Mouse Motor Nerve Research Articles

A proteomic view on the differential phenotype of Schwann cells derived from mouse sensory and motor nerves.

Schwann cells (SCs) are myelin-forming glial cells of the peripheral nervous system. Recent studies suggested that SCs comprise two phenotypes: sensory SCs and motor SCs, which are associated with the modality-specific promotion of sensory and motor axon growth during peripheral neuronal regeneration. However, the molecular basis of the two phenotypic SCs is unclear. We established a workflow to obtain highly purified SCs derived from sensory nerve (SNdSCs) and motor nerve (MNdSCs) from B6; D2-Tg(s100B-EGFP)1Wjt/J mice. Subsequently, a quantitative proteomic analysis based on iTRAQ labeling was performed to compare the proteome of SNdSCs and MNdSCs. A total of 6,567 proteins were identified, of which 63 and 11 proteins were overexpressed in SNdSCs and MNdSCs, respectively. Three of the overexpressed proteins were further validated by western blot and immunocytochemistry: GMFB and CNPase, which were overexpressed in sensory SNdSCs, and histone H4, which was overexpressed in MNdSCs. The expression pattern of the three proteins was also validated in the dorsal roots and ventral roots. Bioinformatics analysis indicated that proteins highly expressed in SNdSCs are mainly involved in RNA processing and protein synthesis, while those overexpressed in MNdSCs are related to cell proliferation. Real-time cell analysis confirmed that the proliferation activity of MNdSCs is higher than that of SNdSCs. This study is the first to provide a proteomic view of the differential phenotype of mouse SNdSCs and MNdSCs. The data may serve as a valuable source for the study of the biological characteristics of these two SC phenotypes and their roles in nerve-specific regeneration.

The Contribution of L-Type Calcium Channels to Acetylcholine Secretion in Frog and Mouse Neuromuscular Junctions with Active and Inactivated Voltage-Gated Potassium Channels

We report here experiments addressing the contribution of L-type Ca2+ channels to evoked acetylcholine secretion from frog and mouse motor nerve endings with active and inactivated voltage-gated K+ channels. These studies evaluated the effects of the specific L-type Ca2+ channel blocker nitrendipine on the quantum composition of endplate currents and the time course of the secretion of acetylcholine quanta in intact preparations and after preliminary blockade of voltage-gated K+ channels with 4-aminopyridine (4-AP) in medium with depressed and physiological Ca2+ levels. A fluorescence method was used to measure calcium transients reflecting the integral influx of Ca2+ into nerve endings; computer modeling was applied to the processes underlying exocytosis in the presence of the two types of Ca2+ channel (N and L) and with different durations of nerve ending action potentials. In frog synapses, L-type Ca2+ channels were found to contribute to evoked acetylcholine secretion in the presence of active K+ channels, but only in the presence of a depressed Ca2+ level in the medium; on inactivation of voltage-gated K+ channels, the contribution of L-type channels to the secretory process became less significant. At a physiological Ca2+ level, the involvement of L-type channels in evoked acetylcholine secretion was apparent, as in mouse synapses, only in conditions of blockade of voltage-gated K+ channels.

Perisynaptic Schwann cells phagocytose nerve terminal debris in a mouse model of Guillain-Barré syndrome.

In mouse models of acute motor axonal neuropathy, anti‐ganglioside antibodies (AGAbs) bind to motor axons, notably the distal nerve, and activate the complement cascade. While complement activation is well studied in this model, the role of inflammatory cells is unknown. Herein we aimed to investigate the contribution of phagocytic cells including macrophages, neutrophils and perisynaptic Schwann cells (pSCs) to distal nerve pathology. To observe this, we first created a subacute injury model of sufficient duration to allow inflammatory cell recruitment. Mice were injected intraperitoneally with an anti‐GD1b monoclonal antibody that binds strongly to mouse motor nerve axons. Subsequently, mice received normal human serum as a source of complement. Dosing was titrated to allow humane survival of mice over a period of 3 days, yet still induce the characteristic neurological impairment. Behaviour and pathology were assessed in vivo using whole‐body plethysmography and post‐sacrifice by immunofluorescence and flow cytometry. ex vivo nerve‐muscle preparations were used to investigate the acute phagocytic role of pSCs following distal nerve injury. Following complement activation at distal intramuscular nerve sites in the diaphragm macrophage localisation or numbers are not altered, nor do they shift to a pro‐ or anti‐inflammatory phenotype. Similarly, neutrophils are not significantly recruited. Instead, ex vivo nerve‐muscle preparations exposed to AGAb plus complement reveal that pSCs rapidly become phagocytic and engulf axonal debris. These data suggest that pSCs, rather than inflammatory cells, are the major cellular vehicle for axonal debris clearance following distal nerve injury, in contrast to larger nerve bundles where macrophage‐mediated clearance predominates.

