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I Mody

Publications and source records attributed to I Mody.

At least 73 records · Page 4Linked to original sources
PubMed1993

Properties of NMDA receptor channels in neurons acutely isolated from epileptic (kindled) rats.

The hyperexcitability accompanying chronic epileptiform activity may result from long-term alterations of ligand- and voltage-gated channels. Previous studies have indicated that NMDA responses and other electrophysiological characteristics of dentate gyrus granule cells are profoundly altered following chronic epilepsy (kindling). We have now investigated channels activated by NMDA using whole-cell patch-clamp and cell-attached single-channel recordings in granule cells acutely isolated from control and epileptic (kindled) rats. In control neurons, the amplitude of whole-cell NMDA currents was not sensitive to the presence of an intracellular ATP regeneration system, whereas NMDA currents in kindled cells showed a great variability, with larger amplitudes consistently recorded in the presence of intracellular high-energy phosphates. The ratio of peak to steady-state NMDA current (desensitization) was comparable (approximately 51%) in control and kindled neurons. Single-channel conductance determined from fluctuation analysis of whole-cell NMDA currents ranged between 21 and 35 pS in control and between 17 and 37 pS in kindled cells. Whole-cell NMDA channel noise power spectra yielded a single normal distribution of long channel lifetimes (mean, 4.3 msec) in control neurons, and the sum of two normal distributions (means, 4.6 and 7.1 msec) in kindled cells. The voltage-dependent Mg2+ block of NMDA channels was altered following kindling. From curves fitted to voltage-ramp-evoked currents in the presence of NMDA, the calculated affinity for Mg2+ of kindled channels at 0 mV was lower (12 mM) than that of controls (1.7 mM). Cell-attached recordings in the absence of Mg2+ have substantiated the lack of effect of kindling on single-channel conductance (approximately 50 pS), and have demonstrated large increases in mean open times (from 1.26 msec in control to 2.05 msec in kindled), burst lengths (from 1.91 msec to 4.18 msec), and cluster lengths (from 9.11 msec to 20.86 msec) of NMDA channels in kindled neurons. In summary, kindling, an NMDA receptor-dependent form of activity-dependent neuronal plasticity induced in vivo, results in lasting modifications in the function of single NMDA receptor channels that can be studied in acutely dissociated neurons. Kindling-induced epilepsy predominantly affects the mean open time, burst, and cluster duration of NMDA channels, their sensitivity to intracellular high-energy phosphates, and their block by Mg2+, but not the desensitization or single-channel conductance. Such alterations may reflect a change in the molecular structure of NMDA channels and may underlie the maintenance of the epileptic state.

Adenosine Triphosphate↗
PubMed1992

Modulation of decay kinetics and frequency of GABAA receptor-mediated spontaneous inhibitory postsynaptic currents in hippocampal neurons.

Inhibitory postsynaptic currents mediated by spontaneous activation of GABAA receptors were studied using whole-cell voltage-clamp recordings in granule cells of the adult rat (postnatal day 60+) dentate gyrus in 400-microns-thick coronal half-brain slices maintained at 34-35 degrees C. The average amplitude of spontaneous inhibitory postsynaptic currents remained constant during a given recording period (i.e. no rundown was noted). The spontaneous currents had an average conductance between 200-400 pS, were mediated by Cl- flux through GABAA receptor/channels since they reversed at the Cl- equilibrium potential and were blocked by bicuculline or picrotoxin. Their mono-exponential decay time-constants (range: 4.2-7.2 ms) were prolonged by midazolam and pentobarbital in a dose-dependent manner. The effect of midazolam was reversed by the benzodiazepine receptor antagonist flumazenil (RO 15-1788) which, by itself, had no effect on the decay time-constant. The decay time-constant was also dependent on membrane voltage and on temperature. A 132-mV change in membrane potential produced an e-fold prolongation of the decay while the Q10 (between 22-37 degrees C) of the decay rate was 2.1. Within a given neuron, the frequency of spontaneous GABAergic events was remarkably constant over long time-periods, though the mean frequency among different cells showed large variability. Spontaneous miniature inhibitory postsynaptic currents also persisted under experimental conditions such as the presence of extracellular tetrodotoxin (1 microM), Cd2+ (200 microM) or lowered extracellular Ca2+/elevated Mg2+, which effectively abolished all stimulus-evoked GABAergic neurotransmission. The frequency of tetrodotoxin-resistant miniature events was increased by elevating extracellular K+ concentration and was diminished by the GABAB receptor agonist (-)baclofen only at a dose (50 microM) which was an order of magnitude larger than that required to depress stimulus-evoked responses. These findings are consistent with different mechanisms being responsible for the spontaneous and stimulus-evoked release of GABA from interneuron terminals and also identify pre- and postsynaptic modulatory factors of the endogenous, action-potential-independent, GABAergic neurotransmission as being important determinants of the excitability level of mammalian CNS neurons.

