The time course of transmitter release in mouse motor nerve terminals is differentially affected by activation of muscarinic M1 or M2 receptors.

At endplates of mouse diaphragms the effects of activation of presynaptic muscarinic M1 and M2 autoreceptors on the time courses of monoquantal releases have been investigated at 20 degrees C. Quantal excitatory postsynaptic currents (qEPSCs) were elicited and recorded with a perfused macropatch electrode, through which control- and drug-containing solutions were applied to 10 microm phi regions of a neuromuscular junction. M2 receptors were activated with muscarine, while the M1 receptors were blocked by pirenzepine. M2 activation presented a slight, but highly significant augmentation of early releases. Analogously, M1 receptors were activated with muscarine, while M2 receptors were blocked by methoctramine. M1 activation elicited a highly significant small shift of the time course of release towards longer delays. In controls, the number of late releases decayed with a time constant of 0.3 ms. This time constant did not change appreciably when methoctramine or methoctramine + muscarine were applied. However, methoctramine + muscarine reduced the amplitude of qEPSCs and shortened their decay by a partial block of postsynaptic channels. Double blocks with pirenzepine + methoctramine allowed no presynaptic effect of muscarine, showing that the blocker concentrations were sufficient. Neither the addition of methoctramine to pirenzepine, nor the further addition of muscarine changed the time constant of decay of the number of late releases. The results are very similar to that of autoreceptor activations in the glutamatergic crayfish synapse: activation of inhibitory receptors augmented early releases, and that of facilitatory receptors depressed early releases [J. Dudel (2006a) Eur. J. Neurosci., 23, 2695-2700], which may suggest a general presynaptic mechanism.

Glutamatergic chloride currents associated to glutamate transport?

Especially in arthropod glutamatergic synaptic systems, microM l-glutamate (Glu) concentrations often elicit Cl- currents, in addition to the excitatory cationic currents that are triggered by much higher Glu concentrations. In crayfish, Ibotenate (Ibo) is a specific agonist of the Glu-ergic Cl- currents. Application of Glu to Glu-transporters opens associated Cl- currents that inhibit quantal release presynaptically and by occupying the transporter prevents removal of released Glu. The latter prolongs the decay of postsynaptic EPSCs. It was tested whether the Ibo-elicited Cl- currents show the same pre- and post-synaptic effects as the transporter elicited ones, suggesting that also this current component arises through transporter activation. Indeed, Ibo applied to single synaptic junctions produced inhibition of quantal release and prolongation of EPSCs, very similar to the effects of Glu. It seems probable, therefore, that at least in crayfish Glu-ergic Cl- currents are generated by activation of transporters. Since generally such transporters are located around Glu-ergic synapses, this is likely to be a general mechanism. The toxin Ivermectin also elicits Cl- currents. However, while Ivermectin inhibits release too, it does not prolong the decay of EPSCs and is probable to activate GABAergic channels.

Use of knockout mice reveals involvement of M2-muscarinic receptors in control of the kinetics of acetylcholine release.

We have previously suggested that presynaptic M(2)-muscarinic receptors (M(2)R) are involved in the control of the time course of evoked acetylcholine release in the frog neuromuscular junction. The availability of knockout mice lacking functional M(2)R (M(2)-KO) enabled us to address this issue in a more direct way. Using the phrenic diaphragm preparation, we show that in wild-type (WT) mice experimental manipulations known to affect Ca(2+) entry and removal, greatly affected the amount of acetylcholine released (quantal content). However, the time course of release remained unaltered under all these experimental treatments. On the other hand, in the M(2)-KO mice, similar experimental treatments affected both the quantal content and the time course of release. In general, a larger quantal content was accompanied by a longer duration of release. Similarly, the rise time of the postsynaptic current produced by axon stimulation was sensitive to changes in [Ca(2+)](o) or [Mg(2+)](o) in M(2)-KO mice but not in WT mice. Measurements of Ca(2+) currents revealed that the shorter rise time of the postsynaptic current seen in high [Mg(2+)](o) in M(2)-KO mice was not produced by a shorter wave of the presynaptic Ca(2+) current. These results support our earlier findings and provide direct evidence for the major role that presynaptic M(2)-muscarinic receptors play in the control of the time course of evoked acetylcholine release under physiological conditions.

Pertussis toxin does not affect the time course of quantal release in crayfish and mouse muscle, but has other post- and presynaptic effects, especially on adenosine autoreceptors.

