Amplitude Of Endplate Currents Research Articles

Homeostatic Synaptic Plasticity of Miniature Excitatory Postsynaptic Currents in Mouse Cortical Cultures Requires Neuronal Rab3A.

Following prolonged activity blockade, amplitudes of miniature excitatory postsynaptic currents (mEPSCs) increase, a form of plasticity termed "homeostatic synaptic plasticity." We previously showed that a presynaptic protein, the small GTPase Rab3A, is required for full expression of the increase in miniature endplate current amplitudes following prolonged blockade of action potential activity at the mouse neuromuscular junction in vivo (Wang et al., 2011), but it is unknown whether this form of Rab3A-dependent homeostatic plasticity shares any characteristics with central synapses. We show here that homeostatic synaptic plasticity of mEPSCs is impaired in mouse cortical neuron cultures prepared from Rab3A-/- and mutant mice expressing a single point mutation of Rab3A, Rab3A Earlybird mice. To determine if Rab3A is involved in the well-established homeostatic increase in postsynaptic AMPA-type receptors (AMPARs), we performed a series of experiments in which electrophysiological recordings of mEPSCs and confocal imaging of synaptic AMPAR immunofluorescence were assessed within the same cultures. We found that Rab3A was required for the increase in synaptic AMPARs following prolonged activity blockade, but the increase in mEPSC amplitudes was not always accompanied by an increase in postsynaptic AMPAR levels, suggesting other factors may contribute. Finally, we demonstrate that Rab3A is acting in neurons because only selective loss of Rab3A in neurons, not glia, disrupted the homeostatic increase in mEPSC amplitudes. This is the first demonstration that neuronal Rab3A is required for homeostatic synaptic plasticity and that it does so partially through regulation of the surface expression of AMPA receptors.

Role of α7 Nicotinic Acetylcholine Receptors in Synaptic Transmission in Frog Neuromuscular Contacts

The role of α7 nicotinic acetylcholine receptors in coupling of synaptic activity and muscle contractions was studied in frog (Rana ridibunda) neuromuscular synapses. The amplitude of endplate currents, the probability of action potential generation, and the strength of muscle contractions decreased in the presence of selective α7 antagonist methyllycaconitine. The effects of nicotinic acetylcholine receptor blockade depended on the pattern of the motor nerve stimulation. It can be assumed that the muscle action potential is a factor of retrograde control of neurosecretion, which modulates activity of α7 nicotinic receptors and the release of acetylcholine from motor nerve endings.

Depressed neuromuscular transmission causes weakness in mice lacking BK potassium channels.

Mice lacking functional large-conductance voltage- and Ca2+-activated K+ channels (BK channels) are viable but have motor deficits including ataxia and weakness. The cause of weakness is unknown. In this study, we discovered, in vivo, that skeletal muscle in mice lacking BK channels (BK-/-) was weak in response to nerve stimulation but not to direct muscle stimulation, suggesting a failure of neuromuscular transmission. Voltage-clamp studies of the BK-/- neuromuscular junction (NMJ) revealed a reduction in evoked endplate current amplitude and the frequency of spontaneous vesicle release compared with WT littermates. Responses to 50-Hz stimulation indicated a reduced probability of vesicle release in BK-/- mice, suggestive of lower presynaptic Ca2+ entry. Pharmacological block of BK channels in WT NMJs did not affect NMJ function, surprisingly suggesting that the reduced vesicle release in BK-/- NMJs was not due to loss of BK channel-mediated K+ current. Possible explanations for our data include an effect of BK channels on development of the NMJ, a role for BK channels in regulating presynaptic Ca2+ current or the effectiveness of Ca2+ in triggering release. Consistent with reduced Ca2+ entry or effectiveness of Ca2+ in triggering release, use of 3,4-diaminopyridine to widen action potentials normalized evoked release in BK-/- mice to WT levels. Intraperitoneal application of 3,4-diaminopyridine fully restored in vivo nerve-stimulated muscle force in BK-/- mice. Our work demonstrates that mice lacking BK channels have weakness due to a defect in vesicle release at the NMJ.

