Presynaptic Voltage-gated Calcium Channels Research Articles

Functional remodeling of presynaptic voltage-gated calcium channels in superficial layers of the dorsal horn during neuropathic pain

N- and P/Q-type voltage-gated Ca2+ channels are critical for synaptic transmission. While their expression is increased in the dorsal root ganglion (DRG) neuron cell bodies during neuropathic pain conditions, less is known about their synaptic remodeling. Here, we combined genetic tools with 2-photon Ca2+ imaging to explore the functional remodeling that occurs in central presynaptic terminals of DRG neurons during neuropathic pain. We imaged GCaMP6s fluorescence responses in an exvivo spinal cord preparation from mice expressing GCaMP6s in Trpv1-Cre lineage nociceptors. We show that Ca2+ transient amplitude is increased in central terminals of these neurons after spared nerve injury, and that this increase is mediated by both N-and P/Q-type channels. We found that GABA-B receptor-dependent inhibition of Ca2+ transients was potentiated in the superficial layer of the dorsal horn. Our results provide direct evidence toward nerve injury-induced functional remodeling of presynaptic Ca2+ channels in Trpv1-lineage nociceptor terminals.

The synergic effects of presynaptic calcium channel antagonists purified from spiders on memory elimination of glutamate-induced excitotoxicity in the rat hippocampus trisynaptic circuit

The hippocampus is a complex area of the mammalian brain and is responsible for learning and memory. The trisynaptic circuit engages with explicit memory. Hippocampal neurons express two types of presynaptic voltage-gated calcium channels (VGCCs) comprising N and P/Q-types. These VGCCs play a vital role in the release of neurotransmitters from presynaptic neurons. The chief excitatory neurotransmitter at these synapses is glutamate. Glutamate has an essential function in learning and memory under normal conditions. The release of neurotransmitters depends on the activity of presynaptic VGCCs. Excessive glutamate activity, due to either excessive release or insufficient uptake from the synapse, leads to a condition called excitotoxicity. This pathological state is common among all neurodegenerative disorders, such as Alzheimer’s and Parkinson’s diseases. Under these conditions, glutamate adversely affects the trisynaptic circuitry, leading to synaptic destruction and loss of memory and learning performance. This study attempts to clarify the role of presynaptic VGCCs in memory performance and reveals that modulating the activity of presynaptic calcium channels in the trisynaptic pathway can regulate the excitotoxic state and consequently prevent the elimination of neurons and synaptic degradation. All of these can lead to an improvement in learning and memory function. In the current study, two calcium channel blockers—omega-agatoxin-Aa2a and omega-Lsp-IA—were extracted, purified, and identified from spiders (Agelena orientalis and Hogna radiata) and used to modulate N and P/Q VGCCs. The effect of omega-agatoxin-Aa2a and omega-Lsp-IA on glutamate-induced excitotoxicity in rats was evaluated using the Morris water maze task as a behavioral test. The local expression of synaptophysin (SYN) was visualized for synaptic quantification using an immunofluorescence assay. The electrophysiological amplitudes of the field excitatory postsynaptic potentials (fEPSPs) in the input-output and LTP curves of the mossy fiber and Schaffer collateral circuits were recorded. The results of our study demonstrated that N and P/Q VGCC modulation in the hippocampus trisynaptic circuit of rats with glutamate-induced excitotoxicity dysfunction could prevent the destructive consequences of excitotoxicity in synapses and improve memory function and performance.

A cross-sectional study of ageing at the mouse neuromuscular junction and effects of an experimental therapeutic approach for dynapenia.

