Rat Neuromuscular Junction Research Articles

Increased Frequency of Giant Miniature End-Plate Potentials at the Neuromuscular Junction in Diabetic Rats.

There is a need for research addressing the functional characteristics of the motor end-plate in diabetes to identify mechanisms contributing to neuromuscular dysfunction. Here, we investigated the effect of diabetes on spontaneous acetylcholine release in the rat neuromuscular junction. We studied two randomized groups of male Wistar rats (n = 7 per group, 350 ± 50 g, 12-16 weeks of age): one with streptozotocin-induced experimental diabetes, and a healthy control group without diabetes. After 8 weeks of monitoring after diabetes induction, rats in both groups were anesthetized with pentobarbital. Then, the diaphragm muscle was dissected for electrophysiological recordings of miniature end-plate potentials (MEPPs) using a single electrode located at the region of the muscle end-plate. All experiments were conducted at environmental temperature (20-22 °C) in rat Ringer solution with constant bubbling carbogen (95% O2, 5% CO2). Compared to healthy controls, in the diaphragm neuromuscular end-plate derived from diabetic rats, the MEPPs were higher in amplitude and frequency, and the proportion of giant MEPPs was elevated (7.09% vs. 1.4% in controls). Our results showed that diabetes affected the acetylcholine MEPP pattern and increased the number of giant potentials compared to healthy controls.

Acute sleep deprivation induces synaptic remodelling at the soleus muscle neuromuscular junction in rats.

Sleep is important for cognitive and physical performance. Sleep deprivation not only affects neural functions but also results in muscular fatigue. A good night's sleep reverses these functional derangements caused by sleep deprivation. The role of sleep in brain function has been extensively studied. However, its role in neuromuscular junction or skeletal muscle morphology is sparsely addressed although skeletal muscle atonia and suspended thermoregulation during rapid eye movement sleep possibly provide a conducive environment for the muscle to rest and repair; somewhat similar to slow-wave sleep for synaptic downscaling. In the present study, we have investigated the effect of 24h sleep deprivation on the neuromuscular junction morphology and neurochemistry using electron microscopy and immunohistochemistry in the rat soleus muscle. Acute sleep deprivation altered synaptic ultra-structure viz. mitochondria, synaptic vesicle, synaptic proteins, basal lamina, and junctional folds needed for neuromuscular transmission. Further acute sleep deprivation showed the depletion of the neurotransmitter acetylcholine and the overactivity of its degrading enzyme acetylcholine esterase at the neuromuscular junction. The impact of sleep deprivation on synaptic homeostasis in the brain has been extensively reported recently. The present evidence from our studies shows new information on the role of sleep on neuromuscular junction homeostasis and its functioning.

Anatomical and Functional Changes to the Colonic Neuromuscular Compartment after Experimental Spinal Cord Injury.

A profound reduction in colorectal transit time accompanies spinal cord injury (SCI), yet the colonic alterations after SCI have yet to be understood fully. The loss of descending supraspinal input to lumbosacral neural circuits innervating the colon is recognized as one causal mechanism. Remodeling of the colonic enteric nervous system/smooth muscle junction in response to inflammation, however, is recognized as one factor leading to colonic dysmotility in other pathophysiological models. We investigated the alterations to the neuromuscular junction in rats with experimental high-thoracic (T3) SCI. One day to three weeks post-injury, both injured and age-matched controls underwent in vivo experimentation followed by tissue harvest for histological evaluation. Spontaneous colonic contractions were reduced significantly in the proximal and distal colon of T3-SCI rats. Histological evaluation of proximal and distal colon demonstrated significant reductions of colonic mucosal crypt depth and width. Markers of intestinal inflammation were assayed by qRT-PCR. Specifically, Icam1, Ccl2 (MCP-1), and Ccl3 (MIP-1α) mRNA was acutely elevated after T3-SCI. Smooth muscle thickness and collagen content of the colon were increased significantly in T3-SCI rats. Colonic cross sections immunohistochemically processed for the pan-neuronal marker HuC/D displayed a significant decrease in colonic enteric neuron density that became more pronounced at three weeks after injury. Our data suggest that post-SCI inflammation and remodeling of the enteric neuromuscular compartment accompanies SCI. These morphological changes may provoke the diminished colonic motility that occurs during this same period, possibly through the disruption of intrinsic neuromuscular control of the colon.

