem cells show that in PD patients, grafted neurons show PD-related pathology. In this manuscript we review the latest findings regarding PD pathophysiology and give an outlook on the implications of these findings in how cell replacement strategies for PD treatment should be tested. These include changes in the type of animal models used, the preparation/conditioning of the cells before intracerebral injection, specially regarding backbone chronic diseases in iPS cells and determining the optimal proliferation, survival, differentiation and migration capacity of the grafted cells.

Muscle biopsy substantiates long-term MRI alterations one year after a single dose of botulinum toxin injected into the lateral gastrocnemius muscle of healthy volunteers.

Despite numerous clinical and experimental studies on botulinum toxin type A (BoNT/A), long-term alterations of muscle texture and fine structure following BoNT/A treatment have thus far not been studied in normal human skeletal muscle. After obtaining institutional review board approval, we performed a prospective, placebo-controlled, double-blinded follow-up study on two healthy adults using magnetic resonance imaging (MRI) and muscle biopsy to visualize long-term alterations after a single BoNT/A injection into the lateral head of the gastrocnemius muscle. MRI disclosed a high-signal-intensity pattern in short tau inversion recovery sequences, and a reduction of the cross-sectional area in the BoNT/A-injected, but not in the saline-injected contralateral control muscle (at 6 to 9 months in volunteer A: 73%, in B: 62%; at 12 months in A: 88%, and in B: 78%). Enzyme histochemistry, 12 months after injection, confirmed neurogenic atrophy of muscle fibers only in the BoNT/A-injected muscle. Electron microscopy revealed additional degenerative changes at the neuromuscular junction. The data confirm that MRI is a suitable tool to monitor the long-term effect of BoNT/A on skeletal muscle. Neurogenic muscle atrophy following a single BoNT/A injection should be taken into consideration when repeated BoNT/A injections into the same muscles are proposed.

Botulinum toxin in paediatric urology: a systematic literature review.

The use of botulinum toxin in paediatric urology has become increasingly widespread during recent years; however, the evidence base for its clinical use has been from adult urology centres and trials. The mechanism of action of this potent neurotoxin is discussed and evidence for its efficacy and safety is reviewed in both the adult and paediatric literature. Second, an electronic literature search was performed using the keywords paediatric urology, botulinum toxin, bladder, sphincter, and voiding dysfunction (excluding abstracts, letters and case reports). A total of 225 children in 10 peer-reviewed publications were evaluated with 165 paediatric patients with neurogenic overactivity (6 studies), 21 patients with idiopathic overactivity (1 study) and 39 patients with voiding dysfunction (3 studies). Specifically, we discuss the main indications of intravesical injections for detrusor overactivity and of intrasphincteric injections for refractory voiding dysfunctions in the paediatric population.

Botulinum neurotoxin: evolution from poison, to research tool--onto medicinal therapeutic and future pharmaceutical panacea.

Botulinum neurotoxin (BoNT), for more than a hundred years, has been a recognized poisonous principle in spoiled food. As its chemical structure became unraveled, and as more knowledge was gained over its mechanism of toxicity, it became clear that BoNT had the potential to act therapeutically as a targeted toxin that could inactivate specific nerve populations, and thus achieve a therapeutic goal. BoNT has evolved over the past 25 years into a viable therapeutic, now being a first line treatment for dystonia, overtly altering the course of progression of this disorder. BoNT is used for hyperhidrosis and gustatory sweating syndrome, alleviation of pain, as a treatment for overactive bladder, achalasia and anal fissure; and it has gained popularity as a cosmetic aid. Many other possible uses are being explored. The greatest potential for BoNT may lie in its being a molecular Trojan Horse - able to carry a specific enzyme or specific drug to the inside of a cancer or other type of cell while bypassing other cells and thereby having little or no ill effect. BoNT's pharmaceutical potential is boundless.

