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Biomedical subjects

A Marty

Publications and source records attributed to A Marty.

At least 37 records · Page 2Linked to original sources
PubMed1999

Somatic recording of GABAergic autoreceptor current in cerebellar stellate and basket cells.

Patch-clamp recordings were performed from stellate and basket cells in rat cerebellar slices. Under somatic voltage clamp, short depolarizing pulses were applied to elicit action potentials in the axon. After the action potential, a bicuculline- and Cd2+-sensitive current transient was observed. A similar response was obtained when eliciting axonal firing by extracellular stimulation. With an isotonic internal Cl- solution, the peak amplitude of this current varied linearly with the holding potential, yielding an extrapolated reversal potential of -20 to 0 mV. Unlike synaptic or autaptic GABAergic currents obtained in the same preparation, the current transient had a slow rise-time and a low variability between trials. This current was blocked when 10 mM BAPTA was included in the recording solution. In some experiments, the current transient elicited axonal action potentials. The current transient was reliably observed in animals aged 12-15 d, with a mean amplitude of 82 pA at -70 mV, but was small and rare in the age group 29-49 d. Numerical simulations could account for all properties of the current transient by assuming that an action potential activates a distributed GABAergic conductance in the axon. The actual conductance is probably restricted to release sites, with an estimated mean presynaptic current response of 10 pA per site (-70 mV, age 12-15 d). We conclude that in developing rats, stellate and basket cell axons have a high density of GABAergic autoreceptors and that a sizable fraction of the corresponding current can be measured from the soma.

Action Potentials↗
PubMed1999

Presynaptic effects of NMDA in cerebellar Purkinje cells and interneurons.

NMDA receptors (NMDARs) are generally believed to mediate exclusively postsynaptic effects at brain synapses. Here we searched for presynaptic effects of NMDA at inhibitory synapses in rat cerebellar slices. In Purkinje cells, application of NMDA enhanced the frequency of miniature IPSCs (mIPSCs) but not that of miniature EPSCs (mEPSCs). This increase in frequency was dependent on the external Mg2+ concentration. In basket and stellate cells, NMDA induced an even larger mIPSC frequency increase than in Purkinje cells, whereas mEPSCs were again not affected. Moreover, NMDA induced an inward current in both types of interneuron, which translated into a small depolarization (approximately 10 mV for 30 microM NMDA) under current-clamp conditions. In paired recordings of connected basket cell-Purkinje cell synapses, depolarizations of 10-30 mV applied to the basket cell soma enhanced the frequency of postsynaptic mIPSCs, suggesting that somatic depolarization was partially transmitted to the terminals in the presence of tetrodotoxin. However, this effect was small and unlikely to account fully for the effects of NMDA on mIPSCs. Consistent with a small number of dendritic NMDARs, evoked EPSCs in interneurons had a remarkably small NMDA component. Evoked IPSCs at interneuron-interneuron synapses were inhibited by NMDA, and the rate of failures was increased, indicating again a presynaptic site of action. We conclude that activation of NMDARs in interneurons exerts complex presynaptic effects, and that the corresponding receptors are most likely located in the axonal domain of the cell.

Animals↗
PubMed1998

Water activity control: a way to improve the efficiency of continuous lipase esterification.

During continuous lipase-catalyzed oleic acid esterification by ethanol in n-hexane, the oleic acid conversion, initially at 95%, decreases to 20% after 2 h. This decrease is caused by the accumulation of the water produced in the course of the reaction in the packed-bed reactor (PBR). In order to improve the PBR efficiency, it is necessary to evacuate the water produced. In this study, different approaches have been tested to control the water content in the PBR during continuous esterification. The first approach consisted in improving the water solubility by increasing the reaction medium polarity. The addition of polar additives to n-hexane, the use of more polar solvents, and the use of solvent-free reaction medium were tested as a means to favor the water evacuation from the PBR. First of all, the use ofn-hexane supplemented with acetone (3 M) or 2-methyl-2-propanol (1 M) enabled the conversion to be maintained at higher values than those obtained in pure n-hexane. The replacement of n-hexane by a more polar solvent, like the 5-methyl-2-hexanone, resulted in the same effect. In all cases, conversions at steady-state were always less than 95%, as obtained in pure n-hexane. This is explained by a decrease in the enzyme activity due to the increase in the medium polarity. Nevertheless, an increase in enzyme quantity allowed 90% conversion to be maintained during 1 week using 3 M acetone amended n-hexane. Good results (a steady-state conversion of about 80%) were obtained when esterification was carried out in a solvent-free reaction medium containing 2 M 2-methyl-2-propanol as a polar additive. The second approach consisted in the evaporation of the accumulated water by use of an intermittent airflow. Although this process did not enable constant esterification rate to be maintained, it did enable the initial conversion (95%) to be restored intermittently.