The Participation of Endocannabinoid Receptors in the Regulation of Spontaneous Synaptic Activity at Neuromuscular Junctions of Mice

The mechanisms underlying changes in spontaneous release of acetylcholine (ACh) induced by exogenous agonists and antagonists of endocannabinoid receptors were investigated by recording miniature endplate potentials (MEPPs) in motor synapses of mouse diaphragm preparations with intracellular microelectrodes. There have not been observed any statistically significant changes either in the membrane potential of muscle fibers or in the frequency, amplitude, and time parameters of the MEPPs under the action of AM-251 (1 μM), antagonist/inverse agonist of CB1 receptors, for 60 min. Activation of CB receptors by their exogenous agonist WIN 55,212-2 (WIN) (20 μM) for 1 h did not cause statistically significant changes in the amplitude of the MEPPs. However, MEPP frequency increased by more than 50% as compared to the control under the action of WIN for 30–60 min. Blocking of CB1 receptors by AM-251 (1 μM) prevented the WIN-induced increase in MEPP frequency. A protein kinase A inhibitor H-89 (1 μM) did not prevent the enhancement of the ACh secretion upon activation of CB receptors by WIN. WIN lost the ability to increase MEPP frequency in the presence of a phospholipase C inhibitor U73122 (5 μM), which did not affect by itself the amplitude and temporal characteristics or MEPP frequency. A protein kinase C inhibitor, chelerythrin (4 μM) or ryanodine, which blocks the ryanodine receptors (RyRs) at a concentration of 5 μM, also prevented the WIN-induced increase in MEPP frequency. Thus, a signaling cascade triggered by stimulation of presynaptic CB1 receptors with subsequent activation of phospholipase C, protein kinase C, and the release of stored calcium, leading to an increase in the frequency of spontaneous quantal release of ACh, has been detected for the first time in mouse motor synapses. This suggests the presence of CB1 receptors on the presynaptic membrane in mouse motor nerve terminals and the possibility of activation of these receptors by endocannabinoids for potentiating the spontaneous secretion of ACh.

Вклад кальциевых каналов L-типа в секрецию ацетилхолина в нервно-мышечных соединениях лягушки и мыши при активных и инактивированных потенциал-зависимых калиевых каналах

In this work, the contribution of L-type Ca2+ channels to the evoked secretion of acetylcholine from the frog and mouse motor nerve endings with active and inactivated potential-dependent K+ channels was studied. The effects of the specific Ca2+ blocker of the L-type channels nitrendipine on the quantal content of endplate currents and the timing of acetylcholine quanta secretion in intact preparations and after the preliminary blockade of potential-activated K+ channels with 4-aminopyridine (4-AP) were evaluated under conditions of a decreased and physiological level of ambient Ca2+. Fluorescent measurement of calcium transient reflecting the integral input of Ca2+ into the nerve ending was performed as well as the computer simulation of the processes underlying exocytosis assuming the presence of two types of Ca2+ channels (N- and L-types) and varying duration of the nerve action potential. It was shown that in the frog synapses, L-type calcium channels contribute to the evoked secretion of acetylcholine at active K+ channels only under conditions of decreased Ca2+ level; after inactivation of potential-dependent K+ channels, the contribution of L-type channels to the secretory process becomes less significant. At the physiological level of ambient Ca2+, the involvement of L-type channels in the evoked acetylcholine secretion, as in mice synapses, is manifested only under conditions of inactivated potential-dependent K+ channels.

Ryanodine- and CaMKII-dependent release of endogenous CGRP induces an increase in acetylcholine quantal size in neuromuscular junctions of mice.