Modulation of Kv2.1 channel gating and TEA sensitivity by distinct domains of SNAP-25.

Distinct domains within the SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) proteins, STX1A (syntaxin 1A) and SNAP-25 (synaptosome-associated protein-25 kDa), regulate hormone secretion by their actions on the cell's exocytotic machinery, as well as voltage-gated Ca2+ and K+ channels. We examined the action of distinct domains within SNAP-25 on Kv2.1 (voltage gated K+ 2.1) channel gating. Dialysis of N-terminal SNAP-25 domains, S197 (SNAP-25(1-197)) and S180 (SNAP-25(1-180)), but not S206 (full-length SNAP-25(1-206)) increased the rate of Kv2.1 channel activation and slowed channel inactivation. Remarkably, these N-terminal SNAP-25 domains, acting on the Kv2.1 cytoplasmic N-terminus, potentiated the external TEA (tetraethylammonium)-mediated block of Kv2.1. To further examine whether these are effects of the channel pore domain, internal K+ was replaced with Na+ and external K+ was decreased from 4 to 1 mM, which decreased the IC50 of the TEA block from 6.8+/-0.9 mM to >100 mM. Under these conditions S180 completely restored TEA sensitivity (7.9+/-1.5 mM). SNAP-25 C-terminal domains, SNAP-25(198-206) and SNAP-25(181-197), had no effect on Kv2.1 gating kinetics. We conclude that different domains within SNAP-25 can form distinct complexes with Kv2.1 to execute a fine allosteric regulation of channel gating and the architecture of the outer pore structure in order to modulate cell excitability.

Fyn is required for haloperidol-induced catalepsy in mice.

Fyn-mediated tyrosine phosphorylation of N-methyl-D-aspartate (NMDA) receptor subunits has been implicated in various brain functions, including ethanol tolerance, learning, and seizure susceptibility. In this study, we explored the role of Fyn in haloperidol-induced catalepsy, an animal model of the extrapyramidal side effects of antipsychotics. Haloperidol induced catalepsy and muscle rigidity in the control mice, but these responses were significantly reduced in Fyn-deficient mice. Expression of the striatal dopamine D(2) receptor, the main site of haloperidol action, did not differ between the two genotypes. Fyn activation and enhanced tyrosine phosphorylation of the NMDA receptor NR2B subunit, as measured by Western blotting, were induced after haloperidol injection of the control mice, but both responses were significantly reduced in Fyn-deficient mice. Dopamine D(2) receptor blockade was shown to increase both NR2B phosphorylation and the NMDA-induced calcium responses in control cultured striatal neurons but not in Fyn-deficient neurons. Based on these findings, we proposed a new molecular mechanism underlying haloperidol-induced catalepsy, in which the dopamine D(2) receptor antagonist induces striatal Fyn activation and the subsequent tyrosine phosphorylation of NR2B alters striatal neuronal activity, thereby inducing the behavioral changes that are manifested as a cataleptic response.

Botulinum toxin: mechanisms of action

O propósito deste artigo é uma revisão dos mecanismos de ação da toxina botulínica (TB) relevantes para a compreensão do seu uso terapêutico. A ação da TB a nível molecular consiste na sua ligação extracelular a estruturas glicoprotéicas em terminais nervosos colinérgicos e no bloqueio intracelular da secreção de acetilcolina. A TB interfere no reflexo espinal de estiramento através do bloqueio de fibras musculares intrafusais causando redução da sinalização aferente veiculada por fibras Ia e II e do tono muscular. Portanto, o efeito da TB pode estar relacionado não somente à paresia muscular mas também à inibição reflexa espinal. A TB promove ainda o bloqueio de fibras autonômicas para músculos lisos e glândulas exócrinas. Apesar de ocorrer alguma difusão sistêmica após a aplicação intramuscular a TB não atinge o sistema nervoso central (SNC) devido ao seu peso molecular (não atravessa a barreira hematoencefálica) e à lentidão do seu transporte axonal retrógrado que permite a sua inativação. Os efeitos indiretos sobre o SNC são: inibição reflexa, reversão das alterações da inibição recíproca, da inibição intracortical e de potenciais evocados somatosensoriais. A redução da dor induzida por formalina sugere que a TB tenha efeito analgésico direto possivelmente mediado por bloqueio da substância P, do glutamato e do peptídeo relacionado ao gene da calcitonina.