(Invited) Vesicular Exocytosis in Neuronal and Neuroendocrine Cells: Quantifying Fusion Pore Size from Amperometric Data

It is well-known that amperometric measurements of vesicular exocytosis in artificial synapse configuration [1] provide two important advantages: unsurpassed temporal resolution of single exocytotic events and possibility to obtain massive data. These two advantages allow statistical analysis of exocytosis and its trends in different cell types and/or under various physico-chemical conditions (osmotic shocks, effect of drugs etc.). However, generally statistical analysis is restricted to the examination of some shape features of the individual spikes (half-time, charge released etc.) even though all relevant physico-chemical parameters are intricately convoluted in the monitored current. Extraction of these thought parameters is extremely difficult due to the fact that each exocytotic event is unique in terms of vesicle size, its internal composition, neurotransmitter load etc. During several last years we developed a theoretical framework providing means to extract statistically sound fusion pore sizes as well as pore dynamics from individual exocytotic spikes [2-4], that is information hardly accessible or not accessible by other approaches. This permits us to analyze and quantify vesicle pore sizes from amperometric data obtained at chromaffin cells [4] and rat neuro-muscular junctions [5]. Recently we dramatically simplified the fusion pore size extraction procedure (without sacrificing its accuracy) so that it can be easily implemented by the experimentalists, e.g. in spreadsheet programs. This advance allow us to address a larger data set of spikes obtained at chromaffin cells and reveal changes in fusion pores topology under modified conditions (osmotic stress, membrane lipid modification) with respect to control conditions. Of high interest was the finding that in all considered cases the fusion pore radii was never larger 30 nm, that is much smaller to the average radius of the chromaffin cell vesicle 156 nm. Taking into account significant size of the data set (more than 1000 spikes) this questions the ‘inevitable full fusion’ paradigm and statistically support a mode of exocytosis where the pore size is significantly smaller the vesicle size [6].

Severity of Myasthenia Gravis Influences the Relationship between Train-of-four Ratio and Twitch Tension and Run-down of Rat Endplate Potentials.

Train-of-four ratio (TOFR) is often used to evaluate muscle relaxation caused by neuromuscular-blocking agents (NMBAs). However, it is unknown whether TOFR reliably correlates with the first twitch tension (T1) in patients with myasthenia gravis (MG). By using rat models of experimental autoimmune MG (EAMG), the authors verified the hypothesis that the severity of MG influences the relationship between TOFR and T1. EAMG rats were divided into sham, moderate MG, and severe MG groups. Isometric twitch tension of the hemidiaphragm was elicited by phrenic nerve stimulation with and without use of the NMBA rocuronium to measure TOFR and T1, and run-down of endplate potentials was estimated in the three groups. Changes around the neuromuscular junction in EAMG rats were investigated by observation of electron micrographs. With similar attenuation of T1, TOFR was significantly (n = 6) different among the three groups in the presence of 50% inhibitory concentrations of rocuronium (IC50). Run-down in the sham group was significantly (n = 8) greater with exposure to IC50, whereas that in the severe MG group was statistically insignificant. Width of the primary synaptic cleft in the severe MG group was significantly (n = 80) greater than that in the other groups. Severity of MG influences the relationship between TOFR and T1, together with changes in run-down of endplate potentials and those around the neuromuscular junction in rats. TOFR may, therefore, not be an accurate indicator of recovery from NMBAs in MG patients.

The novel protein kinase C epsilon isoform modulates acetylcholine release in the rat neuromuscular junction.

BackgroundVarious protein kinase C (PKC) isoforms contribute to the phosphorylating activity that modulates neurotransmitter release. In previous studies we showed that nPKCε is confined in the presynaptic site of the neuromuscular junction and its presynaptic function is activity-dependent. Furthermore, nPKCε regulates phorbol ester-induced acetylcholine release potentiation, which further indicates that nPKCε is involved in neurotransmission. The present study is designed to examine the nPKCε involvement in transmitter release at the neuromuscular junction.ResultsWe use the specific nPKCε translocation inhibitor peptide εV1-2 and electrophysiological experiments to investigate the involvement of this isoform in acetylcholine release. We observed that nPKCε membrane translocation is key to the synaptic potentiation of NMJ, being involved in several conditions that upregulate PKC isoforms coupling to acetylcholine (ACh) release (incubation with high Ca2+, stimulation with phorbol esters and protein kinase A, stimulation with adenosine 3′,5′-cyclic monophosphorothioate, 8-Bromo-, Rp-isomer, sodium salt -Sp-8-BrcAMP-). In all these conditions, preincubation with the nPKCε translocation inhibitor peptide (εV1-2) impairs PKC coupling to acetylcholine release potentiation. In addition, the inhibition of nPKCε translocation and therefore its activity impedes that presynaptic muscarinic autoreceptors and adenosine autoreceptors modulate transmitter secretion.ConclusionsTogether, these results point to the importance of nPKCε isoform in the control of acetylcholine release in the neuromuscular junction.

