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N S Veselovsky

Publications and source records attributed to N S Veselovsky.

17 recordsLinked to original sources

Regulation of GABA release by depolarisation-evoked Ca2+ transients at a single hippocampal terminal.

We correlated dynamic changes in free cytosolic [Ca2+] ([Ca2+]i) within single presynaptic terminals of cultured hippocampal neurones with the postsynaptic GABA-mediated currents. The local changes in [Ca2+]i and evoked inhibitory postsynaptic currents (eIPSCs) were recorded simultaneously using Fura-2 fluorescence and whole-cell patch-clamp respectively. The Ca2+ signals and eIPSCs were evoked by direct extracellular electrical stimulation of a single presynaptic terminal by short depolarising pulses. The presynaptic Ca2+ transient was graded by varying the amplitude of extracellular stimulating pulses. The probability of the release event, P, estimated for each stimulation strength, reached a maximum (P=1) when the Ca2+ signal became maximal and remained at this level at higher stimulation strength, despite the subsequent decrease in the amplitude of the Ca2+ transient. A gradual, linear increase in stimulation amplitude (Vstim) resulted in a bell-shaped dependence of the averaged amplitudes of Ca2+ signals and corresponding averaged amplitudes of eIPSCs. Analysis of the eIPSC demonstrated that the decrease in both the mean eIPSC amplitude and the mean quantal content of release resulted from a reduction in the probability of multivesicular release, i.e. in the disappearance of failures and in the decrease of individual eIPSC amplitude. The Ca2+ signals of similar amplitude resulted in both random and determinate (non-random) neurotransmitter release. We conclude that depolarisation-induced elevation of [Ca2+]i within the terminal is necessary but not sufficient for activation of vesicular release of neurotransmitter.

Animals↗

Activation of ryanodine receptors influences the paired-pulse depression in cultured rat hippocampal neurons.

Role of intraterminal calcium stores in modulation of short-term plasticity of evoked inhibitory postsynaptic currents (IPSCs) was studied in synaptically connected cultured hippocampal neurons using patch-clamp technique in whole-cell configuration. Currents were induced by voltage stimulation which were applied externally to presynaptic fiber. Paired stimuli resulted in paired-pulse depression (n=18) or facilitation (n=7) of the second IPSC at interpulse intervals 150 and 500 ms. Calcium release from intracellular calcium stores was activated by local application of caffeine and ryanodine, ryanodine receptor agonists. One of the characteristics of short-term plasticity, the pair-pulsed ratio (ratio of amplitudes of second IPSC to first IPSC), decreased during addition of ryanodine (50 nM) from 0.79 +/- 0.02 to 0.71 +/- 0.04 (n=10). This change was observed only for cells that demonstrated pair-pulsed depression. We also studied the influence of caffeine and ryanodine on spontaneous currents. Attenuation of the mean amplitude to 0.71 +/- 0.06 and frequency to 0.42 +/- 0.08 (n=7) of spontaneous IPSCs was observed during application of caffeine (10 mM). Upon ryanodine application the mean amplitude did not change but frequency of spontaneous events decreased to 0.74 +/- 0.09 (n=12). The amplitudes of currents evoked by fast local application of gamma-aminobutyric acid (GABA 100 mM) were diminished in the presence of caffeine (10 mM) to 0.59 +/- 0.03 (n=5) and in the presence of ryanodine (50 nM) to 0.56 +/- 0.11 (n=7). Thus we conclude that endoplasmic reticular calcium stores are able to modulate synaptic transmission from both presynaptic (in assumption that short-term plasticity and spontaneous activity are believed to have presynaptic nature) and postsynaptic (since GABA-receptors are situated on postsynaptic cell) sides.

Animals↗

Quantal GABA release in hippocampal synapses: role of local Ca2+ dynamics within the single terminals.