Urologic applications of botulinum toxin therapy for voiding dysfunction.

Botulinum toxin is primarily a presynaptic neuromuscular blocking agent inducing selective and reversible muscle weakness up to several months when injected intramuscularly in small quantities. The clinical use of botulinum toxin type-A has gained widespread acceptance and application for numerous adult and pediatric spasticity syndromes. This has led to the urologic adoption of this minimally invasive therapy for the treatment of idiopathic and neurogenic detrusor overactivity, interstitial cystitis, detrusor-sphincter dyssynergia, urinary retention, and prostatic conditions. Outlined below is an overview of the clinical adoption of this therapy for the treatment of various dysfunctions of the lower urinary tract.

Experiencia con toxina botulínica en el tratamiento de la disfonia espasmódica / Experience with botulinic toxine in the treatment of spasmodic dysphonia

Disfonía espasmódica o distonía laríngea son los términos clínicos utilizados para referirse a una alteración neurológica central, de carácter crónico y progresivo. Históricamente estos enfermos han sido tratados por numerosos especialistas, sin resultados satisfactorios. Respecto al manejo otorrinolaringológico se ha intentado múltiples tratamientos, incluyendo la sección del nervio recurrente laríngeo, técnicas fonoquirúrgicas y el uso de fármacos, todos ellos con numerosos efectos adversos y malos resultados vocales. La terapia de elección, desde 1984, es la infiltración de toxina botulínica en la musculatura laríngea intrínseca distónica. El presente trabajo muestra la experiencia clínica con la inyección de toxina botulínica en 20 pacientes con distonía laríngea, realizada en el Servicio de Otorrinolaringología del Hospital Clínico de la Universidad de Chile. Quince de ellos fueron infiltrados con esta toxina. Los restantes 5 pacientes no se inyectaron porque rechazaron este tratamiento. Se comparan los resultados vocales en ambos grupos, utilizando la escala de ranking vocal abreviada (Blitzer y cols., 1992), concluyéndose que los pacientes infriltrados mejoraron su voz; en cambio, quienes no se infiltraron permanecieron o progresaron en su compromiso vocal.

New therapeutic indications for botulinum toxins.

The efficacy of botulinum toxin (BTX) without systemic effects has led to the rapid development of applications in neuromuscular disorders, hyperactivity of sudomotor cholinergic-mediated glandular function, and pain syndromes. The successful use of BTX in conditions with muscle overactivity, such as dystonia and spasticity, has been established and new areas in the field of movement disorders such as tics, tremor, myoclonic jerks, and stuttering has been explored with satisfactory results. Strategies to temporarily inactivate muscle function after orthopaedic or neurosurgery have also been developed. BTX treatment of hyperhidrosis was followed by its application in other hypersecretory conditions (hyperlacrimation and nasal hypersecretion) and in excessive drooling. Studies are in progress, aimed at optimising the technique and protocol of administration. Other applications for BTX have been proposed in gastroenterological and urogenital practice; it appears to be effective in replacing standard surgical procedures. Trials of BTX in painful conditions are ongoing mainly on refractory tension headache, migraine, and backache as well as dystonia-complex regional pain syndrome and myofascial pain with promising results. Recently, the fastest growing use for BTX toxin has been in the cosmetic applications. Clearly, the indications for the use of BTX are expanding, but further clinical trials will be needed in many different areas.

[Detrusor-sphincter dyssynergia and botulinum toxin]. / Dyssynergie vésico-sphinctérienne et toxine botulique.