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

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

Immunohistochemical evidence of the presence of metabotropic receptors for γ-aminobutyric acid at the rat neuromuscular junctions.

in the synapses of the "fast" (m. EDL) and "slow" (m. soleus) skeletal muscles of the rat GABABR1 and GABABR2 subunits of metabotropic receptors for γ-aminobutyric acid (GABA), located primarily on the motor nerve ending membrane were detected by immunohistochemistry and fluorescence microscopy methods.

Effects of IgG anti-GM1 monoclonal antibodies on neuromuscular transmission and calcium channel binding in rat neuromuscular junctions.

Guillain-Barré syndrome is a type of acute inflammatory neuropathy that causes ataxia and is associated with the IgG anti-GM1 antibody. However, the pathogenic role of the IgG anti-GM1 antibody and calcium channels in neuromuscular junctions (NMJs) remains unclear. Thus, the aim of the present study was to investigate the effects of the IgG anti-GM1 monoclonal antibody (mAb) on spontaneous muscle action potentials (SMAPs), and the effects of calcium channel blockers, in a rat spinal cord-muscle co-culture system. In addition, the binding of IgG anti-GM1 mAb to calcium channels was investigated in the rat hemidiaphragm. The frequency of SMAPs in the innervated muscle cells was acutely inhibited by the IgG anti-GM1 mAb; however, this effect was blocked by the N-type calcium channel blocker, ω-conotoxin GVIA (30 nM). Furthermore, the P/Q-type calcium channel blocker, ω-agatoxin IVA (10 nM), was found to partially block the IgG anti-GM1 mAb-induced inhibitory effect in the spinal cord-muscle co-culture system. Immunohistochemical analysis of the rat hemidiaphragm indicated that IgG anti-GM1 mAb binding overlapped with anti-Cav2.2 (α1B) antibody binding in the nerve terminal. In addition, IgG anti-GM1 mAb binding partially overlapped with anti-Cav2.1 (α1A) antibody binding. Thus, the results demonstrated that the IgG anti-GM1 mAb binds to calcium channels in the nerve terminals of NMJs. Therefore, the inhibitory effect of IgG anti-GM1 mAb on SMAPs may involve N-type and P/Q-type calcium channels in motor nerve terminals at the NMJ.

Effect of sepsis on vecuronium-induced inhibition of acetylcholine release in neuromuscular junction in rats

Objective To investigate the effect of sepsis on vecuronium-induced inhibition of acetylcholine release in neuromuscular junction in rats. Methods Thirty-six adult male SPF Sprague-Dawley rats, aged 2-3 months, weighing 200-220 g, were randomly divided into 3 groups (n=12 each) using a random number table: control group (group C), sham operation group (group S) and sepsis group (group Sep). Sepsis was induced by cecum ligation and puncture (CLP) in rats anesthetized with intraperitoneal chloral hydrate 350 mg/kg. At 12 h after CLP, the sciatic nerve-pretibial muscle was prepared. Vecuronium was added to the culture medium with the final concentration of 0.08 μg/ml, and the sciatic nerve-pretibial muscle was incubated for 15 min. Before and after administration, evoked endplate potentials (EPPs) and miniature endplate potentials (MEPPs) were recorded by using intracellular microelectrode. EPP/MEPP ratio was calculated. Results Compared to C and S groups, EPPs, MEPPs and EPP/MEPP ratio were significantly increased before and after administration in group Sep. EPPs, MEPPs and EPP/MEPP ratio were significantly lower after administration than before administration in the three groups. Conclusion Sepsis can promote acetylcholine release in neuromuscular junction, thus weakening vecuronium-induced inhibition of acetylcholine release in neuromuscular junction in rats. Key words: Sepsis; Acetylcholine; Neuromuscular nondepolarizing agents

The expression and function of gelatinolytic activity at the rat neuromuscular junction upon physical exercise.