ObjectiveThe aim of this study was to identify the mechanism responsible for an increase in miniature endplate potentials (MEPPs) amplitude, induced by ryanodine as an agonist of ryanodine receptors in mouse motor nerve terminals.MethodsUsing intracellular microelectrode recordings of MEPPs and evoked endplate potentials (EPPs), the changes in spontaneous and evoked acetylcholine release in motor synapses of mouse diaphragm neuromuscular preparations were studied.ResultsRyanodine (0.1 μM) increased both the amplitudes of MEPPs and EPPs to a similar extent (up to 130% compared to control). The ryanodine effect was prevented by blockage of receptors of calcitonin gene‐related peptide (CGRP) by a truncated peptide CGRP 8‐37. Endogenous CGRP is stored in large dense‐core vesicles in motor nerve terminals and may be released as a co‐transmitter. The ryanodine‐induced increase in MEPPs amplitude may be fully prevented by inhibition of vesicular acetylcholine transporter by vesamicol or by blocking the activity of protein kinase A with H‐89, suggesting that endogenous CGRP is released in response to the activation of ryanodine receptors. Activation of CGRP receptors can, in turn, upregulate the loading of acetylcholine into synaptic vesicles, which will increase the quantal size. This new feature of endogenous CGRP activity looks similar to recently described action of exogenous CGRP in motor synapses of mice. The ryanodine effect was prevented by inhibitors of Ca/Calmodulin‐dependent kinase II (CaMKII) KN‐62 or KN‐93. Inhibition of CaMKII did not prevent the increase in MEPPs amplitude, which was caused by exogenous CGRP.ConclusionsWe propose that the activity of presynaptic CaMKII is necessary for the ryanodine‐stimulated release of endogenous CGRP from motor nerve terminals, but CaMKII does not participate in signaling downstream the activation of CGRP‐receptors followed by quantal size increase.

Neurofilament depletion improves microtubule dynamics via modulation of Stat3/stathmin signaling.

In neurons, microtubules form a dense array within axons, and the stability and function of this microtubule network is modulated by neurofilaments. Accumulation of neurofilaments has been observed in several forms of neurodegenerative diseases, but the mechanisms how elevated neurofilament levels destabilize axons are unknown so far. Here, we show that increased neurofilament expression in motor nerves of pmn mutant mice, a model of motoneuron disease, causes disturbed microtubule dynamics. The disease is caused by a point mutation in the tubulin-specific chaperone E (Tbce) gene, leading to an exchange of the most C-terminal amino acid tryptophan to glycine. As a consequence, the TBCE protein becomes instable which then results in destabilization of axonal microtubules and defects in axonal transport, in particular in motoneurons. Depletion of neurofilament increases the number and regrowth of microtubules in pmn mutant motoneurons and restores axon elongation. This effect is mediated by interaction of neurofilament with the stathmin complex. Accumulating neurofilaments associate with stathmin in axons of pmn mutant motoneurons. Depletion of neurofilament by Nefl knockout increases Stat3–stathmin interaction and stabilizes the microtubules in pmn mutant motoneurons. Consequently, counteracting enhanced neurofilament expression improves axonal maintenance and prolongs survival of pmn mutant mice. We propose that this mechanism could also be relevant for other neurodegenerative diseases in which neurofilament accumulation and loss of microtubules are prominent features.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-016-1564-y) contains supplementary material, which is available to authorized users.

Synaptosome-associated protein 25 (SNAP25) synthesis in terminal buttons of mouse motor neuron.

Previously, we formulated the hypothesis of compartmentalized protein synthesis in axons of motor neurons. In the axon hillock, along the entire length of the axon and in its ending, specific proteins are locally synthesized, which ensure the function of each compartment. In support of this hypothesis, in this work we studied the local protein synthesis in mouse motor nerve ending.

Different dynamin blockers interfere with distinct phases of synaptic endocytosis during stimulation in motoneurones.