Results of recent studies dedicated to the mechanisms of neurotransmission at a single inhibitory synaptic terminal in cultured neurones support the hypothesis that multiple quanta of neurotransmitter are released during excitation of inhibitory and excitatory central synapses. This is an important consideration as previous less direct measurements have suggested that a synapse can release no more than one quantum. Neurotransmitter release during long stimuli may occur at certain times with maximal probability, keeping the mean inter-release interval constant. This interval is not determined directly by vesicle depletion and moreover, each release event is independent of previous ones. The recent data also suggest that constant Ca(2+) influx is an important determinant of neurotransmitter release. It is speculated that the neurotransmitter release is regulated by a superposition of two processes: a continuous homogeneous process, (i.e. background Ca(2+) influx), and a periodic process that acts as a synchronizing factor of the release at definite moments.

Animals↗

Temporal regularity of neurotransmitter release at single terminal in cultured hippocampal neurons.

The whole-cell GABA-mediated inhibitory postsynaptic currents were studied using the patch-clamp technique on synaptically connected cultured hippocampal neurons. The stimulus-evoked inhibitory postsynaptic currents were recorded in the tetrodotoxin-containing solution in response to the low-amplitude long (10-20ms) extracellular depolarization of a single presynaptic terminal. During each depolarization the postsynaptic response in a form of several superimposed independent events was recorded. The amplitudes of these responses fluctuated randomly, irrespective of the number of the event. In all the investigated neurons the distributions of delays revealed regularly spaced multiple peaks. The number of peaks increased with the duration of stimulus. The distance between the peaks was on average 2.97+/-0.86ms (n=58). The mean intervals between successive releases were distributed exponentially indicating the independence of the release sites. Thus neurotransmitter release might occur with maximal probability at the most probable times irrespective of the presence or failure of the previous event. The increase in stimulating pulse amplitude led to a decrease in the number of clearly detectable peaks in distributions. The decrease in the number of peaks in the distribution of delays was not accompanied with a decrease in the distance between peaks within the range of reliable resolution of the peaks. The amplitude distribution also revealed regularly spaced multiple peaks. The absence of significant correlation between the amplitude of the first and the second event demonstrated the independence of the succeeding release on the preceding release during long stimulation. Results of statistical analysis of our experimental data supports the hypothesis of multiquantal neurotransmitter release in a single inhibitory hippocampal synapse. Neurotransmitter release during long stimulation may occur at certain times with maximal probability, keeping the mean inter-release interval constant. Thus the interval is not determined directly by the depletion of vesicles and the number of vesicles which may be released at the most probable time is random.

Animals↗

Possibility of multiquantal transmission at single inhibitory synapse in cultured rat hippocampal neurons.

Miniature, spontaneous and evoked inhibitory postsynaptic currents were studied using the whole-cell patch-clamp technique on synaptically connected cultured hippocampal neurons, at a holding potential of -75 mV. All experiments were done in tetrodotoxin-containing solution to exclude an action potential generation. Spontaneous miniature inhibitory postsynaptic currents were observed in Ca2+-free solution. The distribution of miniature inhibitory postsynaptic currents was skewed to larger current amplitudes and could be fitted reliably by one Gaussian with the mean at 10.0 +/- 1.2 pA (n = 7). Spontaneously occurring whole-cell spontaneous inhibitory postsynaptic currents were recorded in physiological solution (Ca2+ 2 mM). The average amplitude of spontaneously occurring currents depended on membrane potential and reversed at -18 +/- 5 mV (n = 5). The amplitude distribution of spontaneous inhibitory postsynaptic currents had one peak clearly detectable with the mean of 20.0 +/- 2.0 pA (n = 6) or 10.0 +/- 2.0 pA (n = 2). Inhibitory postsynaptic stimulus-evoked currents arose in responses to gradual activation of neurotransmitter release by direct extracellular electrical stimulation of a single presynaptic bouton by short depolarizing pulses. The current-voltage relation of the averaged amplitudes of evoked inhibitory postsynaptic currents was linear and reversed at potential predicted by the Nernst equation for corresponding intra- and extracellular Cl- concentrations. The time-course of decay of miniature, spontaneous and evoked inhibitory postsynaptic currents was fitted by a sum of two exponents and their time-constants were the same in the range of standard deviation. The stimulus-evoked inhibitory postsynaptic currents fluctuated with regard to the discrete aliquot values of their peak amplitudes in all the investigated synapses from a measurable minimum of about 8 pA to 200 pA. The evoked inhibitory postsynaptic currents were assumed as superimposition of statistically independent quantal events. Fitting amplitude histograms of evoked inhibitory postsynaptic currents with several Gaussian curves resulted in peaks that were equidistant with the mean space of 20 +/- 3 pA (n = 10), which was assumed as one quantum (quantum size) to construct the Poisson's distribution. A possibility of simultaneous multiquantal release at single inhibitory synapses of rat hippocampal neurons was discussed.