Neuromuscular junction transmission failure is a late phenotype in aging mice

Neurodegeneration has increasingly been considered an important factor in the pathogenesis of sarcopenia or age-related loss of muscle mass and strength. Experiments were designed to investigate the fidelity of neuromuscular junction (NMJ) transmission across the lifespan in hindlimb muscles of male and female C57BL/6J mice (at 12, 20, 24, 27, and 29 months of age). Single-fiber electromyography recordings demonstrated abrupt onset of NMJ transmission failure at 27 months of age. Failed NMJ transmission was a later onset phenotype as compared with other assessments of motor unit numbers, muscle contractility, and frailty which showed alterations at 20 months of age. Ex vivo NMJ recordings demonstrated no reduction of endplate current amplitude in support of reduced muscle fiber excitability as the cause of failed NMJ transmission in aged mice. Improved understanding of age-related neurodegeneration will likely have important implications in designing novel therapeutic interventions specific for different stages of sarcopenia. Our findings suggest reduced muscle excitability may be a potential therapeutic target for improvement of physical function in older adults.

Prenatal hyperhomocysteinemia induces oxidative stress and accelerates ‘aging’ of mammalian neuromuscular synapses

Enhanced levels of homocysteine during pregnancy induce oxidative stress and contribute to many age-related diseases. In this study, we analyzed age-dependent synaptic modifications in developing neuromuscular synapses of rats with prenatal hyperhomocysteinemia (hHCY). One of the main findings indicate that the intensity and the timing of transmitter release in synapses of neonatal (P6 and P10) hHCY rats acquired features of matured synaptic transmission of adult rats. The amplitude and frequency of miniature end-plate currents (MEPCs) and evoked transmitter release were higher in neonatal hHCY animals compared to the control group. Analysis of the kinetics of neurotransmitter release demonstrated more synchronized release in neonatal rats with hHCY. At the same time lower release probability was observed in adults with hHCY. Spontaneous transmitter release in neonates with hHCY was inhibited by hydrogen peroxide (H2O2) whereas in controls this oxidant was effective only in adult animals indicating a higher susceptibility of motor nerve terminals to oxidative stress. The morphology and the intensity of endocytosis of synaptic vesicles in motor nerve endings was assessed using the fluorescence dye FM 1-43. Adult-like synapses were found in neonates with hHCY which were characterized by a larger area of presynaptic terminals compared to controls. No difference in the intensity of FM 1-43 fluorescence was observed between two groups of animals. Prenatal hHCY resulted in reduced muscle strength assessed by the Paw Grip Endurance test. Using biochemical assays we found an increased level of H2O2 and lipid peroxidation products in the diaphragm muscles of hHCY rats. This was associated with a lowered activity of superoxide dismutase and glutathione peroxidase. Our data indicate that prenatal hHCY induces oxidative stress and apparent faster functional and morphological "maturation" of motor synapses. Our results uncover synaptic mechanisms of disrupted muscle function observed in hHCY conditions which may contribute to the pathogenesis of motor neuronal diseases associated with enhanced level of homocysteine.

Optical Monitoring of Individual Release Sites Tests a New Mechanism for Synaptic Depression

We recently used paired motoneuron muscle recordings in zebrafish to explore frequency-dependent synaptic depression. Most models for synaptic depression focus on vesicle heterogeneity. However, our variance analysis of steady state endplate current (EPC) amplitudes has turned up a new mechanism that assigns depression to the dropout of a release site class with ultra-slow vesicle reloading rates. This assignment was made on the basis of 1) high release probability of all release sites at low frequency; 2) low variance of EPC amplitude at steady state transmission, suggesting that only a subset of release sites can sustain fast release at high frequencies; 3) greater than 60 fold difference in vesicle reloading rate of for the two classes of release sites. As a direct test of these processes, we generated a fish line where the calcium indicator GCaMP6f was fused to postsynaptic rapsyn to optically monitor the activity of postsynaptic ACh receptors. Both the density and calcium permeability of the receptors provide detection of single release events. Because the ∼15 synaptic boutons are physically separated along the length of the muscle, each can be individually monitored for optical response to a motoneuron action potential. Data thus far indicates that stimulus frequencies that produce no depression (e.g., 0.2 Hz) show faithful GCaMP6f fluorescence responses to each stimulus. Stimulation at frequencies associated with synaptic depression has been more difficult to analyze due to the slow kinetics of GCaMP6f. However, preliminary recordings obtained at 10 Hz revealed all synapses responding to the first stimulation and with select synapses consistently dropping out during the stimulus train. These results are consistent with two classes of active sites bearing distinct release kinetics underlying frequency-dependent depression. Firm conclusions require further testing at high frequencies.

