Postsynaptic depolarisation enhances transmitter release and causes the appearance of responses at "silent" synapses in rat hippocampus.

Recent data indicate that most "silent" synapses in the hippocampus are "presynaptically silent" due to low transmitter release rather than "postsynaptically silent" due to "latent" receptors of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid type (AMPARs). That synapses bearing only N-methyl-d-aspartate (NMDAR) receptors do exist is suggested by the decreased number of transmission failures during postsynaptic depolarisation and by the presence of NMDA-mediated excitatory postsynaptic currents (EPSCs) in synapses silent at rest. We tested whether these effects could be due to potentiated transmitter release at depolarised postsynaptic potentials rather than removal of Mg(2+) block from NMDARs. Using whole-cell recordings of minimal EPSCs from CA1 and CA3 neurones of hippocampal slices we confirmed decreased incidence of failures at +40 mV as compared with -60 mV. This effect was associated with a gradual increase of EPSC amplitude after switching to +40 mV and with a decrease of paired-pulse facilitation. In initially silent synapses, potentiation of pharmacologically isolated AMPAR-mediated EPSCs was still observed at +40 mV and this persisted after stepping back to -60 mV. All above effects were blocked when the cell was dialysed with the Ca(2+) chelator BAPTA (20 mM). These observations are difficult to reconcile with the "latent AMPAR" hypothesis and suggest an alternative explanation, namely that the reduction in failure rates at positive potentials is due to potentiation of transmitter release following Ca(2+) influx through NMDARs. Our results suggest that silent synapses can be mainly "presynaptically" rather than "postsynaptically silent" and thus increased transmitter release rather than insertion of AMPARs is a major mechanism of early long-term potentiation maintenance.

Dendritic spatial flicker of local membrane potential due to channel noise and probabilistic firing of hippocampal neurons in culture.

Whole-cell recordings and imaging of dissociated hippocampal neurons stained with voltage sensitive dye provide a new microscopic picture of neuronal excitation. This is the first attempt to combine imaging of active channel clusters on the geometry of live neurons and a theoretical approach. During single somatic action potentials and the back-invasion into the neurites, local mean potentials are generated at sites of active channel clusters which are unevenly distributed in the neuronal membrane. Similar mean membrane potentials are observed in the neurites and at the soma. Identical action potentials produce different spatial patterns of mean membrane potentials from trial to trial. This spatial variability is explained by the stochastic behavior of the channels in the clusters. When hippocampal neurons are excited by synaptic inputs, their evoked responses are probabilistic and generate variable spatial patterns of mean membrane potential trial after trial. Our stochastic model reproduces this random behavior by assuming that the voltage fluctuations generated by channel noise are added to the synaptic potentials reaching the soma. We demonstrate that the probability of action potential initiation depends on the strength of the synaptic input, the diameter of the dendrites and the relative positions of the channel clusters, of the synapse and of the soma.

Imaging stochastic spatial variability of active channel clusters during excitation of single neurons.

Topographical maps of membrane voltages were obtained during action potentials by imaging, at 1 microm resolution, live dissociated neurons stained with the voltage sensitive dye RH237. We demonstrate with a theoretical approach that the spatial patterns in the images result from the distribution of net positive charges condensed in the inner sites of the membrane where clusters of open ionic channels are located. We observed that, in our biological images, this spatial distribution of open channels varies randomly from trial to trial while the action potentials recorded by the microelectrode display similar amplitudes and time-courses. The random differences in size and intensity of the spatial patterns in the images are best evidenced when the time of observation coincides with the duration of single action potentials. This spatial variability is explained by the fact that only part of the channel population generates an action potential and that different channels open in turn in different trials due to their stochastic operation. Such spatial flicker modifies the direction of lateral current along the neuronal membrane and may have important consequences on the intrinsic processing capabilities of the neuron.

Effect of voltage drop within the synaptic cleft on the current and voltage generated at a single synapse.

In a model of a single synapse with a circular contact zone and a single concentric zone containing receptor-gated channels, we studied the dependence of the synaptic current on the synaptic cleft width and on the relative size of the receptor zone. During synaptic excitation, the extracellular current entered the cleft and flowed into the postsynaptic cell through receptor channels distributed homogeneously over the receptor zone. The membrane potential and channel currents were smaller toward the cleft center if compared to the cleft edges. This radial gradient was due to the voltage drop produced by the synaptic current on the cleft resistance. The total synaptic current conducted by the same number of open channels was sensitive to changes in the receptor zone radius and the cleft width. We conclude that synaptic geometry may affect synaptic currents by defining the volume resistor of the cleft. The in-series connection of the resistances of the intracleft medium and the receptor channels plays the role of the synaptic voltage divider. This voltage dividing effect should be taken into account when the conductance of single channels or synaptic contacts is estimated from experimental measurements of voltage-current relationships.