The gelatinases MMP-9 and MMP-2 have been implicated in skeletal muscle adaptation to training; however, their specific role(s) in the different muscle types are only beginning to be unraveled. Recently, we found that treadmill running increased the activity and/or expression of these enzymes in myonuclei and in activated satellite cells of the soleus (Sol), but not extensor digitorum longus (EDL) muscles on the fifth day of training of adult rats. Here, we asked whether the gelatinases can be involved in physical exercise-induced adaptation of the neuromuscular compartment. To determine the subcellular localization of the gelatinolytic activity, we used high-resolution in situ zymography and immunofluorescence techniques. In both control and trained muscles, strong gelatinolytic activity was associated with myelin sheaths within intramuscular nerve twigs. In EDL, but not Sol, there was an increase in the gelatinolytic activity at the postsynaptic domain of the neuromuscular junction (NMJ). The increased activity was found within punctate structures situated in the vicinity of synaptic cleft of the NMJ, colocalizing with a marker of endoplasmic reticulum. Our results support the hypothesis that the gelatinolytic activity at the NMJ may be involved in NMJ plasticity.

Detection and identification of huwentoxin-IV interacting proteins by biotin-avidin chemistry combined with mass spectrometry.

BackgroundNumerous spider toxins are of interest as tools for neurophysiological research or as lead molecules for the development of pharmaceuticals and insecticides. Direct detection and identification of the interacting proteins of a spider toxin are helpful for its action-mechanism analysis and practical application. The present study employed a combinative strategy for the analysis of interacting proteins of huwentoxin-IV (HWTX-IV), a peptidic neurotoxin from the venom of the spider Selenocosmia huwena.ResultsHWTX-IV was first lightly labeled with biotin under the optimized mild experimental conditions and the toxin labeled with a single biotin group (monobiotinylated HWTX-IV) was demonstrated by electrophysiological experiments to retain its original bioactivity and was used in combination with far-western blotting to detect its interacting proteins. Comparative experiments indicated that some membrane proteins from rat neuromuscular junction preparations bind to monobiotinylated HWTX-IV after being transferred onto a PVDF membrane from the SDS-gel. With capillary high performance liquid chromatography-tandem mass spectrometry, several membrane proteins with which HWTX-IV potentially interacted were identified from the preparations and then bioinformatically analyzed.ConclusionsThis work has provided not only a new insight into the action mechanism of HWTX-IV but also a reference technology for the relevant researches.

Neurophysiological and immunohistochemical studies of IgG anti-GM1 monoclonal antibody on neuromuscular transmission: effects in rat neuromuscular junctions

Guillain-Barré syndrome, which is a variant of acute inflammatory neuropathy, is associated with anti-GM1 antibodies and causes ataxia. We investigated the effects of IgG anti-GM1 monoclonal antibody (IgG anti-GM1 mAb) on spontaneous muscle action potentials in a rat spinal cord-muscle co-culture system and the localization of IgG anti-GM1 mAb binding in the rat hemi-diaphragm. The frequency of spontaneous muscle action potentials in innervated muscle cells was acutely inhibited by IgG anti-GM1 mAb. When cultures were pretreated with GM2 synthase antisense oligodeoxynucleotide, IgG anti-GM1 mAb failed to inhibit spontaneous muscle action potentials, demonstrating the importance of the GM1 epitope in the action of IgG anti-GM1 mAb. Immunohistochemistry of rat hemi-diaphragm showed that IgG anti-GM1 mAb binding overlapped with neurofilament 200 (NF200) antibodies staining, but not α-bungarotoxin (α-BuTx) staining, demonstrating that IgG anti-GM1 mAb was localized at the presynaptic nerve terminal. IgG anti-GM1 mAb binding overlapped with syntaxin antibody and S-100 antibody in the nerve terminal. After collagenase treatment, IgG anti-GM1 mAb and NF200 antibodies did not show staining, but α-BuTx selectively stained the hemi-diaphragm. IgG anti-GM1 mAb binds to the presynaptic nerve terminal of neuromuscular junctions. Therefore, we suggest that the inhibitory effect of IgG anti-GM1 mAb on spontaneous muscle action potentials is related to the GM1 epitope in presynaptic motor nerve terminals at the NMJs.