While G-proteins are involved in the synaptic release machinery and also can mediate inhibition of presynaptic Ca2+ channels, we find that pertussis toxin (PTX) does not affect the amount and the time course of quantal release from motor nerve terminals on crayfish or mouse muscle. Monoquantal excitatory currents (qEPSCs) were recorded that were elicited by constant depolarisation pulses to a terminal by means of a perfused macro-patch electrode. Although presynaptic effects of PTX on output and time course of release of quanta were absent, postsynaptically the rise time of qEPCs was increased and their decay time constant reduced. Adenosine (Ad) is known to inhibit quantal release in vertebrate motor nerve terminals via PTX sensitive G-proteins, and Ad is generated during nicotinic synaptic transmission by breakdown of the co-transmitter adenosine triphosphate (ATP). As reported by others, we found in mouse muscle inhibition of quantal release after application of Ad, but in addition late facilitation. Both these effects of Ad were blocked when the muscle was pre-incubated with PTX.

Combined pre- and postsynaptic action of IgG antibodies in Miller Fisher syndrome.

BACKGROUND: Miller Fisher syndrome (MFS), a variant of the Guillain-Barré syndrome, is associated with the presence of neuromuscular blocking antibodies, some of which may be directed at the ganglioside GQ1b. MATERIALS AND METHODS: The authors investigated the in vitro effects of serum and purified immunoglobulin (Ig) G in a total of 11 patients with typical MFS during active disease, and in three of those patients after recovery. From one patient's serum, we prepared an IgG fraction enriched in anti-GQ1b antibodies by affinity chromatography. For combined pre- and postsynaptic analysis, endplate currents were recorded by a perfused macro-patch clamp electrode. Postsynaptic nicotinic acetylcholine receptor channels were investigated by an outside-out patch clamp technique in cultured mouse myotubes. RESULTS: AllMFS-sera depressed evoked quantal release and reduced the amplitude of postsynaptic currents. Five of the 11 sera were additionally examined by outside-out patch clamp analysis and caused a concentration-dependent and reversible decrease in acetylcholine-induced currents. The time course of activation and desensitization of nicotinic acetylcholine receptor channels was not altered by MFS-IgG. Nine patients (82 %) were positive for anti-GQ1b antibodies in ELISA and dot-blot. The enriched anti-GQ1b antibody fraction had a similar effect as whole serum. After recovery from MFS, blocking activity was lost and sera originally positive for anti-GQ1b antibodies became negative. CONCLUSION: Circulating IgG antibodies induce both pre- and postsynaptic blockade and may play a pathogenic role in acute MFS.

Activation of rat recombinant alpha(1)beta(2)gamma(2S) GABA(A) receptor by the insecticide ivermectin.

In the present study, the activation of rat recombinant alpha(1)beta(2)gamma(2S) gamma-aminobutyric acid (GABA)-ergic Cl(-) channel expressed in human embryonic kidney (HEK) 293 cells by ivermectin was investigated. Maximal activation of the channel occurred with GABA concentrations of 10 mM or 20 microM ivermectin both achieving about the same current amplitudes. With those saturating concentrations, the currents rose with GABA within 1 ms to the maximal values, whereas the rise time for ivermectin was about 500 times longer. In contrast to activation with GABA, no desensitisation in the presence of the agonist was observed with ivermectin. With both agonists, two different open states were detected. On simultaneous application of GABA and ivermectin the current amplitudes and the kinetics were determined by the agonist applied in the concentration eliciting the higher open probability. It is concluded that GABA and ivermectin activated the channel independently resulting in different kinetic properties.

Autoreceptors, membrane potential and the regulation of transmitter release.

It has been suggested that depolarization per se can control neurotransmitter release, in addition to its role in promoting Ca2+ influx. The 'Ca2+ hypothesis' has provided an essential framework for understanding how Ca2+ entry and accumulation in nerve terminals controls transmitter release. Yet, increases in intracellular Ca2+ levels alone cannot account for the initiation and termination of release; some additional mechanism is needed. Several experiments from various laboratories indicate that membrane potential has a decisive role in controlling this release. For example, depolarization causes release when Ca2+ entry is blocked and intracellular Ca2+ levels are held at an elevated level. The key molecules that link membrane potential with release control have not yet been identified: likely candidates are presynaptic autoreceptors and perhaps the Ca2+ channel itself.

Desensitization characteristics of rat recombinant GABA(A) receptors consisting of alpha1beta2gamma2S and alpha1beta2 subunits expressed in HEK293 cells.