Despite prior efforts to understand and target dynapenia (age-induced loss of muscle strength), this condition remains a major challenge that reduces the quality of life in the aged population. We have focused on the neuromuscular junction (NMJ) where changes in structure and function have rarely been systematically studied as a dynamic and progressive process. Our cross-sectional study found neurotransmission at the male mouse NMJ to be biphasic, displaying an early increase followed by a later decrease, and this phenotype was associated with structural changes to the NMJ. A cross-sectional characterization showed that age-induced alterations fell into four age groups: young adult (3-6months), adult (7-18months), early aged (19-24months), and later aged (25-30months). We then utilized a small molecule therapeutic candidate, GV-58, applied acutely during the later aged stage to combat age-induced reductions in transmitter release by increasing calcium influx during an action potential, which resulted in a significant increase in transmitter release. This comprehensive study of neuromuscular ageing at the NMJ will enable future research to target critical time points for therapeutic intervention. KEY POINTS: Age-induced frailty and falls are the leading causes of injury-related death and are caused by an age-induced loss of muscle strength due to a combination of neurological and muscular changes. A cross-sectional approach was used to study age-induced changes to the neuromuscular junction in a mouse model, and physiological changes that were biphasic over the ageing time course were found. Changes in physiology at the neuromuscular junction were correlated with alterations in neuromuscular junction morphology. An acutely applied positive allosteric gating modifier of presynaptic voltage-gated calcium channels was tested as a candidate therapeutic strategy that could increase transmitter release at aged neuromuscular junctions. These results provide a detailed time course of age-induced changes at the neuromuscular junction in a mouse model and test a candidate therapeutic strategy for weakness.

Depolarization-induced bursts of miniature synaptic currents in individual synapses of developing cerebellum.

In central synapses, spontaneous transmitter release observed in the absence of action potential firing is often considered as a random process lacking time or space specificity. However, when studying miniature glutamatergic currents at cerebellar synapses between parallel fibers and molecular layer interneurons, we found that these currents were sometimes organized in bursts of events occurring at high frequency (about 30 Hz). Bursts displayed homogeneous quantal size amplitudes. Furthermore, in the presence of the desensitization inhibitor cyclothiazide, successive events within a burst displayed quantal amplitude occlusion. Based on these findings, we conclude that bursts originate in individual synapses. Bursts were enhanced by increasing either the external potassium concentration or the external calcium concentration, and they were strongly inhibited when blocking voltage-gated calcium channels by cadmium. Bursts were prevalent in elevated potassium concentration during the formation of the molecular layer but were infrequent later in development. Since postsynaptic AMPA receptors are largely calcium permeant in developing parallel fiber-interneuron synapses, we propose that bursts involve presynaptic calcium transients implicating presynaptic voltage-gated calcium channels, together with postsynaptic calcium transients implicating postsynaptic AMPA receptors. These simultaneous pre- and postsynaptic calcium transients may contribute to the formation and/or stabilization of synaptic connections.

Microphysiological Modeling of the Structure and Function of Neuromuscular Transmitter Release Sites.

The general mechanism of calcium-triggered chemical transmitter release from neuronal synapses has been intensely studied, is well-known, and highly conserved between species and synapses across the nervous system. However, the structural and functional details within each transmitter release site (or active zone) are difficult to study in living tissue using current experimental approaches owing to the small spatial compartment within the synapse where exocytosis occurs with a very rapid time course. Therefore, computer simulations offer the opportunity to explore these microphysiological environments of the synapse at nanometer spatial scales and on a sub-microsecond timescale. Because biological reactions and physiological processes at synapses occur under conditions where stochastic behavior is dominant, simulation approaches must be driven by such stochastic processes. MCell provides a powerful simulation approach that employs particle-based stochastic simulation tools to study presynaptic processes in realistic and complex (3D) geometries using optimized Monte Carlo algorithms to track finite numbers of molecules as they diffuse and interact in a complex cellular space with other molecules in solution and on surfaces (representing membranes, channels and binding sites). In this review we discuss MCell-based spatially realistic models of the mammalian and frog neuromuscular active zones that were developed to study presynaptic mechanisms that control transmitter release. In particular, these models focus on the role of presynaptic voltage-gated calcium channels, calcium sensors that control the probability of synaptic vesicle fusion, and the effects of action potential waveform shape on presynaptic calcium entry. With the development of these models, they can now be used in the future to predict disease-induced changes to the active zone, and the effects of candidate therapeutic approaches.

