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H Parnas

Publications and source records attributed to H Parnas.

At least 19 recordsLinked to original sources
PubMed2006

Role of NSF in neurotransmitter release: a peptide microinjection study at the crayfish neuromuscular junction.

Peptides that inhibit the SNAP-stimulated ATPase activity of N-ethylmaleimide-sensitive fusion protein (NSF-2, NSF-3) were injected intra-axonally to study the role of this protein in the release of glutamate at the crayfish neuromuscular junction. Macropatch recording was used to establish the quantal content and to construct synaptic delay histograms. NSF-2 or NSF-3 injection reduced the quantal content, evoked by either direct depolarization of a single release bouton or by axonal action potentials, on average by 66 +/- 12% (mean +/- SD; n = 32), but had no effect on the time course of release. NSF-2 had no effect on the amplitude or shape of the presynaptic action potential nor on the excitatory nerve terminal current. Neither NSF-2 nor NSF-3 affected the shape or amplitude of single quantal currents. Injection of a peptide with the same composition as NSF-2, but with a scrambled amino acid sequence, failed to alter the quantal content. We conclude that, at the crayfish neuromuscular junction, NSF-dependent reactions regulate quantal content without contributing to the presynaptic mechanisms that control the time course of release.

Animals↗
PubMed2005

Depolarization initiates phasic acetylcholine release by relief of a tonic block imposed by presynaptic M2 muscarinic receptors.

The role of presynaptic muscarinic autoreceptors in the initiation of phasic acetylcholine (ACh) release at frog and mouse neuromuscular junctions was studied by measuring the dependency of the amount (m) of ACh release on the level of presynaptic depolarization. Addition of methoctramine (a blocker of M2 muscarinic receptors), or of acetylcholinesterase (AChE), increased release in a voltage-dependent manner; enhancement of release declined as the depolarizing pulse amplitude increased. In frogs and wild-type mice the slope of log m/log pulse amplitude (PA) was reduced from about 7 in the control to about 4 in the presence of methoctramine or AChE. In M2 muscarinic receptor knockout mice, the slope of log m/log PA was much smaller (about 4) and was not further reduced by addition of either methoctramine or AChE. The effect of a brief (0.1 ms), but strong (-1.2 microA) depolarizing prepulse on the dependency of m on PA was also studied. The depolarizing prepulse had effects similar to those of methoctramine and AChE. In particular, it enhanced release of test pulses in a voltage-dependent manner and reduced the slope of log m/log PA from about 7 to about 4. Methoctramine + AChE occluded the prepulse effects. In knockout mice, the depolarizing prepulse had no effects. The cumulative results suggest that initiation of phasic ACh release is achieved by depolarization-mediated relief of a tonic block imposed by presynaptic M2 muscarinic receptors.

Acetylcholine↗
PubMed2003

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.

Acetylcholine↗
PubMed2001

Presynaptic M(2) muscarinic receptors are involved in controlling the kinetics of ACh release at the frog neuromuscular junction.

1. Macropatch recording was used to study release of acetylcholine in the frog neuromuscular junction evoked by either direct local depolarization or by an action potential. 2. The quantal content was established by directly counting the released quanta. The time course of release was obtained by constructing synaptic delay histograms. 3. Perfusion of the neuromuscular junction with methoctramine, a selective M(2)/M(4) muscarinic antagonist, increased the quantal content and slowed the exponential decay of the synaptic delay histograms. Addition of the agonist muscarine reversed these effects. 4. Addition of acetylcholinesterase prolonged the decay of the delay histogram, and muscarine reversed this effect. 5. Methoctramine slowed the rise time of the postsynaptic current produced by axon stimulation without affecting either the excitatory nerve terminal current or the presynaptic Ca(2+) current. 6. These results show that presynaptic M(2) muscarinic receptors are involved in the process which terminates evoked ACh release.

Acetylcholine↗
PubMed2001

One-vesicle hypothesis for neurotransmitter release: a possible molecular mechanism.

Neurotransmitter-containing vesicles are clustered in release sites. Although a given site can contain tens of vesicles, there is evidence that under a wide range of conditions, following an action potential, rarely is more than one vesicle released from each site. Such findings led to the one vesicle hypothesis, for which this paper suggests a molecular mechanism. The release of a vesicle from a site provides a transient high concentration of transmitter in that site. It is proposed here that the local high transmitter concentration interrupts further vesicle releases from the same release site. The suggested mechanism for this 'release interruption' is based on a theory of release control by the authors wherein inhibitory transmitter autoreceptors play a central role. (That transmitter binding to these autoreceptors can inhibit release on a fast time scale has recently been shown experimentally.) A detailed kinetic scheme is presented for the proposed mechanism. Stochastic simulations of this scheme demonstrate how the mechanism accounts for the one vesicle hypothesis. In agreement with recent experiments, the simulations also show that changes in conditions that affect the release process can cause frequent release of more than one vesicle per site.