Desensitization kinetics of rat recombinant typeA GABAergic receptors consisting of the subunits alpha1beta2gamma2S or alpha1beta2 was investigated on application of 10-0.001 mM GABA to whole cell patches using a piezo driven liquid filament switch for fast application and deapplication. At high GABA concentrations desensitization was triphasic showing increasing time constants and a decreasing extent of desensitization on lowering the GABA concentration. Below agonist concentrations of 1 mM for the trimeric receptor and 0.1 mM for the dimeric one desensitization was biphasic switching to monophasic kinetics at GABA concentrations < or = 0.01 mM for the alpha1beta2gamma2S-type and < or = 0.003 mM for the alpha1beta2-type, respectively. Comparison with former studies performed with GABAergic receptors consisting of different subunits revealed differences in the desensitization kinetics.

Quartz glass pipette puller operating with a regulated oxy-hydrogen burner.

Quartz glass electrodes are superior to conventional glass electrodes for low-noise recording. They have better electrical characteristics and hydrophobic surfaces which resist creeping of salt solutions. We used oxy-hydrogen heating with program-controlled gas pressure to melt quartz glass capillaries. Usually, the relative wall thickness (the quotient of the outer and inner diameters do/di) of capillaries is, at best, maintained up to the electrode tip. If tips with thicker walls can be produced, coating and other surface treatments can be avoided. We found that programmed heating periods without pull allowed an fivefold increase of do/di in the tip region. Since do/di is inversely proportional to input capacity, the recording noise was minimized and became insignificant relative to amplifier and holder noise. A sample patch-clamp recording is shown.

Block of quantal end-plate currents of mouse muscle by physostigmine and procaine.

of quantal end-plate currents of mouse muscle by physostigmine and procaine. Quantal endplate currents (qEPCs) were recorded from hemidiaphragms of mice by means of a macro-patch-clamp electrode. Excitation was blocked with tetrodotoxin, and quantal release was elicited by depolarizing pulses through the electrode. Physostigmine (Phys) or procaine (Proc) was applied to the recording site by perfusion of the electrode tip. Low concentrations of Phys increased the amplitude and prolonged the decay time constants of qEPCs from approximately 3 to approximately 10 ms, due to block of acetylcholine-esterase. With 20 microM to 2 mM Phys or Proc, the decay of qEPCs became biphasic, an initial short time constant taus decreasing to <1 ms with 1 mM Phys and to approximately 0.3 ms with 1 mM Proc. The long second time constant of the decay, taul, reached values of

Desensitization reduces amplitudes of quantal end-plate currents after a single preceding end-plate current in mouse muscle.

While in membrane patches nicotinic channels from end-plates desensitize with time constants of 10-100 ms and recover with time constants of 100-200 ms, doubts remain as to whether such rapid reactions are also found in intact neuromuscular junctions. Therefore, the desensitization effected by a single end-plate current (EPC) on monoquantal EPCs (qEPCs) was studied. Using a published reaction scheme for the adult end-plate receptor, the desensitizing effects of an EPC on a following one were simulated. Twenty milliseconds after an EPC, a subsequent qEPC was reduced to 90% of control, and to 70-80% if acetylcholine esterase (AChE) was blocked. EPCs were elicited and recorded through a macropatch electrode. Series of four EPCs (control qEPC, conditioning EPC, test qEPC 1 and 2) were repeated several thousand times and the amplitudes and decay time constants of the qEPCs were evaluated. A highly significant average depression of the qEPC to 96.4% of control was found 20 ms after the conditioning EPC; if the AChE was blocked the depression after 40 ms was to 76% of control. Under both conditions, the time constant of recovery from desensitization was below 100 ms. The time constant of decay of the qEPC, taudecay, tended to be slightly shortened during depression in the presence of AChE. If AChE was blocked, taudecay was lengthened to 130-140% of control after a preceding EPC, and this lengthening outlasted the depression of the EPC amplitude by a second. These changes in taudecay are not predicted by the channel reaction scheme, and possible additional modulatory effects are discussed.

Evoked quantal currents at neuromuscular junctions of wild type Drosophila larvae.

Evoked excitatory postsynaptic currents (EPSC) were recorded with an extracellular macropatch electrode from glutamatergic neuromuscular junctions of Drosophila larvae. At 20 degrees C quantal current amplitude was about -400 pA and the 10-90% rise time was slightly below 0.2 ms for the fastest rising events and on average 0.3+/-0.1 ms in our best recordings. The quantal currents often had 'shoulders' but decayed approximately monoexponentially from half amplitude. The time constant of the exponential fit varied with mean values ranging from 2.5 ms to 7.7 ms in 13 experiments and an average value of 4.4+/-1.6 ms. Comparison of these results with data obtained earlier with outside-out patches of larval muscle membrane (Heckmann, M. and Dudel, J., Biophys. J., 72 (1997) 2160-2169) leads us to conclude that glutamate has to reach a saturating peak concentration of at least 10 mM in the synaptic cleft to allow the observed short quantal current rise times. To account for the time course of the quantal current decay one has to assume that the glutamate concentration in the synaptic cleft remains in the millimolar range for more than a millisecond and that the time course of the decay of the quantal currents is in part due to desensitization of the postsynaptic receptor channels.