2-Amino-5-phosphonovalerate↗
PubMed1992

Differential activation of GABAA and GABAB receptors by spontaneously released transmitter.

1. Whole-cell patch-clamp techniques were used to record from dentate gyrus granule cells in adult rat brain slices when N-methyl-D-aspartate (NMDA) and non-NMDA type glutamate receptors were blocked by D-2-amino-5-phosphonovaleric acid (D-AP5) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), respectively. Spontaneous inhibitory postsynaptic currents (sIPSCs), each presumably due to vesicular release of gamma-aminobutyric acid (GABA), selectively activated GABAA-type receptors. None of the individual sIPSCs showed a slow-onset potassium current characteristic of GABAB receptor activation. 2. In contrast, stimulation in the molecular layer with a bipolar stimulating electrode or bath application of the convulsant drug 4-aminopyridine (4-AP, 10-30 microM) elicited fast GABAA IPSCs followed by slower outward currents that were sensitive to the selective GABAB antagonist CGP 35348 (0.1-1 mM) and that reversed polarity near the potassium equilibrium potential. 3. CGP 35348 (0.5-1 mM) or the GABAB agonist (-)baclofen (1 microM) had no significant effect on the frequency or average amplitude of sIPSCs. However, either bath application of (-)baclofen (1 microM) or a preceding conditioning stimulus caused large reductions in the amplitude of stimulus-evoked IPSCs, suggesting a strong GABAB-mediated presynaptic inhibition of stimulus-evoked GABA release. 4. We conclude that under normal conditions spontaneous transmitter release does not activate GABAB receptors in dentate gyrus slices. These findings are consistent with either of two general possibilities. Separate groups of interneurons with different basal firing rates may selectively form GABAA and GABAB synapses.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Membrane properties of dentate gyrus granule cells: comparison of sharp microelectrode and whole-cell recordings.

