Different Time Courses of Mono- and Bi-Liganded Bursts of Channel Openings of Adult nAChR Molecules Formed by the Reactions of Transmembrane Regions.

We recorded transmembrane currents through single nicotinic acetylcholine receptors (nAChRs) in cell-attached patches at high temporal resolutions from cultured and transiently transfected HEK 293 cells. Receptor activation was elicited by acetylcholine (ACh) or epibatidine (Ebd) at concentrations ranging from 0.01 to 100 µM, binding to one (R or R) or both extracellular ligand binding sites (R).

α-Conotoxin M1 (CTx) blocks αδ binding sites of adult nicotinic receptors while ACh binding at αε sites elicits only small and short quantal synaptic currents.

In 'embryonic' nicotinic receptors, low CTx concentrations are known to block only the αδ binding site, whereas binding of ACh at the αγ-site elicits short single channel openings and short bursts. In adult muscles the αγ- is replaced by the αε-site. Quantal EPSCs (qEPSCs) were elicited in adult muscles by depolarization pulses and recorded through a perfused macropatch electrode.

Agonists binding nicotinic receptors elicit specific channel-opening patterns at αγ and αδ sites.

'Embryonic' muscle-type nicotinic acetylcholine receptor channels (nAChRs) bind ligands at interfaces of α- and γ- or δ-subunits. αγ and αδ sites differ in affinity, but their contributions to opening the channel have remained elusive. We compared high-resolution patch clamp currents evoked by epibatidine (Ebd), carbamylcholine (CCh) and acetylcholine (ACh).

Depolarization amplitude and Ca2+ -inflow control the time course of quantal releases at murine motor nerve terminals.

In order to test whether the time courses of quantal releases after a depolarization pulse are affected by the depolarization amplitude, time courses were measured for small depolarization pulses that elicited close to threshold releases and for large depolarizations that elicited releases approaching saturation level. Diaphragms of young mice were excised and superfused with Bretag's solution at 18 degrees C. Synaptic currents were elicited and recorded through a perfused macropatch pipette.

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.

Glutamatergic autoinhibition of quantal release augments the early phase of releases after a depolarization pulse.

At the crayfish neuromuscular junction, glutamatergic autoinhibition of quantal excitatory postsynaptic current (qEPSC) release is mediated by a presynaptic DL-glutamate transporter and its associated Cl- conductance. I investigated whether it also affects the time course of release. qEPSCs were recorded with a perfused macroelectrode through which depolarization pulses and D- or L-glutamate could be applied to a terminal.

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.

Short openings in high resolution single channel recordings of mouse nicotinic receptors.

The temporal fine structure of single channel currents was studied to obtain information on how agonists open nicotinic acetylcholine receptor channels. Currents were recorded from mouse myoballs with quartz pipettes in the on-cell mode of the patch-clamp technique. With 62 kHz filter cut-off and root mean square (r.

Both d- and l-glutamate induce transporter-mediated presynaptic autoinhibition of transmitter release.

In crayfish motor nerve terminals l-glutamate (Glu) is the excitatory transmitter and low l-Glu concentrations exert autoinhibition by inhibiting release of Glu quanta from the terminals. This autoinhibition has been shown to be mediated by binding and transport of l-Glu by Glu transporters in the presynaptic membrane. Activated transporters open an associated Cl(-) channel and inhibit release [J.

A receptor for presynaptic glutamatergic autoinhibition is a glutamate transporter.

Monoquantal excitatory postsynaptic currents were recorded by means of a perfused macropatch electrode from 9 to 15 micro m stretches of crayfish neuromuscular junctions. The excitatory transmitter l-glutamate superfused to a terminal inhibits quantal release by activating autoreceptors [Parnas et al. (1996) Eur.

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).

Quantal endplate currents from newborn to adult mice and the switch from embryonic to adult channel type.

Quantal endplate currents (qEPCs) were recorded extracellularly by a macropatch electrode from excised diaphragms of mice. During the first 3 days after birth, the mean rise time t(r) was 0.5 ms (0.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Neuromuscular glutamatergic and GABAergic channels.

Our laboratory has worked extensively on glutamatergic and GABA-ergic channels, predominantly in crayfish, but also in locust, Drosophila and recently Ascaris. Channel currents were recorded in the different modes of the patch-clamp technique (Hamill et al., 1981).

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.