Algorithms↗

Voltage-operated potassium currents in the somatic membrane of rat dorsal root ganglion neurons: ontogenetic aspects.

Whole-cell transmembrane potassium currents were studied in somatic membrane of freshly isolated rat dorsal root ganglion neurons. We defined three types of potassium currents, which were separated on the basis of their different potential dependence of activation and sensitivity to external tetraethylammonium and 4-aminopyridine. The potential dependence of kinetic and steady-state properties of a fast inactivating potassium current, a slow inactivating potassium current and a non-inactivating delayed rectifier current were described by the Hodgkin-Huxley equations. A transient fast inactivating potassium current was activated at the most negative membrane potentials and was not reduced in the presence of 10 mM tetraethylammonium in the external solution. 4-Aminopyridine (2 mM) caused an 80% inhibition of this current. The activation of the fast inactivating potassium current was properly described by fitting a single exponent raised to the fourth power. The time constant of activation changed from 4 to 1 ms in the voltage range between -30 and +40 mV. The time constant of inactivation decreased from 35 to 15 ms over the same range of potentials. Parameters for the fit of a Boltzmann equation to mean values for steady-state activation were V1/2=-20mV, k=11.8mV, and for steady-state inactivation V1/2= -85 mV, k=-9.8 mV. A transient slow inactivating potassium current had an activation threshold between -40 and -30 mV. At 2 mM 4-aminopyridine, the depression of the slow potassium current was 55%. The extracellular application of 10 mM tetraethylammonium was less effective and evoked a 40% reduction. The activation of the slow inactivating potassium current was also described by a single exponential function raised to the fourth power. The time constant of activation decreased from 12 ms at a membrane potential of -10 mV to 4 ms at the potential of 60 mV. The inactivation of slow inactivating potassium current was described by two exponents. The time constant for the fast exponent ranged from 300 ms at -20 mV to 160 ms at +60 mV. The slower exponent was also potential dependent and its time constant ranged from approximately 2600 to 1600 ms over the same potentials. Parameters for the Boltzmann equation fittings to mean values were V1/2= -12.8 mV, k=13.4 mV and V1/2= -54.6 mV, k= -12 mV for steady-state activation and inactivation, respectively. A non-inactivating delayed rectifier potassium current was activated at the most positive membrane potentials. This non-inactivating current did not change in the presence of 4-aminopyridine. Extracellular tetraethylammonium (10 mM) caused a 70% reduction of this current. The activation of the non-inactivating potassium current was described by one exponent raised to the fourth power. The time constant for activation ranged from 85 ms at -5 mV to 30 ms at 45 mV. No time-dependent inactivation was observed during 15-s testing potentials in the voltage range between 10 and +60 mV. The activation behavior was characterized by V1/2=15.3 mV, k=12.5 mV. The densities of these potassium currents were studied for three groups of animals: one, five to six and 14-15 days of postnatal development. Fifty cells were examined in each age group. All three types of potassium currents were found in each investigated neuron. The mean densities of slow and fast inactivating potassium currents increased during ontogenetic development. The densities of non-inactivating delayed rectifier potassium current decreased in the first week of ontogenetic development and did not change thereafter.

4-Aminopyridine↗

Fast local superfusion technique.

A system for rapid, local superfusion of cultured neurones and their neurites with various different test drugs is elucidated. An area of down to 30 micron diameter was superfused with the aid of two micropipettes, one for delivering the test solution and the other for its removal. Active removal of solution within the deadspace of the delivery pipette guarantees, on the one hand, fast and flexible pressure control and, on the other, enables the quick exchange (<1 s) of multiple solutions. By increasing the pressure in the superfusion pipette, the laminar stream between the pipettes was forced down onto the cell layer. The change from bath to superfusion solutions, evaluated by liquid junction potential changes, occurs in the order of 1 ms.