Synaptic abnormalities of mice lacking toll-like receptor (TLR)-9

Motor, sensory, and autonomic abnormalities are reported for toll-like receptor 9 (TLR9) knock-out (KO) mice. However, a physiological role of TLR9 in the nervous system is largely unknown. Since altered synaptic transmission can contribute to sensory and motor abnormalities, we evaluated neuromuscular junction (NMJ) function and morphology of TLR9 KO mice. Triangularis sterni nerve-muscle preparations were dissected from TLR9 KO and age-matched control mice. Two-electrode voltage clamp of the motor endplate revealed that the amplitude and frequency of miniature end plate currents (mEPCs) for TLR9 KO NMJs were significantly greater than control. In contrast, mean endplate current (EPC, 1Hz) amplitude was equivalent to control. The ratio of mean EPC to mean mEPC amplitude indicated a decline of quantal content (m) for TLR9 KO NMJs. Furthermore, m declined more rapidly than control in response to 50-Hz stimulus trains. A rightward shift of the mEPC amplitude distribution suggested formation of vesicles containing larger amounts of acetylcholine (ACh). Staining with rhodamine α-bungarotoxin revealed a significant decline of endplate size in TLR9 KO mice. This alteration may result from ACh-induced decline of acetylcholine receptor (AChR) expression resulting from increased frequency and amplitude of mEPCs. At the same time, excessive spontaneous vesicular ACh release may initiate retrograde suppression of excitation-secretion coupling. These data suggest a novel role of TLR9 in the development of the NMJ.

Synaptic Abnormalities of Mice Lacking Toll Like Receptor (TLR)‐9

TLR9 is abundant in the central as well as the peripheral nervous systems. Although, motor and sensory abnormalities are reported for TLR9 knock‐out (KO) mice, a physiological role of TLR9 in the nervous system is largely unknown. Since altered synaptic transmission can contribute to sensory and motor abnormalities, we evaluated neuromuscular junction (NMJ) function and morphology of TLR9 KO mice. Triangularis sterni nerve‐muscle preparations were dissected from TLR9 KO and age‐matched control mice. Two electrode voltage clamp of the motor endplate revealed that the amplitude (nAmp) and frequency (sec‐1) of miniature end plate currents (mEPCs) for TLR9 KO NMJs (N=22) increased (p<0.001) to 4.7 ± 0.21 (mean ± SEM) and 52.7 ± 15.04 from the control (N=31) values of 3.5 ± 0.12 and 10.4 ± 1.03. In contrast, mean endplate current (EPC, 1Hz) amplitude was equivalent to control. The ratio of mean EPC to mean mEPC amplitude indicated a decline (p<0.006) of quantal content (m) from the control value (N=14) of 104 ± 5.9 to 80 ± 5.3 for TLR9 KO NMJs (N=13); furthermore, m declined quicker during 50 Hz stimulus trains. A rightward shift of the mEPC amplitude distribution (6240 mEPCs from 22 NMJs of both groups) suggested formation of vesicles containing larger amounts of acetylcholine (ACh). Staining with rhodamine α‐bungarotoxin revealed a decline of endplate volume from 2201 ± 116.0 µm3 (N=36) in control to 1751 ± 89.6 µm3 (N=44) in TLR9 KO mice. This decline of AChRs may result from ACh‐induced decline of AChR expression resulting from increased frequency and amplitude of mEPCs at TLR9 NMJs. At the same time, excessive ACh may induce retrograde suppression of the exocytosis process. Overall, these data suggest a novel role of TLR9 in the maintenance of motor nerve terminal and endplate properties. Supported by The Kirby Foundation

Effects of 5α-cholestan-3-one on the synaptic vesicle cycle at the mouse neuromuscular junction