1. Whole-cell and sharp electrode recordings from adult rat dentate gyrus GCs were performed in the 400-microns-thick hippocampal slice preparation maintained at 34 +/- 1 degrees C. Intrinsic membrane properties of granule cells (GCs) were evaluated with the use of a switching current-clamp amplifier. 2. With the whole-cell technique, the average resting membrane potential (RMP) of GCs was -85 mV when a potassium gluconate electrode solution was used versus -74 mV measured with potassium acetate-filled sharp microelectrodes. The membrane voltage response to injected current was linear over two membrane potential ranges, greater than 10 mV hyperpolarized from RMP and between 10 mV more negative than RMP and -62 mV. The average input resistances (RN) calculated over these ranges were 107 and 228 M omega in the whole-cell recordings versus 37 and 54 M omega in the sharp electrode recordings. There was no correlation between RMP and RN with either recording technique. The membrane time constant (tau m) determined at the RMP was 26.9 ms for whole-cell recordings and 13.9 ms for sharp electrode recordings. 3. There was no evidence of time-dependent changes in RMP, RN, and tau m in whole-cell recordings, although the slow inward rectification seen at hyperpolarized potentials decreased over 30-60 min. Addition of calcium buffers to the whole-cell recording solution did not result in a significant change in the average RMP, the average RN, or the average tau m. 4. Action potential threshold was comparable in whole-cell (-49 mV) and sharp electrode (-52 mV) recordings, but action potential amplitude was larger in whole-cell (126 mV) than in sharp electrode (106 mV) recordings. Spike frequency adaptation was present in the whole-cell recordings and could be abolished by addition of calcium buffers to the electrode solution. 5. We estimated rho, the ratio of dendritic to somatic conductance, to be 5.1 for the whole-cell records and 2.1 for sharp electrode recordings. The electrotonic length of the equivalent cylinder representing the cell processes was estimated to be 0.49 from the whole-cell data and 0.79 from the sharp electrode recordings. This implies that at rest there is only a 10% decrement in steady-state membrane voltage along the length of the dendrite due to shunting across the membrane resistance; small synaptic events occurring in the distal dendritic tree will therefore have a more substantial influence on the soma than previous analyses suggested.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Shunting of excitatory input to dentate gyrus granule cells by a depolarizing GABAA receptor-mediated postsynaptic conductance.

1. Stimulation of the perforant path in the outer molecular layer of the adult rat dentate gyrus produced a depolarizing post-synaptic potential (DPSP) in granule cells when recorded using whole-cell techniques in the standard hippocampal slice preparation at 34 degrees C. The postsynaptic currents (PSCs) contributing to the DPSP were analyzed using specific receptor antagonists in current- and voltage-clamp recordings. 2. The DPSP reversal potential was dependent on the intracellular chloride concentration, and the amplitude of the DPSP was increased 55% after perfusion of the gamma-aminobutyric acid-A (GABAA) receptor antagonist bicuculline methiodide (BMI). The GABAA receptor-mediated PSC reversed at -66 mV, which was 19 mV positive to the resting membrane potential (-85 mV) but hyperpolarized relative to action potential threshold. At -35 mV, the GABAA PSC had a latency to peak of 12.9 ms after the stimulus and decayed monoexponentially with an average time constant of 23.4 ms. 3. The component of the PSC blocked by the Quis/AMPA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) had a latency to peak of 7.1 ms and decayed monoexponentially with a time constant of 9.9 ms at -35 mV. The N-methyl-D-aspartate (NMDA) receptor-mediated PSC, which was blocked by D-amino-5-phosphonovaleric acid (D-AP5), had a waveform that was similar to the GABAA PSC: the latency to peak was 16 ms and the decay was monoexponential with a time constant of 24.5 ms at -35 mV. 4. The ratio of the peak PSCs mediated by GABAA, Quis/AMPA, and NMDA receptors measured at -35 mV with cesium gluconate electrode solutions was 1:0.2:0.1. This ratio was essentially constant over the range of stimulus intensities that produced compound PSC amplitudes of 80-400 pA. 5. Measured at its reversal potential, the GABAA receptor-mediated postsynaptic conductance (GGABA-A) decreased the peak DPSP amplitude by 35%, shunted 50% of the charge transferred to the soma by the excitatory PSC, and completely inhibited the NMDA receptor-mediated component of the DPSP. 6. Simultaneous stimulation of presynaptic fibers from both the perforant path and interneurons results in a large depolarizing GGABA-A that inhibits the granule cell by shunting the excitatory PSCs. As predicted by models of shunting, the similar kinetics of the GABAA and NMDA PSCs leads to particularly effective inhibition of the NMDA PSC. The more rapid Quis/AMPA PSC is less affected by the GGABA-A, so that granule cell excitation under these conditions is primarily due to Quis/AMPA receptor activation.

2-Amino-5-phosphonovalerate↗
PubMed1992

The electrophysiology of dentate gyrus granule cells in whole-cell recordings.