Neuromuscular Active Zone Structure and Function in Healthy and Lambert-Eaton Myasthenic Syndrome States.

The mouse neuromuscular junction (NMJ) has long been used as a model synapse for the study of neurotransmission in both healthy and disease states of the NMJ. Neurotransmission from these neuromuscular nerve terminals occurs at highly organized structures called active zones (AZs). Within AZs, the relationships between the voltage-gated calcium channels and docked synaptic vesicles govern the probability of acetylcholine release during single action potentials, and the short-term plasticity characteristics during short, high frequency trains of action potentials. Understanding these relationships is important not only for healthy synapses, but also to better understand the pathophysiology of neuromuscular diseases. In particular, we are interested in Lambert-Eaton myasthenic syndrome (LEMS), an autoimmune disorder in which neurotransmitter release from the NMJ decreases, leading to severe muscle weakness. In LEMS, the reduced neurotransmission is traditionally thought to be caused by the antibody-mediated removal of presynaptic voltage-gated calcium channels. However, recent experimental data and AZ computer simulations have predicted that a disruption in the normally highly organized active zone structure, and perhaps autoantibodies to other presynaptic proteins, contribute significantly to pathological effects in the active zone and the characteristics of chemical transmitters.

Editors' Note: What Is the Mechanism of Therapeutic and Adverse Effects of Gabapentinoids?

Dr. Benarroch reviewed the mechanisms underlying the therapeutic and adverse effects of gabapentinoids such as gabapentin and pregabalin. The author noted that the efficacy of these drugs for neuropathic pain, restless legs syndrome, and focal seizures is primarily attributed to their inhibition of the α2δ subunit of presynaptic voltage-gated calcium channels, which decreases neurotransmitter release. He discussed the relevance of this inhibitory action in the potential induction of cerebellar ataxia by gabapentinoids. In response, Dr. Gazulla et al. argue that GABAergic effects of these medications are also important, noting that gabapentin has been shown to increase the expression of δGABAA receptors, inhibitory tone in the cerebellum, and brain GABA concentration in patients, whereas pregabalin has been shown to increase neuronal calcium influx, which facilitates neurotransmission. They argue that gabapentinoids could actually help treat ataxia in diseases involving GABA deficiency. Responding to these comments, Dr. Benarroch notes that he did cite work finding increased expression of GABAA receptors with gabapentin use but did not cite a pilot study of pregabalin for ataxia because the review was focused on mechanisms relating to the current clinical use of gabapentinoids. He agrees that increased GABAergic transmission by these drugs may benefit disorders affecting Purkinje cells and other inhibitory neurons such as periodic alternating nystagmus but cautions that the mechanisms of ataxia are complex and that further studies are needed to confirm potential beneficial effects of gabapentinoids in cerebellar ataxia. This exchange highlights uncertainties regarding the potential therapeutic vs adverse effects of gabapentinoids in ataxia and other cerebellar disorders. Dr. Benarroch reviewed the mechanisms underlying the therapeutic and adverse effects of gabapentinoids such as gabapentin and pregabalin. The author noted that the efficacy of these drugs for neuropathic pain, restless legs syndrome, and focal seizures is primarily attributed to their inhibition of the α2δ subunit of presynaptic voltage-gated calcium channels, which decreases neurotransmitter release. He discussed the relevance of this inhibitory action in the potential induction of cerebellar ataxia by gabapentinoids. In response, Dr. Gazulla et al. argue that GABAergic effects of these medications are also important, noting that gabapentin has been shown to increase the expression of δGABAA receptors, inhibitory tone in the cerebellum, and brain GABA concentration in patients, whereas pregabalin has been shown to increase neuronal calcium influx, which facilitates neurotransmission. They argue that gabapentinoids could actually help treat ataxia in diseases involving GABA deficiency. Responding to these comments, Dr. Benarroch notes that he did cite work finding increased expression of GABAA receptors with gabapentin use but did not cite a pilot study of pregabalin for ataxia because the review was focused on mechanisms relating to the current clinical use of gabapentinoids. He agrees that increased GABAergic transmission by these drugs may benefit disorders affecting Purkinje cells and other inhibitory neurons such as periodic alternating nystagmus but cautions that the mechanisms of ataxia are complex and that further studies are needed to confirm potential beneficial effects of gabapentinoids in cerebellar ataxia. This exchange highlights uncertainties regarding the potential therapeutic vs adverse effects of gabapentinoids in ataxia and other cerebellar disorders.