Acetylcholine↗
PubMed2000

Theory for the feedback inhibition of fast release of neurotransmitter.

Autoinhibition of neurotransmitter release occurs via binding of transmitter to appropriate receptors. Experiments have provided evidence suggesting that the control of neurotransmitter release in fast systems is mediated by these inhibitory autoreceptors. Earlier, the authors formulated and analysed a mathematical model for a theory of release control in which these autoreceptors played a key role. The key experimental findings on which the release-control theory is based are: (i) the inhibitory autoreceptor has high affinity for transmitter under rest potential and shifts to low affinity upon depolarization; (ii) the bound (with transmitter) autoreceptor associates with exocytotic machinery Ex and thereby blocks it, preventing release of neurotransmitter. Release commences when depolarization shifts the autoreceptor to a low-affinity state and thereby frees Ex from its association with the autoreceptors. Here we extend the model that describes control of release so that it also accounts for release autoinhibition. We propose that inhibition is achieved because addition of transmitter, above its rest level, causes transition of the complex of autoreceptor and Ex to a state of stronger association. Relief of Ex from this state requires higher depolarization than from the weakly associated complex. In contrast to the weakly associated complex that only requires binding of transmitter to the autoreceptor to be formed, the transition to the strongly associated complex is induced by a second messenger, which is produced as a result of the receptor binding to transmitter. The theory explains the following experimental results (among others): for inhibition via transmitter or its agonists, the magnitude of inhibition decreases with depolarization; a plot of inhibition as a function of the concentration of muscarine (an acetylcholine agonist) yields an S-shaped curve that shifts to the right for higher depolarizations; the time course of release does not change when transmitter is added; the time course of release also does not change when transmitter antagonists are added, although quantal content increases; however, addition of acetylcholine esterase (an enzyme that hydrolyses acetylcholine) prolongs release.

Acetylcholine↗
PubMed2000

Evaluation of the number of agonist molecules needed to activate a ligand-gated channel from the current rising phase.

We propose a new method for calculating the number of agonist binding sites (n) in ligand-gated receptor channels from the initial phase of the current. This method is based on the fact that the relation between the current (I) and its first-time derivative (I') at the beginning of the current reflects the number of transitions that lead to channel opening. We show that, for constant agonist concentration, the above relationship at t --> 0 provides the number of steps leading to channel opening. When the agonist concentration is not constant but rather increases linearly with time, the corresponding value can be obtained using a slightly modified procedure. The analytical results were compared with computer simulations and a good match between the two was obtained. The theoretical procedure was then validated experimentally using the nicotinic receptor, because, for this receptor, the number of binding sites is well established. Indeed, the expected number of two binding sites was obtained. The method was then tested for the quisqualate-type glutamate receptor channel from the opener muscle of crayfish. The number of this receptor's binding sites is not fully resolved. Our results suggest that, for this glutamate receptor as well, two binding sites must be occupied to open the channel.

Animals↗
PubMed2000

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.

Animals↗
PubMed2000

On the mechanism of desensitization in quisqualate-type glutamate channels.

Desensitization of crayfish glutamate channels was studied in outside-out patches employing an improved fast drug-application technique. Low concentrations of glutamate produced substantial desensitization without correlation with the detected number of open channels. The desensitization time constant (tau(D)) was found to be independent of glutamate concentration (0.3-20 mM). These results suggest that in addition to desensitization from a state of fully liganded channels, a substantial fraction of desensitization occurs also from channels in a partly-liganded state. A kinetic model was developed. The model accounts for the multifaceted behavior of desensitization as well as for resensitization.

Animals↗
PubMed1999

Depolarization affects the binding properties of muscarinic acetylcholine receptors and their interaction with proteins of the exocytic apparatus.