The whole-cell patch-clamp recording technique was used both in neurons acutely dissociated from the dentate gyri of adult Wistar rats and in 400-microns-thick brain slices to examine passive membrane properties, voltage- and neurotransmitter-gated currents and synaptic physiology of granule cells. Voltage-dependent calcium currents and biophysical properties of N-methyl-D-aspartate channels were examined in acutely isolated granule cells and revealed significant differences between control and epileptic (kindled) neurons. In the slice preparation, the input resistance of granule cells recorded in the whole-cell mode was about 5-6 times larger than that obtained with sharp microelectrode recordings. The membrane time constant was longer while the electrotonic length was significantly shorter than previously estimated. Whole-cell recordings in granule cells of hippocampal slices also established the presence of a powerful depolarizing-shunting gamma-aminobutyric acid-A (GABAA) receptor-mediated inhibition which appears to control the NMDA component of synaptic transmission through the perforant path. Furthermore, spontaneous miniature synaptic excitatory and inhibitory postsynaptic currents, occurring with relatively high frequency, could be observed in granule cells. The present findings demonstrate that granule cells of the dentate gyrus have electrophysiological and synaptic properties in many ways different from those previously reported. Our study shows the feasibility of whole-cell recordings from granule cells in slices or acutely dissociated from a chronically altered preparation (e.g. after kindling) enabling the study of plasticity at the level of single neurons or even single channels.

Animals↗
PubMed1991

Perpetual inhibitory activity in mammalian brain slices generated by spontaneous GABA release.

Miniature spontaneous inhibitory postsynaptic currents (sIPSCs) mediated by GABAA receptors were recorded using whole-cell patch clamp recordings in rat brain slices maintained in vitro at 34 +/- 1 degree C. We have found that firing of action potentials by principal neurons or by GABAergic interneurons is not necessary to the generation of sIPSCs since they persist in the presence of 1-5 microM tetrodotoxin (TTX). The average frequency of the discrete sIPSCs exhibits a large cell-to-cell variability and is between 5-15 Hz. The amplitudes of the sIPSCs depend on the difference between the membrane potential and the equilibrium potential for Cl- (ECl). Generally, 70-80 mV away from ECl, sIPSCs have a mean amplitude of 30-80 pA (i.e. peak conductance of 400-1000 pS) with an average decay time constant of 5.8 ms. Accordingly, unitary single sIPSCs arise from the simultaneous activation of no more than 20 GABAA receptor/channels. The perpetual barrage of spontaneous GABAergic activity is very likely to be a critical factor in the regulation of neuronal excitability and the mechanism of action of several neuroactive compounds.

Action Potentials↗
PubMed1991

Halothane enhances tonic neuronal inhibition by elevating intracellular calcium.

Whether the major action of anesthetics is to depress the central nervous system (CNS) by reducing excitation or enhancing inhibition remains unknown. Using whole cell patch-clamp recording in hippocampal slices, halothane and pentobarbital were found to prolong the decay time constant (TAU(D)) of GABAA-mediated spontaneous inhibitory postsynaptic currents (sIPSCs). Intracellular administration of the Ca2+ chelator BAPTA or the Ca2+ release inhibitor dantrolene significantly (ANOVA, P less than 0.005) reduced halothane's effect; in contrast, the pentobarbital effect was unchanged. Halothane induced depression of population spike amplitude was blocked by the GABAA antagonist bicuculline. Together, these findings suggest that a major depressant effect of halothane involves enhancement of GABAA-mediated inhibition through release of intraneuronally stored Ca2+.

Animals↗
PubMed1991

Integrity of perforant path fibers and the frequency of action potential independent excitatory and inhibitory synaptic events in dentate gyrus granule cells.