Lambert Eaton Myasthenic Syndrome masquerading as myopathy in asymptomatic lung malignancy

Introduction: Lambert-Eaton Myasthenic Syndrome (LEMS), is a rare disorder characterized by antibodies against presynaptic voltage-gated calcium channels in the neuromuscular junction. It is regarded as a paraneoplastic syndrome, as most patients have an underlying malignancy, commonly a small-cell lung cancer (SCLC).

Presynaptic Paraneoplastic Disorders of the Neuromuscular Junction: An Update.

The neuromuscular junction (NMJ) is the target of a variety of immune-mediated disorders, usually classified as presynaptic and postsynaptic, according to the site of the antigenic target and consequently of the neuromuscular transmission alteration. Although less common than the classical autoimmune postsynaptic myasthenia gravis, presynaptic disorders are important to recognize due to the frequent association with cancer. Lambert Eaton myasthenic syndrome is due to a presynaptic failure to release acetylcholine, caused by antibodies to the presynaptic voltage-gated calcium channels. Acquired neuromyotonia is a condition characterized by nerve hyperexcitability often due to the presence of antibodies against proteins associated with voltage-gated potassium channels. This review will focus on the recent developments in the autoimmune presynaptic disorders of the NMJ.

Functions of Presynaptic Voltage-gated Calcium Channels.

Voltage-gated calcium channels are the principal conduits for depolarization-mediated Ca2+ entry into excitable cells. In this review, the biophysical properties of the relevant members of this family of channels, those that are present in presynaptic terminals, will be discussed in relation to their function in mediating neurotransmitter release. Voltage-gated calcium channels have properties that ensure they are specialized for particular roles, for example, differences in their activation voltage threshold, their various kinetic properties, and their voltage-dependence of inactivation. All these attributes play into the ability of the various voltage-gated calcium channels to participate in different patterns of presynaptic vesicular release. These include synaptic transmission resulting from single action potentials, and longer-term changes mediated by bursts or trains of action potentials, as well as release resulting from graded changes in membrane potential in specialized sensory synapses.

Preoperative Pregabalin Prolongs Duration of Spinal Anesthesia and Reduces Early Postoperative Pain

Abstract Background Various adjuvants have been used to prolong spinal anesthesia, with the additional advantages of delaying the onset of postoperative pain and reducing postoperative analgesic requirements. Pregabalin is an R-aminobutyric acid analog that binds to the α2-δ subunit of presynaptic voltage-gated calcium channels. Objective The aim of this work is to evaluate the efficacy of a single dose of pregabalin in terms of spinal blockade duration and its potential opioid-sparing effect during the first 24 hours postoperatively Patients and Methods There were limitations to the present study. First, since only 1 dosage of pregabalin was evaluated, we could not determine the most effective dosage. Second, clinically meaningful improvements in recovery were not assessed. Adequate postoperative pain control provides early postsurgical mobilization, shortened hospitalization, and increased patient satisfaction. Third, preoperative pain and anxiety scores were not recorded. Pregabalin might affect the preoperative pain, mood, and anxiety scores, and these factors can be related to the postoperative pain score. Results The mechanisms by which pregabalin premedication prolongs motor and sensory blocks using local anesthetics in spinal anesthesia are not fully understood. There may be several reasons for the prolongation of spinal anesthesia. Gabapentinoids are an r-aminobutyric acid analog that binds to α2-δ subunit of presynaptic voltage-gated calcium channels, and this inhibition decreases postsynaptic excitability by reducing potassium-evoked excitatory transmitter release. These medications provide antiepileptic, anxiolytic, and analgesic features by modulating both GABAergic neurotransmission and calcium influx. Gabapentinoid compounds produce a significant and clinically important improvement in preoperative anxiety scores. Since patients may be anxious in the perioperative period, the anxiolytic effects and euphorigenic effects of pregabalin may be beneficial.