Simulation of the Kinetics of Neuromuscular Block

The onset time for paralysis varies 3-fold among nondepolarizing muscle relaxants. Possible explanations include: (a) pharmacokinetic differences among drugs and (b) buffering of drug molecules by acetylcholine receptors as they diffuse into the neuromuscular junction. Although some pharmacokinetic models consider buffered diffusion, these models do not account for either the high density of receptors or synapse geometry. Here, I used computer simulations to calculate the kinetics of buffered diffusion. The goal was to determine the conditions under which buffered diffusion could account for differences in onset time among nondepolarizing muscle relaxants. Monte Carlo simulation was used along with a realistic 3-dimensional model of the rat neuromuscular junction. Simulations determined the time dependence of the number of drug-bound receptors. A 1000-fold range of drug potency was examined. In some simulations, the drug concentration outside the junction was changed instantaneously. In other simulations, the concentration changed according to predictions of pharmacokinetic models assuming time-dependent changes in plasma drug concentration. The rate constant for equilibration of drug between plasma and muscle, keo, was varied between 0.15 and 0.6 min(-1). Twitch amplitude was calculated from receptor occupancy assuming a high safety margin for neuromuscular transmission. Some simulations used a synaptic model with an increased nerve-muscle contact width. Simulations with instantaneous changes in drug concentration at the synapse, indicated that the time to 50% twitch depression (onset time) was 0.1 to 30 seconds and was proportional to drug potency. This corresponds to iontophoretic application of drug to isolated neuromuscular junctions, but is too fast to explain onset times in humans. When pharmacokinetic models were used to calculate the drug concentration outside the synapse, buffered diffusion increased onset times of potent drugs (drugs for which the effective concentration at 50% twitch height is <600 nM). Simulations using keo = 0.6 min(-1) and a model with a 2- to 3-fold wider nerve-muscle contact width indicated that buffered diffusion could account for the differences in clinical onset times among the nondepolarizing muscle relaxants. Monte Carlo simulation provides a biophysically appropriate way to incorporate buffered diffusion into pharmacokinetic modeling. The simulations indicated that buffered diffusion could account for differences in onset time among drugs. However, a better understanding of the geometry of the human neuromuscular junction is needed before the magnitude of the effect of buffered diffusion can be quantified.

Expression of cAMP-responsive element binding proteins (CREBs) in fast- and slow-twitch muscles: A signaling pathway to account for the synaptic expression of collagen-tailed subunit (ColQ) of acetylcholinesterase at the rat neuromuscular junction

The gene encoding the collagen-tailed subunit (ColQ) of acetylcholinesterase (AChE) contains two distinct promoters that drive the production of two ColQ mRNAs, ColQ-1 and ColQ-1a, in slow- and fast-twitch muscles, respectively. ColQ-1a is expressed at the neuromuscular junction (NMJ) in fast-twitch muscle, and this expression depends on trophic factors supplied by motor neurons signaling via a cAMP-dependent pathway in muscle. To further elucidate the molecular basis of ColQ-1a’s synaptic expression, here we investigated the expression and localization of cAMP-responsive element binding protein (CREB) at the synaptic and extra-synaptic regions of fast- and slow-twitch muscles from adult rats. The total amount of active, phosphorylated CREB (P-CREB) present in slow-twitch soleus muscle was higher than that in fast-twitch tibialis muscle, but P-CREB was predominantly expressed in the fast-twitch muscle at NMJs. In contrast, P-CREB was detected in both synaptic and extra-synaptic regions of slow-twitch muscle. These results reveal, for the first time, the differential distribution of P-CREB in fast- and slow-twitch muscles, which might support the crucial role of cAMP-dependent signaling in controlling the synapse-specific expression of ColQ-1a in fast-twitch muscles.