Whole-cell voltage clamp recordings in 400 microns thick hippocampal slices revealed discrete excitatory and inhibitory postsynaptic currents which persisted at synapses on granule cells following abolition of action potentials with 1 microM tetrodotoxin (TTX). The conductances associated with excitatory amino acid and GABAA receptor mediated events had mean peaks of 200 and 800 pS, and decayed monoexponentially with time constants of 5.6 and 5.3 ms. At a holding potential close to the normal resting membrane potential of granule cells (-80 to -90 mV), the frequency of glutamate/aspartate mediated spontaneous excitatory postsynaptic currents (sEPSCs) was decreased from 2.04 Hz in slices cut parallel to the plane of the perforant path to 0.87 Hz in slices cut in a plane that disrupted the distal perforant path fibres, suggesting that presynaptic integrity influences the rate of action potential independent neurotransmitter release. The orientation of the slicing had no effect on the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs).

Action Potentials↗
PubMed1991

Mammalian neurons in dissociated cultures form clusters in the presence of retinal pigment epithelium.

The objective of this study was to investigate the cellular processes involved in the formation of the cytoarchitectonics of the retina. Neurons derived from the retina, spinal cord, cerebral cortex and hippocampus were grown in dissociated monolayer tissue culture using standard techniques. The cultures of retina were unique in that the neurons actively formed into cell clusters. On the other hand, cultures of neurons from the other regions of the CNS grew without forming any obvious histotypical pattern. Cell clusters consisted of an apparent monolayer of neurons above a population of flat cells and clusters were observed in retinal cultures derived from all species studied (mouse, cat and guinea pig). Each cluster was surrounded by whorls of fibroblasts; astrocytes (GFAP-positive cells) were often closely associated with clusters. Formation of clusters appeared to depend strongly upon the presence of cells derived from the retinal pigment epithelium (RPE) because the ability of retinal cells to form clusters was markedly impaired when the RPE was omitted from the cultures. Interestingly, monolayer cultures of neurons from other regions of the CNS could be induced to form clusters, but only when cells of the RPE layer were included at the time of plating. In cultures grown without the RPE layer, clusters did not form when media taken from cultures expressing clusters was used, indicating that the formation of clusters was not caused by a media-bourne factor. On the other hand, clusters did form when neurons without RPE were grown on feeder plates in which clusters had previously been expressed and the neurons subsequently killed by prolonged culturing or by treatment with kainic acid. Hence, physical contact between neurons and cells derived from the RPE appears critical for the formation of clusters. Our results suggest that the cellular processes underlying the formation of clusters may reflect those in the development of the retina in vivo. Thus, cluster formation may be a useful model for investigating the initial stages in the development of retinal cytoarchitecture.

Animals↗
PubMed1991

Calbindin-D28K (CaBP) levels and calcium currents in acutely dissociated epileptic neurons.

Nerve cells that lack the cytoplasmic Ca2+ binding protein Calbindin-D28K (CaBP) appear to be selectively vulnerable to Ca(2+)-related injury consistent with a postulated intraneuronal Ca(2+)-buffering role of CaBP. We have confirmed the selective loss of CaBP from the dentate gyrus during kindling-induced epilepsy in acutely dissociated granule cells (GCs) from kindled rats. Immunohistochemically stained kindled neurons showed a significant loss of CaBP when compared to controls (p less than 0.001; ANOVA). The Ca(2+)-buffering role of CaBP was assessed in acutely dissociated control and kindled GCs by examining a physiological process highly sensitive to intracellular Ca(2+)-buffering: the Ca(2+)-dependent inactivation of high-voltage activated (HVA or L-type) Ca2+ currents in the absence (or presence) of exogenous Ca(2+)-chelators. Whole-cell patch clamp recordings in kindled GCs demonstrated a markedly enhanced Ca(2+)-dependent inactivation of Ca(2+)-currents. After brief conditioning Ca2+ currents, in the absence of an exogenous intraneuronal Ca(2+)-chelator, subsequent test Ca2+ currents were inactivated by 58.3% in kindled GCs, a significant increase from the 37.4% inactivation observed in control GCs (p less than 0.005; ANOVA). The differential Ca2+ current decay and Ca(2+)-dependent inactivation were prevented in both control and kindled GCs upon loading the neurons with the exogenous Ca(2+)-chelator BAPTA. These experiments demonstrate a high correlation between the loss of CaBP and changes in Ca2+ current inactivation and are consistent with the hypothesis that CaBP contributes to the physiological Ca(2+)-buffering in mammalian neurons.