Amifampridine to treat Lambert-Eaton myasthenic syndrome.

Lambert-Eaton myasthenic syndrome (LEMS) is a presynaptic autoimmune disabling neuromuscular disease caused by antibodies against presynaptic voltage-gated calcium channels. It reduces the quantal release of acetylcholine (Ach), causing muscle weakness, reduced or absent reflex and dysautonomia. About half of LEMS patients have associated small cell lung cancer. For symptomatic treatment, amifampridine (3,4-diaminopyridine [3,4-DAP]) is ideal because it increases the release of Ach at the presynaptic membrane. Since the first use of 3,4-DAP in LEMS patients in the 1980s, 136 LEMS patients were treated with amifampridines in the open-label studies and 208 patients in the eight randomized studies. These studies showed that amifampridine is the most effective drug for symptomatic treatment in LEMS. Now, 3,4-DAPP (3,4-DAP phosphate) is approved for adult LEMS patients and 3,4-DAP for pediatric patients. The recommended dose is 80 mg a day, divided 3 or 4 times a day. Side effects are usually mild, and the most frequently reported are paresthesia.

Gain-of-function mutations in the UNC-2/CaV2α channel lead to excitation-dominant synaptic transmission in Caenorhabditis elegans.

Mutations in pre-synaptic voltage-gated calcium channels can lead to familial hemiplegic migraine type 1 (FHM1). While mammalian studies indicate that the migraine brain is hyperexcitable due to enhanced excitation or reduced inhibition, the molecular and cellular mechanisms underlying this excitatory/inhibitory (E/I) imbalance are poorly understood. We identified a gain-of-function (gf) mutation in the Caenorhabditis elegans CaV2 channel α1 subunit, UNC-2, which leads to increased calcium currents. unc-2(zf35gf) mutants exhibit hyperactivity and seizure-like motor behaviors. Expression of the unc-2 gene with FHM1 substitutions R192Q and S218L leads to hyperactivity similar to that of unc-2(zf35gf) mutants. unc-2(zf35gf) mutants display increased cholinergic and decreased GABAergic transmission. Moreover, increased cholinergic transmission in unc-2(zf35gf) mutants leads to an increase of cholinergic synapses and a TAX-6/calcineurin-dependent reduction of GABA synapses. Our studies reveal mechanisms through which CaV2 gain-of-function mutations disrupt excitation-inhibition balance in the nervous system.

P89-T Lambert-Eaton myasthenic syndrome with hyperthyroidism and demyelinating lesions – Case report, diagnostic and therapeutic difficulties

The Lambert-Eaton myasthenic syndrome (LEMS) is a rare autoimmune disease of the neuromuscular transmission involving the presynaptic voltage-gated calcium channels (VGCCs). In most cases (60%), LEMS presents a paraneoplastic syndrome associated with small cell lung cancer, in the remaining an idiopathic, autoimmune diseases, with frequent overlap with other immune dysfunctions, including myasthenia gravis. The typical clinical triad in LEMS consists of weakness (predominantly affecting proximal muscles), autonomic dysfunctions (including xerostomia, pupillomotor dysfunction, constipation, impaired sweating) and hypo/areflexia. LEMS patients typically present a proximal lower > upper limb weakness that progressively involves axial, oculomotor and pharyngo-laryngeal muscles, thus leading to gait and upright alteration, ocular and bulbar symptoms in severely affected patients. We report the case of a 41-year-old woman diagnosed and treated from 2015. Due to the unusual initial symptoms and coexisting diseases, the patient remains under constant control and observation, including oncological monitoring.

AAV-encoded CaV2.2 peptide aptamer CBD3A6K for primary sensory neuron-targeted treatment of established neuropathic pain.