Bioreactors↗
PubMed1998

Autaptic inhibitory currents recorded from interneurones in rat cerebellar slices.

1. While the presence of autapses in the brain is indicated by a large body of morphological evidence, the functional role of these structures has remained unclear. To probe for autaptic currents, we have recorded current responses following short somatic depolarizing pulses in Cl--loaded interneurones (stellate and basket cells) from rat cerebellar slices (animals aged 27-39 days). 2. In approximately 20 % of the recordings, fluctuating inward current transients were obtained with a latency of 1.15-2.45 ms (measured from the peak of the depolarization-induced Na+ current; n = 10). 3. These transients were blocked by bicuculline and were sensitive to the extracellular Ca2+ concentration. 4. Assuming low release probability, as suggested by the high failure rate (0.65-0.92, n = 10), quantal sizes ranging from 21 to 178 pA (-70 mV; n = 10) were calculated from a variance analysis of autaptic current amplitudes. 5. We conclude that approximately 20 % of interneurones have a functional autapse. Autaptic currents may inhibit firing of interneurones during high frequency discharges.

Animals↗
PubMed1998

Multivesicular release at single functional synaptic sites in cerebellar stellate and basket cells.

The purpose of the present work was to test the hypothesis that no more than one vesicle of transmitter can be liberated by an action potential at a single release site. Spontaneous and evoked IPSCs were recorded from interneurons in the molecular layer of cerebellar slices. Evoked IPSCs were obtained using either extracellular stimulation or paired recordings of presynaptic and postsynaptic neurons. Connections were identified as single-site synapses when evoked current amplitudes could be grouped into one peak that was well separated from the background noise. Peak amplitudes ranged from 30 to 298 pA. Reducing the release probability by lowering the external Ca2+ concentration or adding Cd2+ failed to reveal smaller quantal components. Some spontaneous IPSCs (1.4-2.4%) and IPSCs evoked at single-site synapses (2-6%) were followed within <5 msec by a secondary IPSC that could not be accounted for by random occurrence of background IPSCs. Nonlinear summation of closely timed events indicated that they involved activation of a common set of receptors and therefore that several vesicles could be released at the same release site by one action potential. An average receptor occupancy of 0.70 was calculated after single release events. At some single-site connections, two closely spaced amplitude peaks were resolved, presumably reflecting single and double vesicular release. Consistent with multivesicular release, kinetics of onset, decay, and latency were correlated to IPSC amplitude. We conclude that the one-site, one-vesicle hypothesis does not hold at interneuron-interneuron synapses.

Animals↗
PubMed1998

Synaptic currents at individual connections among stellate cells in rat cerebellar slices.

1. Unitary inhibitory synaptic connections among stellate cells were studied in rat cerebellar slices. Presynaptic action potentials and inhibitory postsynaptic currents (IPSCs) were simultaneously recorded by loose cell-attached and tight-seal whole-cell recording, respectively. 2. Several types of synaptic connections were distinguished on the basis of the shape of the amplitude distribution of successfully evoked currents. For simple synapses, which presumably arise at single release sites, these histograms could be fitted to a single Gaussian (5 cases). In four additional cases a small amplitude component (< 50 pA) was superimposed to a single Gaussian peak. The small events had slow rise times and widely distributed amplitudes. Finally eleven histograms showed two or more Gaussian components and were classified as complex connections. 3. Failure rates ranged from 0.06 to 0.85 for unitary connections (n = 20) and from 0.59 to 0.78 for simple synapses (n = 5). 4. Coefficient of variation values derived from Gaussian fits to simple synapse histograms ranged between 0.20 and 0.38 (n = 5). 5. In simple synapses peak current amplitudes were positively correlated to both current rise time and decay half-width. 6. Intervals between presynaptic action potentials were widely distributed. During stationary periods there was no correlation between interspike interval and amplitude size, success rate or latency. In some experiments, episodes with shorter interspike intervals were observed. During these periods, amplitude and success rate decreased, and the latency increased. Thus, IPSC characteristics depend on the mean frequency of presynaptic spikes, but not on random fluctuations of interspike intervals during stationary periods.