Membrane depolarization is the signal that triggers release of neurotransmitter from nerve terminals. As a result of depolarization, voltage-dependent Ca(2+) channels open, level of intracellular Ca(2+) increases. and release of neurotransmitter commences. Previous study had shown that in rat brain synaptosomes, muscarinic acetylcholine (ACh) receptors (mAChRs) interact with soluble NSF attachment protein receptor proteins of the exocytic machinery in a voltage-dependent manner. It was suggested that this interaction might control the rapid, synchronous release of acetylcholine. The present study investigates the mechanism for such a voltage-dependent interaction. Here we show that depolarization shifts mAChRs, specifically the m2 receptor subtype, to a low affinity state toward its agonists. At resting potential, mAChRs are in a high affinity state (K(d) of approximately 20 nM) and they shift to a low affinity state (K(d) of tens of microM) upon membrane depolarization. In addition, interaction between m2 receptor subtype and the exocytic machinery increases with receptor occupancy. Both phenomena are independent of Ca(2+) influx. We propose that these results may explain control of ACh release from nerve terminals. At resting potential the exocytic machinery is clamped due to its interaction with the occupied mAChR and depolarization relieves this interaction. This, together with Ca(2+) influx, enables release of ACh to commence.

Acetylcholine↗
PubMed1999

Presynaptic effects of muscarine on ACh release at the frog neuromuscular junction.

1. Presynaptic effects of muscarine on neurotransmitter release were studied at the frog neuromuscular junction, using focal depolarization of the presynaptic terminal to different levels. 2. Muscarine (10 microM) had a dual effect on ACh release: concomitant inhibition and enhancement of release at the same patch of presynaptic membrane. 3. These two effects were maximal at low depolarizing pulses and diminished as depolarization increased. 4. At low depolarizing pulses, atropine (1 microM) enhanced release, suggesting that ACh in the synaptic cleft causes a net tonic inhibition of ACh release. 5. In the presence of the M2 antagonist methoctramine (1 microM), muscarine (10 microM) enhanced ACh release. 6. In the presence of the M1 antagonist pirenzepine (10 microM), muscarine (10 microM) produced stronger inhibition. 7. These results show that the M2 receptor is responsible for inhibition of ACh release, while the M1 receptor is responsible for its enhancement. 8. The inhibitory effect of muscarine did not depend on extracellular [Ca2+]. Enhancement of release was abolished at low extracellular [Ca2+]. 9. The muscarine inhibitory effect was not associated with a reduction of Ca2+ current, while release enhancement was associated with an increase of Ca2+ current.

Acetylcholine↗
PubMed1999

Right-left discrimination in a biologically oriented model of the cockroach escape system.

We present a biologically oriented model that accounts for left-right discrimination in the cockroach's escape behavior. The model includes the main groups of neurons found to be involved in the escape response. Each one of the included neurons is described by the actual processes taking place in an individual neuron (formation of an action potential, transmitter release, conductance changes, etc.). Furthermore, realistic chemical synapses (excitatory or inhibitory and able to undergo different types of modulation) connect the various neurons. With this model, we ere able to achieve, for a wide range of inputs representing different wind directions, behavior which resembles that found experimentally. The model indicates that several synaptic properties, in particular postsynaptic inhibition and presynaptic facilitation, play a key role in the discrimination of wind direction.

Animals↗
PubMed1999

Simultaneous measurement of evoked release and [Ca2+]i in a crayfish release bouton reveals high affinity of release to Ca2+.

The opener neuromuscular junction of crayfish was used to determine the affinity of the putative Ca2+ receptor(s) responsible for evoked release. Evoked, asynchronous release, and steady-state intracellular Ca2+ concentration, [Ca2+]ss, were measured concomitantly in single release boutons. It was found that, as expected, asynchronous release is highly correlated with [Ca2+]ss. Surprisingly, evoked release was also found to be highly correlated with [Ca2+]ss. The quantal content (m) and the rate of asynchronous release (S) showed sigmoidal dependence on [Ca2+]ss. The slope log m/log [Ca2+]ss varied between 1.6 and 3.3; the higher slope observed at the lower [Ca2+]o. The slope log S/log [Ca2+]ss varied between 3 and 4 and was independent of [Ca2+]o. These results are consistent with the assumption that evoked release is controlled by the sum of [Ca2+]ss and the local elevation of Ca2+ concentration near the release sites resulting from Ca2+ influx through voltage-gated Ca2+ channels (Y). On the basis of the above, we were able to estimate Y. We found Y to be significantly <10 microM even for [Ca2+]o = 13.5 mM. The dissociation constant (Kd) of the Ca2+ receptor(s) associated with evoked release was calculated to be in the range of 4-5 microM. This value of Kd is similar to that found previously for asynchronous release.