Electrophysiology↗

Glutamate-produced long-term potentiation by selective challenge of presynaptic neurons in rat hippocampal cultures.

Excitatory postsynaptic long-term potentiation (LTP) was observed after restricted glutamate or metabotropic agonist application to somata and proximal neurites of identified presynaptic neurons. LTP-induction in this way entailed delays of minutes that correlated with lengths of afferent fibres. Potentiation failed to occur in cells pretreated with colchicine to degrade their microtubular transport matrix. The results suggest fast axonal transport to be a message carrier in the acquisition process of LTP.

Animals↗

Comparative analysis of ionic currents in the somatic membrane of embryonic and newborn rat sensory neurons.

Inward currents in the somatic membrane of dissociated rat dorsal root ganglion neurons have been studied in two groups of animals (17 days of embryonic development and the first day after birth) by suction pipette (whole-cell configuration) and voltage-clamp techniques. Altogether 157 neurons were examined. Four components in the inward currents have been identified: fast tetrodotoxin-sensitive (INaf) and slow tetrodotoxin insensitive (INas) sodium, low-(ICal) and high-threshold (ICah) calcium currents. The percentage of neurons demonstrating four types of inward currents INaf, INas, ICal, ICah increased from 21% in embryo to 61% in newborn. The percentage of neurons with INaf, INas and ICah increased from 4% in embryonic to 14% in the first day after birth. The percentage of cells with INaf, ICal and ICah (without tetrodotoxin-insensitive INas) decreased from 56 to 11% in embryo and newborn rats, respectively. A statistically significant linear correlation was found between the densities of INaf and ICah currents for both ages. A correlation also occurred between the densities of INas and ICah. A reciprocal relation between the densities of both types of calcium currents and the size of cell soma was found in the neurons with all four types of inward currents from newborn animals. A comparison of these data with previous study of inward currents during postnatal development indicates that the most dramatic changes in their distributions and mean densities takes place some time after the birth of the animals.

Animals↗

Pharmacological characterization of calcium currents and synaptic transmission between thalamic neurons in vitro.

We recorded from pairs of cultured, synaptically connected thalamic neurons. Evoked excitatory postsynaptic currents (EPSCs) reversed at +17 mV and were blocked reversibly by 1 mM kynurenic acid, a glutamate receptor antagonist. NMDA and non-NMDA receptors mediated excitatory post-synaptic responses, as shown by selective block of EPSC components with 50 microM (+/-)-2-amino-5-phosphonopentanoic acid and 10 microM 6,7-dinitroquinoxaline-2,3-dione, respectively. Inhibitory postsynaptic responses were evoked less frequently and were blocked by the GABAA receptor antagonist (-)-bicuculline methochloride. The pharmacological profiles of whole-cell calcium currents and evoked EPSCs were compared. With 50 microM cadmium chloride (Cd), whole-cell low voltage-activated (LVA) calcium currents were reduced in amplitude and high voltage-activated (HVA) calcium currents and excitatory synaptic transmission were completely blocked. This suggests that the residual calcium influx through LVA channels into the presynaptic terminal does not suffice to trigger transmitter release. A saturating concentration of omega-conotoxin GVIA (omega-CgTx) (2.5 microM) blocked one-third of whole-cell HVA calcium currents and evoked EPSCs. The dihydropyridine nifedipine (50 microM) reversibly reduced whole-cell HVA calcium currents in a voltage-dependent manner but not excitatory synaptic transmission. Cd and omega-CgTx did not alter amplitude distributions of miniature EPSCs, demonstrating that the inhibition of synaptic transmission was due to block of presynaptic calcium channels. We conclude that excitatory glutamatergic transmission in thalamic neurons in vitro was mediated mainly by HVA calcium currents, which were insensitive to omega-CgTx and nifedipine.

Animals↗

Ionic mechanisms of electrical excitability in rat sensory neurons during postnatal ontogenesis.