Electrodiffusion of synaptic receptors: a mechanism to modify synaptic efficacy?

We analysed physical forces that act on synaptic receptor-channels following the release of neurotransmitter. These forces are: 1) electrostatic interaction between receptors, 2) stochastic Brownian diffusion in the membrane, 3) transient electric field force generated by currents through open channels, 4) viscous drag force elicited by the flowing molecules and 5) strong in-membrane friction. By considering alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) type receptors, we show that, depending on the size and electrophoretic charge of the extracellular receptor domain, release of an excitatory neurotransmitter (glutamate) can induce receptor clustering towards the release site on a fast time scale (8-100 ms). This clustering progresses whenever repetitive synaptic activation exceeds a critical frequency (20-100 s(-1), depending on the currents through individual channels). As a result, a higher proportion of the receptors is exposed to higher glutamate levels. This should increase by 50-100% the peak synaptic current induced by the same amount of released neurotransmitter. In order for this mechanism to contribute to long-term changes of synaptic efficacy, we consider the possibility that the in-membrane motility of the AMPA receptors is transiently increased during synaptic activity, e. g., through the breakage of receptor anchors in the postsynaptic membrane due to activation of N-methyl-d-aspartic acid receptors.

Predicted acetylcholine layer around embryonic motoneurones.

The secretion of acetylcholine (ACh) from cultures of dissociated rat purified embryonic motoneurones into the medium was measured at different times using a chemiluminescent assay. From these results it is proposed that a motoneurone is able to generate an extracellular ACh layer surrounding its membrane. This layer is expected to modulate the ACh concentration at the membrane in a range where ACh receptors can be sensitive, and thus to affect the response of the motoneurone. The influences of both membrane-bound and soluble-secreted acetylcholinesterase (AChE) were simulated and a local ACh membrane efflux was deduced. Since ACh usually exerts an inhibitory effect on neurite outgrowth, a possible autocrine influence of the ACh layer in motoneurone morphogenesis is discussed.

Glutamatergically induced pattern of Ca2+ driving potential as a mechanism of postsynaptic plasticity.

Simulation studies were performed in a model of neuronal dendrite with Na+ and K+ channels and with ionotropic and metabotropic glutamate receptors. The ionotropic receptors were either N-methyl-D-aspartate (NMDA)-sensitive, voltage-dependent, and permeable to Ca2+, Na+, and K+, or non-NMDA-sensitive, voltage-independent, and permeable to Na+ and K+. The metabotropic receptors provided a catalytic effect on Ca2+-induced Ca2+ release from intracellular stores. Local intracellular concentration [Ca2+]i in the cytoplasm was changed because of exchange with the stores, axial diffusion, and transmembrane inward passive and outward pump fluxes. Tonic activation of ionotropic and metabotropic receptors in a particular range of intensities triggered the formation of spatially periodic [Ca2+]i hot and cold bands arising from an initial uniform state. The period and width of the bands were smaller at higher levels of tonic NMDA activation and higher metabotropically controlled rates of Ca2+-induced Ca2+ release. The bandwidths also depended on the dendrite diameter, the specific membrane, and cytoplasm resistivity. This activity-induced pattern led to long-term, spatially inhomogeneous change in local excitatory postsynaptic potentials (EPSPs) of NMDA synapses phasically activated with the same presynaptic intensity. The phasic EPSPs were potentiated if the synapse occurred in the hot band.

Spatial electrical patterns in simulated neuronal dendrites.

Steady state longitudinal distributions of (a) the density of channels conducting an inward transmembrane current of cations, (b) the submembrane concentrations of these cations, and (c) the resting membrane potential, were investigated in a phenomenological model of a cylinder-shaped dendritic process of the neuron. It was found that spatially non-uniform patterns of these distributions occur only if one of the following conditions held (i) an increase in the intracellular concentration of cations conducting an inward passive transmembrane current amplified the active efflux of those cations by the pump and attenuated their passive influx through the voltage dependent channels, with amplification of the efflux lower than attenuation of the influx; (ii) molecules of mobile channels bore a negative electrophoretic charge exposed to the intracellular space and were subject to lateral electrodiffusion in the membrane; (iii) the cations induced a further release of cations from intracellular stores. Numerical simulation studies of the membrane with Na and K channels and Na/K pumps with conditions (i) and (ii) have demonstrated the possibility of the creation of inhomogeneous patterns in the neurites. These inhomogeneous patterns are dissipative structures (DSs), and they can be spatially periodic.