Transmission of pain signals from primary sensory neurons to secondary neurons of the central nervous system is critically dependent on presynaptic voltage-gated calcium channels. Calcium channel-binding domain 3 (CBD3), derived from the collapsin response mediator protein 2 (CRMP2), is a peptide aptamer that is effective in blocking N-type voltage-gated calcium channel (CaV2.2) activity. We previously reported that recombinant adeno-associated virus (AAV)-mediated restricted expression of CBD3 affixed to enhanced green fluorescent protein (EGFP) in primary sensory neurons prevents the development of cutaneous mechanical hypersensitivity in a rat neuropathic pain model. In this study, we tested whether this strategy is effective in treating established pain. We constructed AAV6-EGFP-CBD3A6K (AAV6-CBD3A6K) expressing a fluorescent CBD3A6K (replacing A to K at position 6 of CBD3 peptide), which is an optimized variant of the parental CBD3 peptide that is a more potent blocker of CaV2.2. Delivery of AAV6-CBD3A6K into lumbar (L) 4 and 5 dorsal root ganglia (DRG) of rats 2 weeks following tibial nerve injury (TNI) induced transgene expression in neurons of these DRG and their axonal projections, accompanied by attenuation of pain behavior. We additionally observed that the increased CaV2.2α1b immunoreactivity in the ipsilateral spinal cord dorsal horn and DRG following TNI was significantly normalized by AAV6-CBD3A6K treatment. Finally, the increased neuronal activity in the ipsilateral dorsal horn that developed after TNI was reduced by AAV6-CBD3A6K treatment. Collectively, these results indicate that DRG-restricted AAV6 delivery of CBD3A6K is an effective analgesic molecular strategy for the treatment of established neuropathic pain.

Preoperative Pregabalin Prolongs Duration of Spinal Anesthesia and Reduces Early Postoperative Pain

Background: Various adjuvants have been used to prolong spinal anesthesia, with the additional advantages of delaying the onset of postoperative pain and reducing postoperative analgesic requirements. Pregabalin is an R-aminobutyric acid analog that binds to the α2-δ subunit of presynaptic voltage-gated calcium channels. Objective: The aim of this work is to evaluate the efficacy of a single dose of pregabalin in terms of spinal blockade duration and its potential opioid-sparing effect during the first 24 hours postoperatively Patients and Methods: There were limitations to the present study. First, since only 1 dosage of pregabalin was evaluated, we could not determine the most effective dosage. Second, clinically meaningful improvements in recovery were not assessed. Adequate postoperative pain control provides early postsurgical mobilization, shortened hospitalization, and increased patient satisfaction. Third, preoperative pain and anxiety scores were not recorded. Pregabalin might affect the preoperative pain, mood, and anxiety scores, and these factors can be related to the postoperative pain score. Results: The mechanisms by which pregabalin premedication prolongs motor and sensory blocks using local anesthetics in spinal anesthesia are not fully understood. There may be several reasons for the prolongation of spinal anesthesia. Gabapentinoids are an r-aminobutyric acid analog that binds to α2-δ subunit of presynaptic voltage-gated calcium channels, and this inhibition decreases postsynaptic excitability by reducing potassium-evoked excitatory transmitter release. These medications provide antiepileptic, anxiolytic, and analgesic features by modulating both GABAergic neurotransmission and calcium influx. Gabapentinoid compounds produce a significant and clinically important improvement in preoperative anxiety scores. Since patients may be anxious in the perioperative period, the anxiolytic effects and euphorigenic effects of pregabalin may be beneficial.