Neurotransmitter release requires a tight coupling between synaptic vesicle exocytosis and endocytosis with dynamin being a key protein in that process. We used imaging techniques to examine the time course of endocytosis at mouse motor nerve terminals expressing synaptopHluorin, a genetically encoded reporter of the synaptic vesicle cycle. We separated two sequential phases of endocytosis taking place during the stimulation train: early and late endocytosis. Freshly released synaptic vesicle proteins are preferentially retrieved during the early phase, which is very sensitive to dynasore, an inhibitor of dynamin GTPase activity. Synaptic vesicle proteins pre-existing at the plasma membrane before the stimulation are preferentially retrieved during the late phase, which is very sensitive to myristyl trimethyl ammonium bromide (MitMAB), an inhibitor of the dynamin-phospholipid interaction. Synaptic endocytosis is essential at nerve terminals to maintain neurotransmitter release by exocytosis. Here, at the neuromuscular junction of synaptopHluorin (spH) transgenic mice, we have used imaging to study exo- and endocytosis occurring simultaneously during nerve stimulation. We observed two endocytosis components, which occur sequentially during stimulation. The early component of endocytosis apparently internalizes spH molecules freshly exocytosed. This component was sensitive to dynasore, a blocker of dynamin 1 GTPase activity. In contrast, this early component was resistant to myristyl trimethyl ammonium bromide (MiTMAB), a competitive agent that blocks dynamin binding to phospholipid membranes. The late component of endocytosis is likely to internalize spH molecules that pre-exist at the plasma membrane before stimulation starts. This component was blocked by MiTMAB, perhaps by impairing the binding of dynamin or other key endocytic proteins to phospholipid membranes. Our study suggests the co-existence of two sequential synaptic endocytosis steps taking place during stimulation that are susceptible to pharmacological dissection: an initial step, preferentially sensitive to dynasore, that internalizes vesicular components immediately after they are released, and a MiTMAB-sensitive step that internalizes vesicular components pre-existing at the plasma membrane surface. In addition, we report that post-stimulus endocytosis also has several components with different sensitivities to dynasore and MiTMAB.

Capsaicin modulates acetylcholine release at the myoneural junction

Transient receptor potential (TRP) proteins are non-selective cation channel proteins that are expressed throughout the body. Previous studies demonstrated the expression of TRP Vanilloid 1 (TRPV1), capsaicin (CAP) receptor, in sensory neurons. Recently, we reported TRPV1 expression in mouse motor nerve terminals [MNTs; (Thyagarajan et al., 2009)], where we observed that CAP protected MNTs from botulinum neurotoxin A (BoNT/A). Phrenic nerve diaphragm nerve muscle preparations (NMP) isolated from isoflurane anesthetized adult mice were analyzed for twitch tension, spontaneous (mEPCs) and nerve stimulus evoked (EPCs) acetylcholine release. When acutely applied to isolated NMP, CAP produced a concentration-dependent decline of twitch tension and produced a significant decline in the amplitude of EPCs and quantal content without any effect on the mEPCs. The suppression of nerve stimulus evoked acetylcholine release by CAP was antagonized by capsazepine (CPZ), a TRPV1 antagonist. CAP did not suppress phrenic nerve stimulus evoked acetylcholine release in TRPV1 knockout mice. Also, CAP treatment, in vitro, interfered with the localization of adapter protein 2 in cholinergic Neuro 2a cells. Wortmannin, (WMN; non-selective phosphoinositol kinase inhibitor), mimicked the effects of CAP by inhibiting the acetylcholine exocytosis. Our data suggest that TRPV1 proteins expressed at the MNT are coupled to the exo-endocytic mechanisms to regulate neuromuscular functions.

P62: Hydrogen sulfide in regulation of synaptic transmission

The role of H2S in regulation of synaptic transmission was studied using electrophysiological and fluorescent methods. In frog and mouse neuromuscular junctions (NMJ) NaHS increased spontaneous and evoked transmitter release in response to single stimuli. During high-frequency stimulation NaHS decreased the depression of end-plate potential amplitudes (EPPs) in frog NMJ and increased the decay of EPPs amplitudes in mouse NMJ. Using the fluorescent dye FM 1–43 we found that NaHS had opposite effects endocytosis of synaptic vesicles during high-frequency stimulation in frog (increased endocytosis) and mouse (decreased endocytosis) motor nerve endings. It was concluded that H2S accelerated the recycling of synaptic vesicles in frog nerve endings during high-frequency stimulation. In mouse NMJ the increase of exocytosis followed by the decrease in endocytosis leads to more pronounced depression of EPPs. The analysis of intracellular mechanisms revealed that H2S activated ryanodine receptors and increased intracellular Ca2+ concentrations in motor nerve ending. In frog and mouse NMJ L-cysteine increased evoked transmitter release similar to H2S and inhibition of CSE or CBS decreased acetylcholine release, suggesting that endogenous H2S modulates synaptic transmission. Using RT PCR we found expression of mRNA of CSE and CBS in mouse diaphragm. We conclude, H2S is synthesized in frog and mouse NMJ and acts to modulate transmitter release. In further experiments using hippocampal slices of neonatal rats it was shown that NaHS influenced the neuronal network activity. NaHS dose-dependently inhibited giant depolarizing potentials affecting GABA–evoked responses without influence on spontaneous GABA-currents.