Animals↗
PubMed1999

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 </=100 ms with these blocker concentrations. The blocking effects of Phys and Proc on the qEPC are due to binding to the open channel conformation. A method is described to extract the rate constants of binding (bp) from the sums 1/taus + 1/taul, and the rates of unbinding (b-p) from tau0. taus-1. taul-1 (tau0 is the decay time constant of the control EPC). For Phys and Proc bp of 1.3 and 5. 10(6) M-1 s-1 and b-p of 176 and 350 s-1, respectively, were found. Using these rate constants and a reaction scheme for the nicotinic receptor together with the respective rate constants determined before, we could model the experimental results satisfactorily.

Acetylcholine↗
PubMed1999

Partial uncoupling of neurotransmitter release from [Ca2+]i by membrane hyperpolarization.

The dependence of evoked and asynchronous release on intracellular calcium ([Ca2+]i) and presynaptic membrane potential was examined in single-release boutons of the crayfish opener neuromuscular junction. When a single bouton was depolarized by a train of pulses, [Ca2+]i increased to different levels according to the frequency of stimulation. Concomitant measurements of evoked release and asynchronous release, from the same bouton, showed that both increased in a sigmoidal manner as a function of [Ca2+]i. When each of the depolarizing pulses was immediately followed by a hyperpolarizing pulse, [Ca2+]i was elevated to a lesser degree than in the control experiments, and the rate of asynchronous release and the quantal content were reduced; most importantly, evoked quantal release terminated sooner. The diminution of neurotransmitter release by the hyperpolarizing postpulse (HPP) could not be entirely accounted for by the reduction in [Ca2+]i. The experimental results are consistent with the hypothesis that the HPP reduces the sensitivity of the release machinery to [Ca2+]i, thereby not only reducing the quantal content but also terminating the quantal release process sooner.

Animals↗
PubMed1999

Geometry of dendritic spines affects calcium dynamics in hippocampal neurons: theory and experiments.

The role of dendritic spine morphology in the regulation of the spatiotemporal distribution of free intracellular calcium concentration ([Ca2+]i) was examined in a unique axial-symmetrical model that focuses on spine-dendrite interactions, and the simulations of the model were compared with the behavior of real dendritic spines in cultured hippocampal neurons. A set of nonlinear differential equations describes the behavior of a spherical dendritic spine head, linked to a dendrite via a cylindrical spine neck. Mechanisms for handling of calcium (including internal stores, buffers, and efflux pathways) are placed in both the dendrites and spines. In response to a calcium surge, the magnitude and time course of the response in both the spine and the parent dendrite vary as a function of the length of the spine neck such that a short neck increases the magnitude of the response in the dendrite and speeds up the recovery in the spine head. The generality of the model, originally constructed for a case of release of calcium from stores, was tested in simulations of fast calcium influx through membrane channels and verified the impact of spine neck on calcium dynamics. Spatiotemporal distributions of [Ca2+]i, measured in individual dendritic spines of cultured hippocampal neurons injected with Calcium Green-1, were monitored with a confocal laser scanning microscope. Line scans of spines and dendrites at a <1-ms time resolution reveal simultaneous transient rises in [Ca2+]i in spines and their parent dendrites after application of caffeine or during spontaneous calcium transients associated with synaptic or action potential discharges. The magnitude of responses in the individual compartments, spine-dendrite disparity, and the temporal distribution of [Ca2+]i were different for spines with short and long necks, with the latter being more independent of the dendrite, in agreement with prediction of the model.

Animals↗
PubMed1998

Depolarization increases the single-channel conductance and the open probability of crayfish glutamate channels.

We have studied the voltage sensitivity of glutamate receptors in outside-out patches taken from crayfish muscles. We found that single-channel conductance, measured directly at the single-channel level, increases as depolarization rises. At holding potentials from -90 mV to approximately 20 mV, the conductance is 109 pS. At holding potentials positive to 20 mV, the conductance is 213 pS. This increase in single-channel conductance was also observed in cell-attached patches. In addition, desensitization, rise time, and the dose-response curve were all affected by depolarization. To further clarify these multifaceted effects, we evaluated the kinetic properties of single-channel activity recorded from cell-attached patches in hyperpolarization (membrane potential around -75 mV) and depolarization (membrane potential approximately 105 mV). We found that the glutamate dissociation rate constant (k_) was affected most significantly by membrane potential; it declined 6.5-fold under depolarization. The rate constant of channel closing (k(c)) was also significantly affected; it declined 1.8-fold. The rate constant of channel opening (k(o)) declined only 1.2-fold. The possible physiological significance of the depolarization-mediated changes in the above rate constants is discussed.

Animals↗