Action Potentials↗
PubMed1998

Differential effects of noradrenaline on evoked, spontaneous and miniature IPSCs in rat cerebellar stellate cells.

1. The modulation by noradrenaline (NA) of synapses among stellate cells was investigated in rat cerebellar slices by using presynaptic loose cell-attached recording and postsynaptic whole-cell recording. 2. NA increased the frequency of spontaneous IPSCs recorded from stellate cells without changing their mean amplitude. 3. NA increased the firing rate of stellate cells. This effect persisted after blocking ionotropic glutamate receptors and GABA receptors, indicating that it was independent of synaptic input. 4. The effects of NA on action potential frequency were mimicked by the beta-receptor agonist isoprenaline but not by the alpha-receptor agonist 6-fluoro noradrenaline, and they were not blocked by the alpha-receptor antagonist phentolamine, indicating that they were mediated by beta-receptors. 5. In paired recordings of connected stellate cells, NA slightly decreased the success rate of synaptic transmission. A small decrease in mean IPSC amplitude (excluding failures) and a slight increase in latency were also observed in NA. 6. These results show that, while NA increases the number of action potential-dependent IPSCs by increasing the firing rate of stellate cells, it actually reduces the probability of evoked release. Since previous studies showed that NA increases the rate of miniature IPSCs in this preparation, we conclude that different mechanisms underly the modulation by NA of action potential-dependent and action potential-independent transmitter release.

Action Potentials↗
PubMed1998

Extrasynaptic vesicular transmitter release from the somata of substantia nigra neurons in rat midbrain slices.

Substantia nigra neurons release dopamine from their somatodendritic regions. A long-unresolved question is whether this release occurs by exocytosis or by a nonvesicular mechanism. We used carbon fiber microelectrodes in a brainstem slice to assay secretion from single cell bodies that had been cleared of connective tissue. Amperometry at the carbon fiber microelectrodes revealed unitary events in approximately 90% of cells in resting conditions. These events had charge integrals ranging from a few femtocoulombs to several hundred femtocoulombs (fC). Local glutamate application enhanced the event frequency by 3.5-fold on average and up to 10-fold in highly responsive cells, although the mean charge integral was not modified. Local application of a high K+-containing saline had effects similar to those of glutamate. The frequency of resting and stimulated amperometric events was much lower at 21-22 degreesC than at 32-35 degreesC. The addition of Cd2+ (50 microM), a blocker of voltage-dependent Ca2+ channels, to the bath solution blocked the stimulatory effects of glutamate. These results suggest that dopamine is released from the somata of substantia nigra neurons by exocytosis and that this mechanism is regulated by neuronal electrical activity. More generally, this study demonstrates the applicability of carbon fiber microelectrodes to the measurement of quantal monoamine secretion in brain slices.

Animals↗
PubMed1997

Protein kinase A-mediated enhancement of miniature IPSC frequency by noradrenaline in rat cerebellar stellate cells.

1. Cellular mechanisms underlying the enhancement by noradrenaline (NA) of inhibitory postsynaptic currents (IPSCs) were studied at inhibitory synapses in the molecular layer of the cerebellum. IPSCs were obtained from stellate cells in rat cerebellar slices using tight-seal whole-cell recording. 2. Miniature IPSCs (mIPSCs) were recorded in the presence of tetrodotoxin (TTX; 100 nM). NA (10 microM) markedly increased the frequency of mIPSCs, but did not alter their mean amplitude. Bath application of the inhibitor of adenylyl cyclase 9-(tetrahydro-2'-furyl) adenine (SQ 22,536; 300 microM), of the wide spectrum protein kinase inhibitor staurosporine (1 microM), and of the Rp-diastereomer of adenosine-3',5'-cyclic monophosphothioate (Rp-cAMPS; 500 microM), a specific inhibitor of cAMP-dependent protein kinase (PKA), inhibited the mIPSC frequency increase induced by NA. 3. The increase in mIPSC frequency was not attenuated by Cd2+ (100 microM), a blocker of voltage dependent calcium channels. However, after a 12-15 min pre-incubation in Ca(2+)-free saline, the effect of NA on mIPSCs was markedly inhibited. If Ca2+ ions were readmitted in the presence of NA, enhancement of the mIPSC frequency was largely restored. 4. Application of the membrane permeant analogue of cAMP, 8-Br-cAMP (1 mM), together with the inhibitor of cAMP phosphodiesterase, 3-isobutyl-1-methylxanthine (IBMX; 100 microM), caused a frequency increase of mIPSCs. Forskolin also mimicked the stimulatory effect of NA on mIPSC frequency. The effects of both 8-Br-cAMP and forskolin persisted in Ca(2+)-free saline, suggesting that the modulation of transmitter release does not require Ca2+ influx. 5. On the whole, the results indicate that the potentiation of mIPSC frequency by NA is mediated through the sequential activation of adenylyl cyclase and protein kinase A (PKA), and that PKA modulates the vesicle release mechanism rather than Ca2+ influx. The lack of effect of NA after prolonged incubation in Ca(2+)-free solution may be due to an inhibition of adenylyl cyclase by a gradual lowering of the cytosolic presynaptic Ca2+ concentration.