The role of nitric oxide in the regulation of neurotransmitter release and processes of exo- and endocytosis of synaptic vesicles in mouse motor nerve endings

In experiments with mouse diaphragm muscle, the effects of a nitric oxide (NO) donor, S-nitroso- N-acetyl-DL-penicillamine (SNAP), and an NO-synthase blocker, NG-nitro-L-arginine methyl ester (LNAME), on transmitter release and processes of exo- and endocytosis of synaptic vesicles in the motornerve ending were studied using electrophysiological and fluorescence techniques. During single stimulation of the motor nerve, SNAP reduced and LNAME did not change the amplitude of the endplate currents and both of the drugs did not affect spontaneous transmitter release. During high-frequency stimulation (20 Hz, 3 min) SNAP increased and LNAME slowed the depression of the amplitudes of endplate potentials (EPPs) compared to the dynamics of EPPs in the control. In experiments using the fluorescent dye FM 1–43, it was shown that the NO donor induced a decrease and LNAME induced an increase in the fluorescence intensity of motor-nerve endings loaded with dye during stimulation at a frequency of 20 Hz for 30 s compared to the control. At the same time, the rate of dye unloading from the terminals that were preloaded with FM 1–43 was higher after preliminary application of SNAP and lower after preliminary application of LNAME. It was suggested that exogenous and endogenous NO in the mouse neuromuscular synapse caused the depression of neurotransmitter release as a result of the suppression of synaptic-vesicle recycling due to a decrease in endocytosis or/and mobilization of synaptic vesicles from a recycling pool to the exocytosis sites.

The effects of hydrogen sulfide on the processes of exo- and endocytosis of synaptic vesicles in the mouse motor nerve endings

The effects of sodium hydrosulfide (NaHS), the donor of hydrogen sulfide (H2S), on the exo/endocytosis cycle of synaptic vesicles in the motor nerve ending of the mouse diaphragm were studied using intracellular microelectrode technique and fluorescent microscopy. NaHS increased the frequency of miniature end-plate potentials (MEPPs), without changing their amplitude-time parameters. NaHS also increased the amplitude of the evoked postsynaptic responses during single stimulation (0.3 Hz), which was the evidence of the enhanced synaptic vesicle exocytosis. During high-frequency stimulation (50 Hz), NaHS induced more significant decline of neurotransmitter release, probably due to the lower rate of synaptic vesicle mobilization from recycling pool to exocytic sites. NaHS also decreased the uptake of the fluorescent endocytic dye FM 1–43, which indicated the reduced endocytosis of synaptic vesicles. Thus, the H2S donor increases exocytosis and decreases the processes of synaptic vesicle endocytosis and mobilization in the mouse motor nerve ending.

Effects and Mechanisms of Action of Nitric Oxide on Transmitter Release in Mouse Motor Nerve Terminals

We examined the mechanisms of NO action on transmitter release in neuromuscular preparations of the mouse diaphragm muscle using a standard microelectrode technique. A donor of NO (SNAP, 100 μM) and a substrate for NO synthesis (L-arginine, 100 μM) reduced the evoked transmitter release from motor nerve endings. At the same time, SNAP did not change the frequency and amplitude of miniature end-plate potentials, while an inhibitor of NO synthase, L-NAME, exerted no effect on evoked and spontaneous transmitter release. Inhibition of soluble guanylate cyclase by ODQ (2.5 μM) abolished the effect of NO on the evoked transmitter release, while the elevation of cGMP-level by its membranepenetrating analog 8BrcGMP did not prevent such effect. The elevation of intracellular concentration of cAMP by 100 μM of its analog 8(4CPT) cAMP or inhibition of phosphodiesterase (PDE) by the action of 100 μM IBMX eliminated NO effects on transmitter release. It is concluded that NO activates soluble guanylate cyclase and intensifies the cGMP synthesis. Activation of the PDE II via an increase in the cGMP level with consequent reduction of the level of intracellular cAMP and decrease in the activity of PKA reduced transmitter release from mouse motor nerve ending.