Neuromuscular blockade by IgG antibodies from patients with Guillain-Barré syndrome: a macro-patch-clamp study.

Guillain-Barré syndrome (GBS) is often associated with serum antibodies to glycoconjugates such as GM1 and GQ1b. The pathogenic role of these antibodies and other serum factors has not yet been clarified. We have investigated the effect of serum, plasma filtrate, and highly purified IgG and IgM from 10 patients with typical GBS on motor nerve terminals in the mouse hemidiaphragm. Quantal endplate currents were recorded by means of a perfused macro-patch-clamp electrode. The plasma filtrate of all GBS patients led to a 5- to 20-fold reduction of evoked quantal release within 7 to 15 minutes of continuous superfusion. In 4 patients, the amplitudes of single quanta were clearly reduced (by 10-66% of control values), indicating an additional postsynaptic action. Blocking effects could be reversed to a variable degree within 15 to 18 minutes after washout. Purified IgG was as effective as native serum, whereas a purified GBS IgM fraction did not block transmission. Sera from convalescent patients and IgG from healthy subjects were without blocking effect. The effects were complement independent and there was no link to the presence (in 6 patients) or absence (in 4 patients) of detectable antibodies to GM1 or GQ1b. In GBS, antibodies to an undetermined antigen depress the presynaptic transmitter release and, in some cases, the activation of postsynaptic channels. We suggest that weakness in the acute stage of GBS may be caused in part by circulating antibodies.

Functional and immunocytochemical identification of glutamate autoreceptors of an NMDA type in crayfish neuromuscular junction.

Functional and immunocytochemical identification of glutamate autoreceptors of an NMDA type in crayfish neuromuscular junction. J. Neurophysiol. 80: 2893-2899, 1998. N-Methyl--aspartate (NMDA) reduces release from crayfish excitatory nerve terminals. We show here that polyclonal and monoclonal antibodies raised against the mammalian postsynaptic NMDA receptor subunit 1 stain specifically the presynaptic membrane of release boutons of the crayfish neuromuscular junction. In crayfish ganglionic membranes, the polyclonal antibody recognizes a single protein band that is somewhat larger (by approximately 30 kD) than the molecular weight of the rat receptor. Moreover, the monoclonal (but not the polyclonal) antibody abolishes the physiological effect of NMDA on glutamate release. The monoclonal antibody did not prevent the presynaptic effects of glutamate, which also reduces release by activation of quisqualate presynaptic receptors. Only when 6-cyano-7-nitroquinoxatine-2,3,dione (CNQX) was added together with the monoclonal antibody was the presynaptic effect of glutamate blocked. These results show that presynaptic glutamate receptors of the crayfish NMDA type are involved in the regulation of neurotransmitter release in crayfish axon terminals. Although the crayfish receptor differs in its properties from the mammalian NMDA receptor, the two receptors retained some structural similarity.

Single channel currents at six microsecond resolution elicited by acetylcholine in mouse myoballs.

1. A patch-clamp set-up was optimized for low noise and high time resolution. An Axoclamp 200B amplifier was modified to incorporate a Teflon connector to the electrode. An electrode puller was equipped with a hydrogen-oxygen burner to produce quartz-glass pipettes with optimally 0.2 micron openings and 20 MOmega resistance. 2. The r.m.s. (root mean square) noise of sealed pipettes in the bath ranged from 3.6 fA with 100 Hz filter cut-off to 1.5 pA with 61 kHz filter cut-off. At these extremes currents of 17 fA and more than 3 ms, or 9 pA and more than 6 micros could be resolved with a negligible error rate. 3. The system was tested on mouse myoballs, recording 9-10 pA single channel currents on-cell at -200 mV polarization which were elicited by 0.1-5000 microM acetylcholine (ACh). 4. Distributions of open and closed times and of correlations of open times to the preceding closed time defined several open states: single openings with mean durations of 1.2 and 25 micros, from single-liganded receptors, and bursts of 10 ms mean duration containing on average 800 micros openings and 16 micros closings, from double liganded receptors. Above 0.1 mM ACh these openings are interrupted increasingly by on average 18 micros and 72 micros channel blocks by ACh.