Animals↗
PubMed1991

Endogenous intracellular calcium buffering and the activation/inactivation of HVA calcium currents in rat dentate gyrus granule cells.

Granule cells acutely dissociated from the dentate gyrus of adult rat brains displayed a single class of high-threshold, voltage-activated (HVA) Ca2+ channels. The kinetics of whole-cell Ca2+ currents recorded with pipette solutions containing an intracellular ATP regenerating system but devoid of exogenous Ca2+ buffers, were fit best by Hodgkin-Huxley kinetics (m2h), and were indistinguishable from those recorded with the nystatin perforated patch method. In the absence of exogenous Ca2+ buffers, inactivation of HVA Ca2+ channels was a predominantly Ca(2+)-dependent process. The contribution of endogenous Ca2+ buffers to the kinetics of inactivation was investigated by comparing currents recorded from control cells to currents recorded from neurons that have lost a specific Ca(2+)-binding protein, Calbindin-D28K (CaBP), after kindling-induced epilepsy. Kindled neurons devoid of CaBP showed faster rates of both activation and inactivation. Adding an exogenous Ca2+ chelator, 1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), to the intracellular solution largely eliminated inactivation in both control and kindled neurons. The results are consistent with the hypothesis that endogenous intraneuronal CaBP contributes significantly to submembrane Ca2+ sequestration at a concentration range and time domain that regulate Ca2+ channel inactivation.

Adenosine Triphosphate↗
PubMed1991

Actions of ketamine, phencyclidine and MK-801 on NMDA receptor currents in cultured mouse hippocampal neurones.

1. Stable N-methyl-D-aspartic acid (NMDA) receptor-mediated currents in cultured mouse hippocampal neurons were evoked by 20 ms pressure pulse applications of L-aspartate, repeatedly applied at 30 or 40 s intervals, to the cell body region of the neurone. We have characterized the voltage- and use-dependent blockade of the currents by three dissociative anaesthetics: ketamine, phencyclidine (PCP) and MK-801 in mouse hippocampal neurones grown in dissociated tissue culture. 2. We have used a simple model of the blockade, based on the 'guarded receptor hypothesis' to interpret our data. The model assumes that receptors are maximally activated at the peak of the response with an open probability (Po) approaching 1, that there is no desensitization and that the blocking drug only associates with, or dissociates from, receptor channels which have been activated by agonist (e.g. open channels). 3. The model allows us to estimate forward and reverse rate constants for binding of the blockers to open channels from measurements of the steady-state level of blockade and the rate of change of the current amplitude per pulse during onset and offset of blockade. As predicted by the model, the estimated reverse rate was independent of blocker concentration while the forward rate increased with concentration. Changing the level of positively charged ketamine (pKa 7.5) tenfold by changing pH from 6.5 to 8.5 caused a corresponding change in the forward rate while having no effect on the reverse rate. Most of the voltage dependence of the blockade could be accounted for by reduction of the reverse rate by depolarization. 4. Estimated forward rate constants for ketamine, PCP and MK-801 were similar to one another when measured under similar conditions and were 3 x 10(4) - 3 x 10(5) M-1 S-1. Most of the differences in potency of the three blockers could be accounted for by differences in the reverse rate constants which were approximately 0.2, 0.03 and 0.003 s-1 for ketamine, PCP and MK-801, respectively. The estimated rate constants actually are the product of the rate constants and 1/Po. Suggestions that maximum Po is much less than 1 for NMDA channels imply that both forward and reverse rate constants of blockade may in fact be larger than we have calculated. However, their magnitudes, relative to one another, are unaffected by this consideration. 5. The reverse rate constant of blockade increased at positive potentials. This increase was prevented when the neurone was loaded with N-methyl-D-glucamine, an impermeant cation which prevented outward currents.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