Evidence for constitutively-active adenosine receptors at mammalian motor nerve endings

A study was made to determine if constitutively active adenosine receptors are present at mouse motor nerve endings. In preparations blocked by low Ca2+/high Mg2+ solution, 8-cyclopentyl-1,3,dipropylxanthine (CPX, 10–100nM), which has been reported to be both an A1 adenosine receptor antagonist and inverse agonist, produced a dose-dependent increase in the number of acetylcholine quanta released by a nerve impulse. Adenosine deaminase, which degrades ambient adenosine into its inactive congener, inosine, failed to alter the response to 100nM CPX. 8-Cyclopentyltheophylline (CPT, 3μM), a competitive inhibitor at A1 adenosine receptors, prevented the increase in acetylcholine release produced by CPX. At normal levels of acetylcholine release, neither adenosine deaminase nor CPX affected acetylcholine release at low frequencies of nerve stimulation in (+)-tubocurarine blocked preparations. The results suggest that a proportion of the acetylcholine release process is controlled by constitutively active adenosine receptors at murine motor nerve endings, providing the first evidence for constitutive activity of G-protein-coupled receptors that modulate the function of mammalian nerve endings.

Calcium dependence of damage to mouse motor nerve terminals following oxygen/glucose deprivation

Motor nerve terminals are especially sensitive to an ischemia/reperfusion stress. We applied an in vitro model of this stress, oxygen/glucose deprivation (OGD), to mouse neuromuscular preparations to investigate how Ca2+ contributes to stress-induced motor terminal damage. Measurements using an ionophoretically-injected fluorescent [Ca2+] indicator demonstrated an increase in intra-terminal [Ca2+] following OGD onset. When OGD was terminated within 20–30min of the increase in resting [Ca2+], these changes were sometimes reversible; in other cases [Ca2+] remained high and the terminal degenerated. Endplate innervation was assessed morphometrically following 22min OGD and 120min reoxygenation (32.5°C). Stress-induced motor terminal degeneration was Ca2+-dependent. Median post-stress endplate occupancy was only 26% when the bath contained the normal 1.8mM Ca2+, but increased to 81% when Ca2+ was absent. Removal of Ca2+ only during OGD was more protective than removal of Ca2+ only during reoxygenation. Post-stress endplate occupancy was partially preserved by pharmacological inhibition of various routes of Ca2+ entry into motor terminals, including voltage-dependent Ca2+ channels (ω-agatoxin-IVA, nimodipine) and the plasma membrane Na+/Ca2+ exchanger (KB-R7943). Inhibition of a Ca2+-dependent protease with calpain inhibitor VI was also protective. These results suggest that most of the OGD-induced motor terminal damage is Ca2+-dependent, and that inhibition of Ca2+ entry or Ca2+-dependent proteolysis can reduce this damage. There was no significant difference between the response of wild-type and presymptomatic superoxide dismutase 1 G93A mutant terminals to OGD, or in their response to the protective effect of the tested drugs.

A model of mouse motor nerve excitability and the effects of polarizing currents

This study was undertaken to develop a theoretical model of mouse motor nerve excitability, and to validate it by application to recordings from mouse axons with membrane potential altered by polarizing currents. Multiple excitability tests were performed on the caudal motor nerves of 12 mice. Membrane potential was artificially modified by injecting polarizing currents through the stimulating electrodes (±20% of threshold for 1 ms pulse). A human motor nerve model was adapted to the unpolarized mouse recordings by adjusting membrane parameters to optimize the fit. To evaluate the usefulness of the model, we then determined the parameter changes which best fitted the recordings from depolarized and hyperpolarized tail nerves. The recordings from the normal axons were well fitted by a model in which axons were hyperpolarized by 4 mV, and nodal sodium and slow potassium conductances were reduced by half, compared with human median nerve motor axons. The changes with polarizing currents were qualitatively similar to those in human axons, and modeling correctly identified a change in current as the best-fitting explanation for the altered excitability. We conclude that this new model should help identify changes in membrane potential and probably in other membrane parameters in mouse models of neurological disease.