Block by picrotoxin of a GABAergic chloride channel expressed on crayfish muscle after axotomy.

Outside-out patches containing a gamma-aminobutyric acid (GABA)-activated chloride channel expressed after axotomy on crayfish deep extensor abdominal muscle were excised. GABA and the blocker picrotoxin (PTX) were applied to the patches using a liquid filament switch to study the effects of picrotoxin on the GABA-elicited currents. Coapplication of GABA and PTX resulted in a reduction of the current amplitude compared with that elicited by the same GABA concentration alone. This reduction of the amplitude was dependent on both the GABA and PTX concentrations. The rise time of the current decreased after coapplication of GABA and PTX. Evaluation of the single channel currents and off-currents in the presence of GABA and PTX showed a dramatic shortening of the burst duration of the channel. The open time distributions were not altered, whereas in the closed time distributions a new closed time was apparent in presence of PTX. Preincubation with PTX prior to the GABA pulse resulted in an increase of the rise time. This effect was dependent on the PTX concentration only. Possible mechanisms are discussed to explain the effects of PTX and are implemented into the existing molecular reaction scheme of the channel.

Pre- and postsynaptic blockade of neuromuscular transmission by Miller-Fisher syndrome IgG at mouse motor nerve terminals.

Miller-Fisher syndrome, a variant of an acute inflammatory neuropathy is often associated with serum antibodies to the ganglioside GQ1b, but the pathogenic role of these antibodies and other serum factors is unclear. We here investigated the effect of highly purified immunoglobulin G (IgG) from patients with typical Miller-Fisher syndrome, recording quantal endplate currents by means of a perfused macro-patch-clamp electrode on hemidiaphragms of adult mice. The GQ1b-positive and the GQ1b-negative Miller-Fisher IgG as well as its monovalent Fab-fragments depressed evoked quantal release in a fast and fully reversible, concentration and voltage dependent manner. The time-course of quantal release was changed with the late releases becoming more frequent. The extent of depression of release followed a Michaelis-Menten kinetic and depended on the extracellular calcium concentration. In addition the amplitude of quanta was reduced postsynaptically. IgG and sera from healthy subjects had no effect. Our results indicate that in Miller-Fisher syndrome, IgG antibodies to an undetermined antigen depress the release process, most likely by interfering with the presynaptic Ca2+ inflow or by interacting with proteins of the exocytotic apparatus, and prevent the activation of postsynaptic channels. Antibodies thus seem to be one pathogenic factor for muscle weakness in Miller-Fisher syndrome and our findings may explain why muscle strength recovers rapidly after therapeutical plasmapheresis.

Modulatory effects of gamma-hydroxybutyric acid on a GABA(A) receptor from crayfish muscle.

The effects of gamma-hydroxybutyric acid (GHB) were evaluated with a gamma-aminobutyric acid (GABA) activated Cl- channel on crayfish deep extensor abdominal muscle. GABA and GHB were applied to outside-out patches using a fast application system. Application of GHB up to 10 mM did not result in detectable activation of the channel. After coapplication of GABA and GHB, a dose-dependent potentiation of the GABA-elicited current by GHB was observed. The maximal effect was obtained with 0.5-1 mM GHB, with which the amplitude was enhanced by about 50% with 0.4 or 1 mM GABA. Simultaneously with the potentiating effect, a decrease of the rise times was seen. Preapplication of GHB, prior to the GABA pulses, resulted in a reduction of the current amplitude elicited by GABA. This block was persistent throughout the application time of GABA. Therefore, two contrasting effects of GHB on this chloride channel, a potentiating one and a blocking one, seemed to occur simultaneously.

Changes in the ultrastructure of surviving distal segments of severed axons of the rock lobster.

Peripheral axons of lobsters can survive for many months after axotomy. We have investigated the structural and ultrastructural changes seen after axotomy using confocal microscopy and electron microscopy. While the proximal stump had a normal appearance, the distal part of the cut axon became lobulated, and glial cells penetrated the original glial tube (axon tube) in which the axon normally runs. The changes proceeded from the cut end towards the muscle. As time elapsed, the axon tube seemed to be filled with glial cells, but interposed small profiles of the original axon could be identified by injection of a fluorescent dye into the axon. The glial cells send cytoplasmic projections deep into folds of the axolemma, and nuclei were found at the end of these long processes. Proliferation of glial cells was also seen.