We have investigated the effects of 5α-cholesten-3-one (5Ch3, 200 nM) on synaptic transmission in mouse diaphragm. 5Ch3 had no impact on the amplitude or frequency of miniature endplate currents (MEPCs, spontaneous secretion), but decreased the amplitude of EPCs (evoked secretion) triggered by single action potentials. Treatment with 5Ch3 increased the depression of EPC amplitude and slowed the unloading of the dye FM1-43 from synaptic vesicles (exocytosis rate) during high-frequency stimulation. The estimated recycling time of vesicles did not change, suggesting that the decline of synaptic efficiency was due to the reduction in the size of the population of vesicles involved in release. The effects of 5Ch3 on synaptic transmission may be related to changes in the phase properties of the membrane. We have found that 5Ch3 reduces the staining of synaptic regions with the B-subunit of cholera toxin (a marker of lipid rafts) and increases the fluorescence of 22-NBD-cholesterol, indicating a phase change within the membrane. Manipulations of membrane cholesterol (saturation or depletion) strongly reduced the influence of 5Ch3 on both FM1-43 dye unloading and staining with the B-subunit of cholera toxin. Thus, 5Ch3 reduces the number of vesicles which are actively recruited during synaptic transmission and alters membrane properties. These effects of 5Ch3 depend on membrane cholesterol.

Altered active zones, vesicle pools, nerve terminal conductivity, and morphology during experimental MuSK myasthenia gravis.

Recent studies demonstrate reduced motor-nerve function during autoimmune muscle-specific tyrosine kinase (MuSK) myasthenia gravis (MG). To further understand the basis of motor-nerve dysfunction during MuSK-MG, we immunized female C57/B6 mice with purified rat MuSK ectodomain. Nerve-muscle preparations were dissected and neuromuscular junctions (NMJs) studied electrophysiologically, morphologically, and biochemically. While all mice produced antibodies to MuSK, only 40% developed respiratory muscle weakness. In vitro study of respiratory nerve-muscle preparations isolated from these affected mice revealed that 78% of NMJs produced endplate currents (EPCs) with significantly reduced quantal content, although potentiation and depression at 50 Hz remained qualitatively normal. EPC and mEPC amplitude variability indicated significantly reduced number of vesicle-release sites (active zones) and reduced probability of vesicle release. The readily releasable vesicle pool size and the frequency of large amplitude mEPCs also declined. The remaining NMJs had intermittent (4%) or complete (18%) failure of neurotransmitter release in response to 50 Hz nerve stimulation, presumably due to blocked action potential entry into the nerve terminal, which may arise from nerve terminal swelling and thinning. Since MuSK-MG-affected muscles do not express the AChR γ subunit, the observed prolongation of EPC decay time was not due to inactivity-induced expression of embryonic acetylcholine receptor, but rather to reduced catalytic activity of acetylcholinesterase. Muscle protein levels of MuSK did not change. These findings provide novel insight into the pathophysiology of autoimmune MuSK-MG.

Role of Ryanodine Receptors in the Effects of Hydrogen Sulfide on Transmitter Release from the Frog Motor Nerve Ending

We studied the role of ryanodine receptors in the effects of hydrogen sulfide on transmitter release from frog motor nerve ending. Sodium hydrosulfide (300 μM), a donor of hydrogen sulfide, reversibly increased the frequency of miniature endplate current without changes in its amplitude-time parameters. These effects were associated with reversible increase in endplate current amplitude, which was abolished by activation of ryanodine receptors of intracellular Ca(2+)stores with caffeine (3 mM) and ryanodine (0.5 μM). Under conditions of ryanodine receptors blockade with ryanodine (10 μM), sodium hydrosulfide had no effect on induced transmitter release, but its effects remained unchanged during ryanodine receptors blockade with dantrolene (25 μM). We concluded that an enhanced acetylcholine release induced by hydrogen sulfide is related to an increase of intracellular Ca(2+)concentration due to activation of ryanodine receptors for intracellular Ca(2+)-pool.

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.