Preoperative Pregabalin Prolongs Duration of Spinal Anesthesia and Reduces Early Postoperative Pain

Background: Various adjuvants have been used to prolong spinal anesthesia, with the additional advantages of delaying the onset of postoperative pain and reducing postoperative analgesic requirements. Pregabalin is an R-aminobutyric acid analog that binds to the α2-δ subunit of presynaptic voltage-gated calcium channels. Objective: The aim of this work is to evaluate the efficacy of a single dose of pregabalin in terms of spinal blockade duration and its potential opioid-sparing effect during the first 24 hours postoperatively Patients and Methods: There were limitations to the present study. First, since only 1 dosage of pregabalin was evaluated, we could not determine the most effective dosage. Second, clinically meaningful improvements in recovery were not assessed. Adequate postoperative pain control provides early postsurgical mobilization, shortened hospitalization, and increased patient satisfaction. Third, preoperative pain and anxiety scores were not recorded. Pregabalin might affect the preoperative pain, mood, and anxiety scores, and these factors can be related to the postoperative pain score. Results: The mechanisms by which pregabalin premedication prolongs motor and sensory blocks using local anesthetics in spinal anesthesia are not fully understood. There may be several reasons for the prolongation of spinal anesthesia. Gabapentinoids are an r-aminobutyric acid analog that binds to α2-δ subunit of presynaptic voltage-gated calcium channels, and this inhibition decreases postsynaptic excitability by reducing potassium-evoked excitatory transmitter release. These medications provide antiepileptic, anxiolytic, and analgesic features by modulating both GABAergic neurotransmission and calcium influx. Gabapentinoid compounds produce a significant and clinically important improvement in preoperative anxiety scores. Since patients may be anxious in the perioperative period, the anxiolytic effects and euphorigenic effects of pregabalin may be beneficial.

Cdk5-mediated CRMP2 phosphorylation is necessary and sufficient for peripheral neuropathic pain

Neuropathic pain results from nerve injuries that cause ectopic firing and increased nociceptive signal transmission due to activation of key membrane receptors and channels. The dysregulation of trafficking of voltage-gated ion channels is an emerging mechanism in the etiology of neuropathic pain. We identify increased phosphorylation of collapsin response mediator protein 2 (CRMP2), a protein reported to regulate presynaptic voltage-gated calcium and sodium channels. A spared nerve injury (SNI) increased expression of a cyclin dependent kinase 5 (Cdk5)-phosphorylated form of CRMP2 in the dorsal horn of the spinal cord and the dorsal root ganglia (DRG) in the ipsilateral (injured) versus the contralateral (non-injured) sites. Biochemical fractionation of spinal cord from SNI rats revealed the increase in Cdk5-mediated CRMP2 phosphorylation to be enriched to pre-synaptic sites. CRMP2 has emerged as a central node in assembling nociceptive signaling complexes. Knockdown of CRMP2 using a small interfering RNA (siRNA) reversed SNI-induced mechanical allodynia implicating CRMP2 expression as necessary for neuropathic pain. Intrathecal expression of a CRMP2 resistant to phosphorylation by Cdk5 normalized SNI-induced mechanical allodynia, whereas mimicking constitutive phosphorylation of CRMP2 resulted in induction of mechanical allodynia in naïve rats. Collectively, these results demonstrate that Cdk5-mediated CRMP2 phosphorylation is both necessary and sufficient for peripheral neuropathic pain.

Use-dependent potentiation of voltage-gated calcium channels rescues neurotransmission in nerve terminals intoxicated by botulinum neurotoxin serotype A

Botulinum neurotoxins (BoNTs) are highly potent toxins that cleave neuronal SNARE proteins required for neurotransmission, causing flaccid paralysis and death by asphyxiation. Currently, there are no clinical treatments to delay or reverse BoNT-induced blockade of neuromuscular transmission. While aminopyridines have demonstrated varying efficacy in transiently reducing paralysis following BoNT poisoning, the precise mechanisms by which aminopyridines symptomatically treat botulism are not understood. Here we found that activity-dependent potentiation of presynaptic voltage-gated calcium channels (VGCCs) underlies 3,4-diaminopyridine (3,4-DAP)-mediated rescue of neurotransmission in central nervous system synapses and mouse diaphragm neuromuscular junctions fully intoxicated by BoNT serotype A. Combinatorial treatments with 3,4-DAP and VGCC agonists proved synergistic in restoring suprathreshold endplate potentials in mouse diaphragms fully intoxicated by BoNT/A. In contrast, synapses fully intoxicated by BoNT serotypes D or E were refractory to synaptic rescue by any treatment. We interpret these data to propose that increasing the duration or extent of VGCC activation prolongs the opportunity for low-efficiency fusion by fusogenic complexes incorporating BoNT/A-cleaved SNAP-25. The identification of VGCC agonists that rescue neurotransmission in BoNT/A-intoxicated synapses provides compelling evidence for potential therapeutic utility in some cases of human botulism.