Neuropathophysiological potential of Guillain-Barré syndrome anti-ganglioside-complex antibodies at mouse motor nerve terminals

Objectives: Anti-ganglioside antibodies are present in approximately half of Guillain–Barré syndrome (GBS) patients. Recently, it has been shown that a considerable proportion of these patients has serum antibodies against antigenic epitopes formed by a complex of two different gangliosides. However, direct experimental evidence for neuropathogenicity of this special category of antibodies is currently lacking. Here, we explored a series of GBS and GBS-variant sera with anti-ganglioside-complex antibodies for their ability to induce complement-dependent deleterious effects at the living neuronal membrane. Methods: The neuropathophysiological potential of 31 GBS sera containing either anti-GM1/GD1a- or anti-GM1/GQ1b-ganglioside-complex antibodies was studied at motor nerve terminal presynaptic membranes in the mouse phrenic nerve/diaphragm muscle ex vivo experimental model. With electrophysiological measurements and confocal fluorescence microscopy, we assessed and quantified the damaging effect on neuronal membranes by anti-ganglioside-complex antibodies. Results: We show that anti-GM1/GD1a- and anti-GM1/GQ1b-ganglioside-complex positive sera can induce complement-mediated functional and morphological injury at mouse motor nerve terminals ex vivo. Of the 31 investigated anti-ganglioside-complex patient sera, 17 sera induced increases in miniature end-plate potential frequency in this experimental model, mostly associated with muscle fibre twitches. Variability in potency was observed, with the anti-GM1/GD1a-complex sera inducing the most outspoken effects. Conclusions: The present study shows the presence of ganglioside-complexes as available antigens in living neuronal membranes and supplies proof-of-principle that anti-ganglioside-complex antibodies in sera from GBS patients can induce complement-mediated damage. This strongly supports the hypothesis that autoimmune targeting of ganglioside-complexes is of pathogenic relevance in a proportion of GBS patients. (Clin. Exp. Neuroimmunol. doi: 10.1111/j.1759-1961.2011.0022.x, September 2011)

Active Zones and the Readily Releasable Pool of Synaptic Vesicles at the Neuromuscular Junction of the Mouse

Synchronous neurotransmitter release is a highly regulated process that takes place at specializations at the presynaptic membrane called active zones (AZs). The relationships between AZs, quantal release, and vesicle replenishment are not well understood in a mature synapse. We have measured the number, distribution, and other properties of AZs in mouse motor nerve terminals and combined these observations with electrophysiological estimates of the size of the readily releasable pool (RRP) of synaptic vesicles. On average, we counted 850 AZs per terminal. Assuming two primary docked vesicles per AZ, we predict a total of ∼1700 vesicles optimally positioned for exocytosis. Electrophysiological estimates of the size of the RRP, using a simple kinetic model that assumes exponential depletion of the initial pool and refilling by recruitment, gave an average value of 1730 quanta during 100 Hz stimulation, in satisfying agreement with the morphology. At lower stimulus frequencies, however, the model revealed that the estimated RRP size is smaller, suggesting that not all AZs participate in release at low stimulation frequencies.

Vesicular ATPase Inserted into the Plasma Membrane of Motor Terminals by Exocytosis Alkalinizes Cytosolic pH and Facilitates Endocytosis

Key components of vesicular neurotransmitter release, such as Ca(2+) influx and membrane recycling, are affected by cytosolic pH. We measured the pH-sensitive fluorescence of Yellow Fluorescent Protein transgenically expressed in mouse motor nerve terminals, and report that Ca(2+) influx elicited by action potential trains (12.5-100 Hz) evokes a biphasic pH change: a brief acidification (∼ 13 nM average peak increase in [H(+)]), followed by a prolonged alkalinization (∼ 30 nM peak decrease in [H(+)]) that outlasts the stimulation train. The alkalinization is selectively eliminated by blocking vesicular exocytosis with botulinum neurotoxins, and is prolonged by the endocytosis-inhibitor dynasore. Blocking H(+) pumping by vesicular H(+)-ATPase (with folimycin or bafilomycin) suppresses stimulation-induced alkalinization and reduces endocytotic uptake of FM1-43. These results suggest that H(+)-ATPase, known to transfer cytosolic H(+) into prefused vesicles, continues to extrude cytosolic H(+) after being exocytotically incorporated into the plasma membrane. The resulting cytosolic alkalinization may facilitate vesicular endocytosis.