Acetylcholinesterase deficiency contributes to neuromuscular junction dysfunction in type 1 diabetic neuropathy

Diabetic neuropathy is associated with functional and morphological changes of the neuromuscular junction (NMJ) associated with muscle weakness. This study examines the effect of type 1 diabetes on NMJ function. Swiss Webster mice were made diabetic with three interdaily ip injections of streptozotocin (STZ). Mice were severely hyperglycemic within 7 days after the STZ treatment began. Whereas performance of mice on a rotating rod remained normal, the twitch tension response of the isolated extensor digitorum longus to nerve stimulation was reduced significantly at 4 wk after the onset of STZ-induced hyperglycemia. This mechanical alteration was associated with increased amplitude and prolonged duration of miniature end-plate currents (mEPCs). Prolongation of mEPCs was not due to expression of the embryonic acetylcholine receptor but to reduced muscle expression of acetylcholine esterase (AChE). Greater sensitivity of mEPC decay time to the selective butyrylcholinesterase (BChE) inhibitor PEC suggests that muscle attempts to compensate for reduced AChE levels by increasing expression of BChE. These alterations of AChE are attributed to STZ-induced hyperglycemia since similar mEPC prolongation and reduced AChE expression were found for db/db mice. The reduction of muscle end-plate AChE activity early during the onset of STZ-induced hyperglycemia may contribute to endplate pathology and subsequent muscle weakness during diabetes.

Effects of hydrogen sulfide on the exo- and endocytosis of synaptic vesicles in frog motor nerve endings

Using electrophysiological and optical methods, we studied the effects of sodium hydrosulfide (NaHS), a hydrogen sulfide donor, on the dynamics of transmitter release and exo- and endocytosis of synaptic vesicles in motor nerve endings during long-term high-frequency stimulation (20 Hz) in experiments with the cutaneous pectoris frog muscle. H2S increased the amplitude of endplate currents under conditions of a single stimulation of the motor nerve and slowed down the depression of the end plate currents during high-frequency stimulation (20 Hz, 3 min). Using the endocytic fluorescent dye FM 1–43, we showed that NaHS increased the dye uptake during high-frequency stimulation as compared to the control. However, after termination of the high-frequency stimulation the fluorescence intensity of nerve terminals was lower in the presence of NaHS than in the control experiments. In addition, NaHS slowed the dye release from the nerve terminals that were loaded during 20 Hz stimulation. The results thus obtained indicate that H2S accelerates the synaptic vesicles cycle in frog motor nerve ending by enhancement of exocytosis and fast endocytosis of synaptic vesicles during high-frequency stimulation.

Different sensitivities of rat skeletal muscles and brain to novel anti‐cholinesterase agents, alkylammonium derivatives of 6‐methyluracil (ADEMS)

The rat respiratory muscle diaphragm has markedly lower sensitivity than the locomotor muscle extensor digitorum longus (EDL) to the new acetylcholinesterase (AChE) inhibitors, alkylammonium derivatives of 6-methyluracil (ADEMS). This study evaluated several possible reasons for differing sensitivity between the diaphragm and limb muscles and between the muscles and the brain. Increased amplitude and prolonged decay time of miniature endplate currents were used to assess anti-cholinesterase activity in muscles. In hippocampal slices, induction of synchronous network activity was used to follow cholinesterase inhibition. The inhibitor sensitivities of purified AChE from the EDL and brain were also estimated. The intermuscular difference in sensitivity to ADEMS is partly explained caused by a higher level of mRNA and activity of 1,3-bis[5(diethyl-o-nitrobenzylammonium)pentyl]-6-methyluracildibromide (C-547)-resistant BuChE in the diaphragm. Moreover, diaphragm AChE was more than 20 times less sensitive to C-547 than that from the EDL. Sensitivity of the EDL to C-547 dramatically decreased after treadmill exercises that increased the amount of PRiMA AChE(G4), but not ColQ AChE(A12) molecular forms. The A12 form present in muscles appeared more sensitive to C-547. The main form of AChE in brain, PRiMA AChE(G4), was apparently less sensitive because brain cholinesterase activity was almost three orders of magnitude more resistant to C-547 than that of the EDL. Our findings suggest that ADEMS compounds could be used for the selective inhibition of AChEs and as potential therapeutic tools.