Atypical Presentation of Lambert Eaton Myasthenic Syndrome

SESSION TITLE: Critical Care 3 SESSION TYPE: Affiliate Case Report Poster PRESENTED ON: Tuesday, October 31, 2017 at 01:30 PM - 02:30 PM INTRODUCTION: Lambert-Eaton Myasthenic Syndrome (LEMS) is a rare disorder in which antibodies against presynaptic voltage-gated calcium channels (VGCC) prevent the flux of calcium into the nerve terminal. This reduces the release of acetylcholine affecting neuromuscular transmission. Here we discuss an atypical presentation of LEMS. CASE PRESENTATION: A 64 year old male was admitted for dysphagia and difficulty ambulating. Chest x-ray showed a mediastinal mass. CT confirmed a 5 x 5 cm anterior mediastinal mass. He had 4/5 strength and 1+ deep tendon reflexes (DTRs) in lower extremities. Sodium was 119 mEq/L. On day 2, the patient had bradycardic arrest. Return of spontaneous circulation was achieved after 3 minutes of chest compressions and he was intubated. During his hospital course, he had tachybradyarrhythmias. Despite immediate recovery to his normal mental status, normal chest x-ray, and arterial blood gas, he continued to fail his spontaneous breathing trials. He had a negative inspiratory force of -6 and vital capacity of 150 mL. Paraneoplastic syndrome was suspected due to neuromuscular respiratory failure, hyponatremia, and mediastinal mass. Intravenous immunoglobulin (IVIG) was given for 5 days. On day 10 of intubation, supraclavicular lymph node biopsy showed small cell lung carcinoma and carboplatin and etoposide was initiated. The patient was extubated on the same day. Serology was positive for P/Q type VGCC antibodies. DISCUSSION: LEMS has a prevalence of 2.3 per million and an incidence of 0.5 per million. There have been 400 reported LEMS cases in the US, of which 17 cases had frank respiratory failure. Sixty percent of LEMS cases are associated with small cell lung cancer. Patients with LEMS have proximal muscle weakness that transiently improves after repetitive exertion, absent DTRs, bulbar dysfunction, and autonomic dysfunction. This patient had dysphagia, a sign of bulbar dysfunction. He ambulated while intubated, which may reflect improvement of weakness after repetitive exertion. If not, this level of weakness is uncommon in LEMS as diaphragmatic weakness often parallels the degree of proximal muscle weakness. In contrast to other cases in which DTRs are absent, this patient had hyporeflexia. Finally, he had dysautonomia manifesting as fluctuating episodes of bradycardia and tachycardia. EMG is used to confirm LEMS, however was not available. CONCLUSIONS: Neuromuscular respiratory failure should lead to evaluation of more common causes of weakness. In patients with malignancy, the possibility of LEMS should be entertained. The early use of IVIG in this patient allowed for earlier extubation. Reference #1: Laghi F, et al. Disorders of the respiratory muscles. Am J Respir Crit Care Med. 2003. 168(1): 10-48. Reference #2: Bekircan-Kurt C, et al. Voltage gated calcium channel antibody-related neurological diseases. World J of Clin Cases: 3.3 (2015): 293. Reference #3: Sanders DB. Lambert-eaton myasthenic syndrome: diagnosis and treatment. Ann NY Acad Sci. 2003. 998: 500-508. DISCLOSURE: The following authors have nothing to disclose: Gauri Behari, Raja Sinha, Vadym Rusnak, Andrea Espinoza, Michael Charlet, Ibrahim El-Abbassi No Product/Research Disclosure Information