Regulation of acetylcholinesterase activity by nitric oxide in rat neuromuscular junction via N‐methyl‐d‐aspartate receptor activation

Acetylcholinesterase (AChE) is an enzyme that hydrolyses the neurotransmitter acetylcholine, thereby limiting spillover and duration of action. This study demonstrates the existence of an endogenous mechanism for the regulation of synaptic AChE activity. At the rat extensor digitorum longus neuromuscular junction, activation of N-methyl-d-aspartate (NMDA) receptors by combined application of glutamate and glycine led to enhancement of nitric oxide (NO) production, resulting in partial AChE inhibition. Partial AChE inhibition was measured using increases in miniature endplate current amplitude. AChE inhibition by paraoxon, inactivation of NO synthase by N(x)-nitro-L-arginine methyl ester, and NMDA receptor blockade by DL-2-amino-5-phosphopentanoic acid prevented the increase in miniature endplate current amplitude caused by amino acids. High-frequency (10 Hz) motor nerve stimulation in a glycine-containing bathing solution also resulted in an increase in the amplitude of miniature endplate currents recorded during the interstimulus intervals. Pretreatment with an NO synthase inhibitor and NMDA receptor blockade fully eliminated this effect. This suggests that endogenous glutamate, released into the synaptic cleft as a co-mediator of acetylcholine, is capable of triggering the NMDA receptor/NO synthase-mediated pathway that modulates synaptic AChE activity. Therefore, in addition to well-established modes of synaptic plasticity (e.g. changes in the effectiveness of neurotransmitter release and/or the sensitivity of the postsynaptic membrane), another mechanism exists based on the prompt regulation of AChE activity.

NMDA receptors at the endplate of rat skeletal muscles: Precise postsynaptic localization

In this study we demonstrate expression of the N-methyl-D-aspartate receptor NR1 subunit in the rat neuromuscular junction of skeletal muscles of different functional types (extensor digitorum longus, soleus, and diaphragm muscles) using fluorescence immunocytochemistry. Electron microscopic immunocytochemistry has shown that the NR1 subunit is localized solely on the sarcolemma in the depths of the postsynaptic folds. These findings suggest participation of the glutamatergic signaling system in functioning of the adult mammalian neuromuscular junction.

Expression and regulation of Homer in human skeletal muscle during neuromuscular junction adaptation to disuse and exercise

Protein calcium sensors of the Homer family have been proposed to modulate the activity of various ion channels and nuclear factor of activated T cells (NFAT), the transcription factor modulating skeletal muscle differentiation. We monitored Homer expression and subcellular localization in human skeletal muscle biopsies following 60 d of bedrest [Second Berlin Bedrest Study (BBR2-2)]. Soleus (SOL) and vastus lateralis (VL) biopsies were taken at start (pre) and at end (end) of bedrest from healthy male volunteers of a control group without exercise (CTR; n=9), a resistive-only exercise group (RE; n=7), and a combined resistive/vibration exercise group (RVE; n=7). Confocal analysis showed Homer immunoreactivity at the postsynaptic microdomain of the neuromuscular junction (NMJ) at bedrest start. After bedrest, Homer immunoreactivity decreased (CTR), remained unchanged (RE), or increased (RVE) at the NMJ. Homer2 mRNA and protein were differently regulated in a muscle-specific way. Activated NFATc1 translocates from cytoplasm to nucleus; increased amounts of NFATc1-immunopositive slow-type myonuclei were found in RVE myofibers of both muscles. Pulldown assays identified NFATc1 and Homer as molecular partners in skeletal muscle. A direct motor nerve control of Homer2 was confirmed in rat NMJs by in vivo denervation. Homer2 is localized at the NMJ and is part of the calcineurin-NFATc1 signaling pathway. RVE has additional benefit over RE as countermeasure preventing disuse-induced neuromuscular maladaptation during bedrest.