TRPV1 Modulates Acetylcholine Release From Motor Nerve Terminals

Transient receptor potential (TRP) proteins are expressed ubiquitously throughout the body. Previous studies demonstrated expression of TRPV1, the capsaicin receptor, in sensory neurons. Recently, we reported TRPV1 expression in mouse motor nerve endings (MNEs; J. Pharmacol. Exp. Ther. Aug 04. 2009) where we observed that capsaicin protected MNEs from botulinum neurotoxin A (BoNT/A). We hypothesized that capsaicin reduced clathrin coated pit (CCP) dependent endocytosis of BoNT/A and demonstrate the regulatory influence of TRPV1 in exo-endocytic processes of MNEs. Phrenic nerve diaphragm muscle preparations isolated from isoflurane anesthetized adult mice were analyzed for the nerve-evoked twitch and transmitter release (TR). Capsaicin produced a concentration-dependent decline of twitch tension (TT), an effect attributed to suppression of stimulus-evoked acetylcholine release (SEAR) since these capsaisin concentrations reduced the amplitude of endplate currents. These effects of capsaicin were antagonized by capsazepine, the TRPV1 antagonist. To understand the mechanism whereby capsaicin reduced TR, we studied cholinergic Neuro 2a cells. Acute exposure to capsaicin altered the subcellular distribution of clathrin heavy chain (CHc) and AP2, two proteins essential to CCP formation. Wortmannin, (non selective PI3K/PI5K inhibitor), inhibited the TT and SEAR of the isolated nerve-muscle preparations and delocalized CHc and AP2 in Neuro 2a cells. Chlorpromazine, an inhibitor of CCP dependent endocytic pathway [Cell Mol. Biol. Lett. 2004; 9 (3): 475-81], mimicked the effects of capsaicin on AP2 delocalization. These data suggest that endogenous TRPV1 proteins are coupled to the exo-endocytic mechanisms that regulate neuromuscular transmission and that activation of TRPV1 with high capsaicin concentrations reduces exocytosis of acetylcholine by down regulating the compensatory CCP dependent endocytic pathways.

Role of cyclic nucleotides in effects of hydrogen sulfide on the mediator release in frog neuromuscular junction

The role of cyclic nucleotides in the effects of hydrogen sulfide (H2S) on transmitter release in the frog neuromuscular junction was studied using focal microelectrode recording of synaptic signals. A donor of H2S, sodium hydrogen sulfide (NaHS) (100 µM) reversibly increased the amplitude and quantum content of end-plate currents (EPCs) without changing the nerve ending response. The effect of H2S on evoked transmitter release was decreased by preliminary application of the membrane-permeable cAMP analogue pCPT-cAMP (100 µM). Inhibition of adenylate cyclase by MDL-12330A (0.8 µM) did not alter the effect of NaHS on transmitter release. Membrane-permeable cGMP analogue pCPT-cGMP (50 µM) and inhibitor of guanylate cyclase ODQ (0.1 µM) did not change the effect of NaHS. Therefore, we conclude that the H2S-induced increase in the transmitter release was not a result of guanylate cyclase activity. It was concluded that the H2S effects are related to the functioning of cAMP-dependent mechanisms; however, these effects are not associated with the direct action of H2S on the adenylate cyclase activity.

Mechanisms of the inhibition of endplate acetylcholine receptors by antiseptic chlorhexidine (experiments and models)

Mechanisms of the inhibition of evoked multiquantal endplate currents (EPC) by chlorhexidine (CHX) were studied in electrophysiological experiments and by mathematical modeling to discriminate between possible channel, receptor, and non-receptor effects of this common antiseptic drug. Experiments were carried out on the isolated neuromuscular preparation of the cut m. sartorius of the frog Rana ridibunda. The nerve-stimulation-evoked endplate currents were measured by standard double microelectrode technique. For the mathematical simulation, a method based on the solution of a system of ordinary differential equations was used. CHX in milimolar concentrations suppressed the amplitude and shortened the evoked EPC. Recovery of the EPC amplitude was very slow, and EPC shortening persisted during 30-40 min washout of the drug. There is no indication that CHX competes for acetylcholine or carbachol binding site(s). A comparison of the experimental data with mathematical simulation made it possible to construct a reliable kinetic scheme, which describes the action of CHX. CHX induces a combined slow blockade of the open ionic channel and long-lasting allosteric inhibition of the nicotinic acetylcholine receptor. The very slow washout of the drug in terms of EPC amplitude and virtually no recovery of the shortened EPC time course might substantiate certain caution to avoid unintentional high-dose application during its antibacterial application.