Inward currents in the somatic membrane of rat dorsal root ganglion neurons have been studied in three groups of animals (8-10, 45-50 and 90-100 days of postnatal development) by intracellular perfusion and voltage clamp techniques. Altogether, 228 neurons have been examined (76 in each age group). Four components have been identified in the inward current: fast tetrodotoxin-sensitive (IfNa) and slow tetrodotoxin-insensitive (IsNa) sodium, low threshold (IlCa) and high threshold (IhCa) calcium currents. The existence of certain types of inward currents in the somatic membrane allowed the heterogeneous population of all the investigated neurons to be divided into six homogeneous subpopulations. A significant decrease in the percentage of neurons was found, demonstrating four types of inward currents (IfNa, IsNa, IlCa, IhCa) during postnatal ontogenesis. The percentage of cells in the subpopulation showing only IfNa and IhCa increased from 26% in the first age group to 43% in the second and 62% in the third age groups. Correlations between densities of various types of inward currents were studied. A linear relationship was found between the densities of IhCa and IsNa in subpopulations of neurons with IsNa. An inverse dependence was observed between the densities of IfNa and IhCa in cells showing only two current components. In all cells studied a "washout" of IhCa was observed during intracellular dialysis with saline solutions. Recovery of calcium conductance produced by intracellular application of an cAMP-ATP-Mg2+ complex was different in neurons with various inward current combinations. Intracellular introduction of cAMP-ATP-Mg2+ failed to restore IhCa in most cells of the second and third age groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗

Two types of calcium channels in the somatic membrane of new-born rat dorsal root ganglion neurones.

Ca2+ inward currents evoked by membrane depolarization have been studied by the intracellular dialysis technique in the somatic membrane of isolated dorsal root ganglion neurones of new-born rats. In about 20% of the investigated cells a hump has been detected on the descending branch of the current-voltage curve, indicating the presence of two populations of Ca2+ channels differing in their potential-dependent characteristics. An initial less regular component of the Ca2+ current was activated at membrane potentials from -75 to -70 mV. Its amplitude reached 0.2-0.9 nA at 14.6 mM-extracellular Ca2+. The activation kinetics of this component could be approximated by the Hodgkin-Huxley equation using the square of the m variable. tau m varied in the range from 8 to 1 ms at potentials between -60 and -25 mV ('fast' Ca2+ current). The second component of the Ca2+ current was activated at membrane depolarizations to between -55 and -50 mV. It could be recorded in all cells investigated and reached a maximum value of 1-7 nA at the same extracellular Ca2+ concentration. This component decreased rapidly during cell dialysis with saline solutions. The decrease could be slowed down by cooling and accelerated by warming the extracellular solution. Intracellular introduction of 3',5'-cAMP together with ATP and Mg2+ not only prevented the decrease but often restored the maximal current amplitude to its initial level. The activation kinetics of this component could also be approximated by a square function, tau m being in the range 16-2.5 ms at membrane potentials between -20 and +3 mV ('slow' Ca2+ current). The fast Ca2+ current inactivated exponentially at sustained depolarizations in a potential-dependent manner, tau h varying from 76 to 35 ms at potentials between -50 and -30 mV. The inactivation of the slow Ca2+ current studied in double-pulse experiments was current-dependent and developed very slowly (time constant of several hundreds of milliseconds). It slowed down even more at low temperature or after substitution of Ba2+ for Ca2+ in the extracellular solution. Both currents could also be carried by Ba2+ and Sr2+, although the ion-selecting properties of the two types of channels showed quantitative differences. Specific blockers of Ca2+ channels (Co2+, Mn2+, Cd2+, Ni2+ or verapamil) exerted similar effects on them. The existence of metabolically dependent and metabolically independent Ca2+ channels in the neuronal membrane and their possible functional role are discussed.

Adenosine Triphosphate↗

Calcium channels in the somatic membrane of the rat dorsal root ganglion neurons, effect of cAMP.

Isolated rat dorsal root ganglion neurons have been perfused with potassium-free solutions containing cAMP, ATP and Mg2+ ions. In these conditions stable inward calcium currents can be recorded in the somatic membrane of all investigated cells. The kinetics of these currents can be approximated by a modified Hodgkin-Huxley equation using a square power of the m-variable; its inactivation is extremely slow. The corresponding channels pass Ba2+ ions about twice more effective than Ca2+.

Adenosine Triphosphate↗