Self-Gating Effect at the Rat Neuromuscular Junction

Event Abstract Back to Event Self-Gating Effect at the Rat Neuromuscular Junction Mohammed Mostafizur Rahman1*, Mufti Mahmud1 and Stefano Vassanelli1 1 University of Padova, NeuroChip Lab, Italy The neuromuscular Junction (NMJ) is the place where the axon terminal of a motor neuron and the motor end plate of a muscle fiber's functionality meet creating a synapse to transmit messages from the brain which cause the muscle to contract. The pulse carried by the presynaptic neuron when reaches the synapses causes release of Acetylcholine into the synaptic cleft. The flow of current along the narrow extracellular space between nerve cell and muscular fiber (cleft) could affect the local extracellular electrical potential. As a consequence, voltage-dependent processes may be affected in the adjacent membranes. According to this hypothesis the voltage drop in the cleft increase the opening rate of voltage-gated ion channels. This phenomenon of current passing through weakly opened sodium channels and decreasing the potential of the extracellular space by a positive feedback was previously described in a general cell-to-substrate adhesion contact and termed “self-gating” [1]. To model the rat NMJ a barrel shaped muscle is considered (50 µm diameter × 500 µm long). To form a synapse, an axon terminal (10 µm radius) is connected to the highly-excitable muscle fiber with a synaptic cleft (distance: 50 nm). The potential at the cleft (Vj) is calculated using total sodium conductance of the endplate motoneuron (Glob) and the resistance of the cleft. The endplate sodium current (EPINa) is calculated using the potential of the junction and the Glob. The Acetylcholine Receptor current (AChR) together with the EPINa act as stimulus current for the Hodgkin-Huxley based membrane potential (Vm) calculation at the muscle fiber. The conductance associated to voltage-gated sodium channels depends on the voltage drop of Vm–Vj across the membrane [1]. The Vj was calculated for different membrane potentials which showed that the voltage-dependent sodium conductance at the NMJ undergoes self-gating due to the drop of Vj in the synaptic cleft and in the following depolarization of the potential (Vm–Vj) across the membrane. During the self-gating condition a switching behavior was noticed in the sodium channels conductance: from low to high during depolarization and from high to low during repolarization of membrane potential. At this time bistability and hysteresis of sodium channels occurred which explains the transmission efficiency of the NMJ under self-gating condition. During self-gating, the channels remain open for a longer period letting inward currents to flow during repolarization. This sustained current flow favors cell depolarization and action potential firing in the muscle. The higher number of open sodium channels during the self-gating increases sodium current intensity during the first phase of action potential which directly affects the depolarization of the cell, thus, making it fire at a lower firing threshold. Also, the muscle unit’s firing rate increases under self-gating condition due to the fact that the longer inactivation time is compensated by the switching behavior of the sodium current. This allows fast and larger entrance of sodium current in every successive spike. [1] P. Fromherz, Self-Gating of Ion Channels in Cell Adhesion. Phys. Rev. Lett., 78(21): 4131-4134, 1997. Figure 1 Keywords: computational neuroscience, General neuroinformatics Conference: 4th INCF Congress of Neuroinformatics, Boston, United States, 4 Sep - 6 Sep, 2011. Presentation Type: Poster Presentation Topic: General neuroinformatics Citation: Rahman M, Mahmud M and Vassanelli S (2011). Self-Gating Effect at the Rat Neuromuscular Junction. Front. Neuroinform. Conference Abstract: 4th INCF Congress of Neuroinformatics. doi: 10.3389/conf.fninf.2011.08.00018 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 17 Oct 2011; Published Online: 19 Oct 2011. * Correspondence: Dr. Mohammed Mostafizur Rahman, University of Padova, NeuroChip Lab, Padova, Italy, mostafiz.math@gmail.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Mohammed Mostafizur Rahman Mufti Mahmud Stefano Vassanelli Google Mohammed Mostafizur Rahman Mufti Mahmud Stefano Vassanelli Google Scholar Mohammed Mostafizur Rahman Mufti Mahmud Stefano Vassanelli PubMed Mohammed Mostafizur Rahman Mufti Mahmud Stefano Vassanelli Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Synaptic activity‐related classical protein kinase C isoform localization in the adult rat neuromuscular synapse

Protein kinase C (PKC) is essential for signal transduction in a variety of cells, including neurons and myocytes, and is involved in both acetylcholine release and muscle fiber contraction. Here, we demonstrate that the increases in synaptic activity by nerve stimulation couple PKC to transmitter release in the rat neuromuscular junction and increase the level of alpha, betaI, and betaII isoforms in the membrane when muscle contraction follows the stimulation. The phosphorylation activity of these classical PKCs also increases. It seems that the muscle has to contract in order to maintain or increase classical PKCs in the membrane. We use immunohistochemistry to show that PKCalpha and PKCbetaI were located in the nerve terminals, whereas PKCalpha and PKCbetaII were located in the postsynaptic and the Schwann cells. Stimulation and contraction do not change these cellular distributions, but our results show that the localization of classical PKC isoforms in the membrane is affected by synaptic activity.