Motor unit number estimate as a predictor of motor dysfunction in an animal model of type 1 diabetes

Peripheral neuropathy is a common complication of diabetes that leads to severe morbidity. In this study, we investigated the sensitivity of motor unit number estimate (MUNE) to detect early motor axon dysfunction in streptozotocin (STZ)-treated mice. We compared the findings with in vitro changes in the morphology and electrophysiology of the neuromuscular junction. Adult Thy1-YFP and Swiss Webster mice were made diabetic following three interdaily intraperitoneal STZ injections. Splay testing and rotarod performance assessed motor activity for 6 wk. Electromyography was carried out in the same time course, and compound muscle action potential (CMAP) amplitude, latency, and MUNE were estimated. Two-electrode voltage clamp was used to calculate quantal content (QC) of evoked transmitter release. We found that an early reduction in MUNE was evident before a detectable decline of motor activity. CMAP amplitude was not altered. MUNE decrease accompanied a drop of end-plate current amplitude and QC. We also observed small axonal loss, sprouting of nerve endings, and fragmentation of acetylcholine receptor clusters at the motor end plate. Our results suggest an early remodeling of motor units through the course of diabetic neuropathy, which can be readily detected by the MUNE technique. The early detection of MUNE anomalies is significant because it suggests that molecular changes associated with pathology and leading to neurodegeneration might already be occurring at this stage. Therefore, trials of interventions to prevent motor axon dysfunction in diabetic neuropathy should be administered at early stages of the disorder.

Pre- and post-synaptic abnormalities associated with impaired neuromuscular transmission in a group of patients with 'limb-girdle myasthenia'

The properties of neuromuscular junctions (NMJs) were studied in motor-point biopsy samples from eight patients with congenital myasthenic syndromes affecting primarily proximal limb muscles ['limb-girdle myasthenia' (LGM)]. All had moderate to severe weakness of the proximal muscles, without short-term clinical fatigability but with marked variation in strength over periods of weeks or months, with little or no facial weakness or ptosis and no ophthalmoplegia. Most had a characteristic gait and stance. All patients showed decrement of the compound muscle action potential (CMAP) on repetitive stimulation at 3 Hz, and increased jitter and blocking was detected by SFEMG, confirming the presence of impaired neuromuscular transmission. None of the patients had serum antibodies against acetylcholine receptors (AChRs). Two of the patients had similarly affected siblings. Intracellular recording from isolated nerve-muscle preparations revealed that the quantal content (the number of ACh quanta released per nerve impulse) was only approximately 50% of that in controls. However, the quantal size (amplitude of miniature end-plate currents) and the kinetic properties of synaptic potentials and currents were similar to control values. The area of synaptic contact and extent of post-synaptic folding were approximately 50% of control values. Thus, the quantal content per unit area of synaptic contact was normal. The number of AChRs per NMJ was also reduced to approximately 50% of normal, so the local AChR density was normal. Immunolabelling studies revealed qualitatively normal distributions and abundance of each of 14 proteins normally concentrated at the NMJ, including components of the basal lamina, post-synaptic membrane and post-synaptic cytoskeleton. DNA analysis failed to detect mutations in the genes encoding any of the following proteins: AChR subunits, rapsyn, ColQ, ChAT or muscle-specific kinase. Response of these patients to treatment was varied: few showed long-term improvement with pyridostigmine and some even deteriorated with treatments, while others had intolerable side-effects. Several patients showed improvement with 3,4-diaminopyridine, but this was generally only transient. Ephedrine was helpful in half of the patients. We conclude that impaired neuromuscular transmission in these LGM patients results from structural abnormalities of the NMJ, including reduced size and post-synaptic folding, rather from any abnormality in the immediate events of neuromuscular transmission.