OBJECTIVE: Botulinum toxin (BT) injection into the external urethral sphincter is a promising therapy for neurogenic voiding disorders due to detrusor-sphincter dyssynergia (DSD). However the optimal treatment protocol remains unclear. METHOD: A PubMed reference search and manual bibliography review were performed, along with a search in the Annales de réadaptation et de médecine physique and in the reports of the International French-language Society of Urodynamics and the International Continence Society, which allowed us to select twelve pertinent articles with PubMed, two articles from the Annales and two conference reports. Our analysis gave special emphasis to assessment criteria, application, dosage and BT injection technique. RESULTS: Used for the first time in 1988 in spinal cord injury patients to reduce outflow obstruction due to DSD, BT injections have been shown to be a valuable alternative management of bladder dysfunction with DSD. They have been proposed in neurological patients unable to perform self-catheterisation, after drug failure and before surgery. Parameters for results assessment are mostly clinical (increased free interval between voiding, decreased post-void residual urine volumes), urodynamic (improvement in bladder emptying, increase in functional bladder capacity and decrease in urethral pressure) and electromyographic (denervation of striated urethral sphincter). The literature data regarding type of BT, dosage and protocol vary widely. Duration of action is from 2 to 12 months. Both transurethral and transperineal injections monitored by EMG are equally effective in improving detrusor-sphincter dyssynergia. CONCLUSION: With few side effects and satisfactory medium-term results, BT should be recommended as a component of DSD therapies. We propose a practical method for BT use.

[Digestive tract and botulinum toxin]. / Tube digestif et toxine botulique.

INTRODUCTION: the aim of this work was to check literature to assess botulinum toxin injection efficacy in gastrointestinal motor disorders with special emphasis on controlled clinical trials. METHODS: literature was carried out with the Medline data bank. RESULTS: seventy three articles in French and in English including a recent general review were retained. Comparative clinical trials only concerned achalasia and anal fissure. The other gastrointestinal motor disorders only gave rise to open non-controlled trials assessed on clinical end points. DISCUSSION: this review of literature helps to determine usefulness and safety of Botulinum toxin injection in the treatment of esophageal achalasia and anal fissure. The main limitation is its brief duration of action. Studies concerning the others spastic motor disorders are based on unknown physiopathology and controlled trials are required to assess its efficacy. CONCLUSION: botulinum toxin is increasingly used for gastrointestinal motor disorders with worthwhile results. Its efficacy has been yet established for only two disorders : it has a valuable palliative role in achalasia and may be curative for anal fissure.

A new extra-vertebral treatment model for incomplete spinal cord injuries.

Advances made in recent times in spinal cord injury repair research will soon take us toward a cure in paraplegics. But what are the prospects for quadriplegics? Certain fundamental issues make treatment approaches to quadriplegia different and difficult. Injury at cervical region poses additional problems for any surgical intervention with life-threatening risks of i) endangering respiratory function, ii) cavitation, cysts, and syringomyelia formation extending cephalad to the injury, and iii) mid-lower cervical injuries, lower motor neuron death, and the resultant degeneration of brachial plexus axons would still leave the upper limbs denervated and paralyzed even as treatment procedures might successfully salvage the lower limbs. With these apparently insurmountable impediments in quadriplegic cord repair, it would be wise to turn to alternative treatment strategies. Conventional treatment models since the days of Ralph Gerard (1940) have all used intra-vertebral procedures. We present here a plausible extra-vertebral repair model suitable for incomplete cord injuries at cervical, thoracic, and lumbar levels. The procedure consists of identifying the extent of viable grey-white matter in the injured area and to utilize it efficiently as a "neural tissue bridge." Next, labile state is induced by using botulinum toxin/colchicine (Krishnan, 1983, 1991; Krishnan et al., 2001 a,b) and Ca+ channel blockers in the motorsensory nerve terminals of polisegmentally innervated skeletal muscles that "bridge" the injured cord segments. This would retrogradely induce a redundant state of intra-spinal growth of nerve terminals and new synaptic connections within those viable neural tissues, as well as promote effective relinking of the injured cord ends and enhance motor-sensory recovery.

Immediate-early gene response to methamphetamine, haloperidol, and quinolinic acid is not impaired in Huntington's disease transgenic mice.

Striatal neurons in symptomatic Huntington's disease (HD) transgenic mice are resistant to a variety of toxic insults, including quinolinic acid (QA), kainic acid and 3-nitropropionic acid. The basis for this resistance is currently unknown. To investigate the possibility that the immediate-early gene (IEG) response is defective in symptomatic HD mice leading to a lack of response to these compounds, we examined the expression of c-Fos and Krox 24 after administration of the indirect dopamine agonist methamphetamine, the dopamine D(2) receptor antagonist haloperidol and the neurotoxin QA in 5- and 10-week-old R6/2 transgenic HD and wild-type mice. Unlike wild-type and pre-symptomatic R6/2 transgenic HD mice, 10-week-old symptomatic HD mice were resistant to methamphetamine-induced gliosis and QA lesion. There was, however, no difference in the number or distribution of c-Fos-immunoreactive nuclei 2 hr after single injections of methamphetamine or haloperidol among 5- and 10-week-old wild-type mice and 5- and 10-week-old R6/2 HD mice. Similarly, despite their resistance to QA-induced lesioning and lower basal levels of krox-24 mRNA, the symptomatic R6/2 mice had equivalent increases in the amount of c-fos and krox-24 mRNA compared to wild-type and pre-symptomatic R6/2 HD mice as determined by in situ hybridization and densitometry 2 hr after QA administration. These data demonstrate that the c-Fos and Krox 24 IEG response to dopamine agonists, dopamine antagonists and neurotoxic insult is functional in symptomatic R6/2 HD mice. Resistance to toxic insult in R6/2 mice may be conferred by interactions of mutant huntingtin with proteins or transcriptional processes further along the toxic cascade.

Clostridium botulinum neurotoxins act with a wide range of potencies on SH-SY5Y human neuroblastoma cells.

We have described, in undifferentiated SH-SY5Y human neuroblastoma cells, the relative potency of Clostridium botulinum neurotoxin (BoNT) serotypes A-F Sensitivity of stimulated [3H]-noradrenaline ([3H]-NA) release to the toxins had a rank order of potency of: C > D > A > B > F after 3 days exposure. The difference between the most potent (BoNT/C: IC50 0.54 nM) and the least (BoNT/F: IC50 > 300 nM) was approximately 1,000-fold. Though fluid phase endocytosis may have been the mechanism of entry for low potency toxins the far higher potency of BoNT/C would suggest receptor-driven entry. Potency was not a reflection of the dependence of the release mechanism on a particular SNARE since the substrate specificities were mixed throughout the potency order. This indicated that the toxins differed in their efficiency of binding/endocytosis or enzymatic activity inside the cell. The serotypes that cleaved vesicle-associated membrane protein (VAMP) isoforms (BoNT/B, D and F) did not fully inhibit [3H]-NA release. Cleavage of the appropriate substrate proteins was observed for all serotypes. SNAP-25 cleavage by BoNT/A was shown to be a dose-dependent and correlated closely with reduction of release, supporting proteolysis as the mechanism by which toxin inhibited secretion. Comparison of the SH-SY5Y cell line sensitivity to BoNT/A with glycine releasing rat primary spinal cord neuron cultures, revealed a massive difference in potency; the primary cultures being approximately 200,000-fold more sensitive. The demonstration, using BoNTs, of the crucial role of SNAP-25, VAMP and syntaxin in SH-SY5Y cells suggests the use of this neuroblastoma as a model in the study of these proteins in neurotransmitter release.