Animals↗
PubMed1997

Heterogeneity of functional synaptic parameters among single release sites.

Following alpha-latrotoxin application to cerebellar slices, bursts of miniature IPSCs (mIPSCs) were observed in interneurons of the molecular layer. Within bursts, mIPSCs had homogeneous amplitudes with a narrow Gaussian distribution. Analysis of successive event amplitudes revealed an interaction between consecutive IPSCs, indicating that bursts originate at single release sites. A mean receptor occupancy of 76% was calculated. IPSCs within a burst were analyzed using nonstationary noise analysis. The results indicate that individual release sites differ in the number, unitary conductance, and peak opening probability of their postsynaptic channels. In addition, the IPSC decay kinetics were very different among release sites. Finally, a significant correlation was found between several pairs of single site synaptic parameters.

Animals↗
PubMed1996

Glutamate as a candidate retrograde messenger at interneurone-Purkinje cell synapses of rat cerebellum.

1. Depolarization-induced suppression of inhibition (DSI) is a form of synaptic plasticity which involves a retrograde messenger. We have performed experiments in Purkinje cells of rat cerebellar slices to determine the nature of this messenger. 2. DSI is mimicked by 2-(2,3-dicarboxycyclopropyl)-glycine (DCG-IV), a specific agonist of group II metabotropic glutamate receptors (mGluRs). 3. DSI is reduced if transmitter release is inhibited by saturating doses of DCG-IV. 4. Both DSI and DCG-IV-induced inhibition are inhibited by L-2-amino-3-phosphonopropionic acid (L-AP3), a drug which interferes with several subtypes of mGluRs. 5. DSI is reduced if synaptic activity is enhanced by application of forskolin. 6. We propose that glutamate or a glutamate-like substance is the retrograde messenger implicated in DSI, and that the inhibition resulting from presynaptic glutamate binding is mediated by a decrease in the presynaptic concentration of cAMP.

2-Amino-5-phosphonovalerate↗
PubMed1996

Fluctuations of inhibitory postsynaptic currents in Purkinje cells from rat cerebellar slices.

1. Paired patch-clamp recordings were performed in cerebellar slices to study IPSCs evoked by interneurones (stellate and basket cells) in Purkinje cells. 2. IPSCs were first examined while keeping the presynaptic cell in the cell-attached mode. In some of these experiments IPSCs had a remarkably broad amplitude distribution. Large variabilities were associated with large mean IPSC amplitudes and were preferentially obtained with presynaptic basket cells. Action currents recorded from the presynaptic interneurone in the cell-attached mode were reproducible indicating that the variability occurs downstream of action potential generation. 3. The variability of IPSC amplitudes was observed independently of the instantaneous firing rate of the presynaptic cell, and could therefore not be ascribed to synaptic fatigue. 4. Highly variable IPSC amplitude were also obtained if the presynaptic interneurone was placed in whole-cell recording with a potassium-based intracellular solution. 5. If the recording pipette were filled with Cs+ ions a large increase in the mean of the IPSCs was observed within seconds after establishing the whole-cell recording. In Cs(+)-dialysed cells it was possible to modulate the mean IPSC amplitudes by altering the characteristics of the presynaptic stimulation. IPSC amplitudes obtained in response to presynaptic voltage pulses to 0 mV were large and had little scatter. IPSCs obtained in response to pulses near +60 mV, close to the reversal potential of presynaptic Ca2+ currents, had a much lower mean amplitude and also had little scatter. Thus presynaptic application of Cs+ ions both increases the mean amplitude and decreases the variability of the postsynaptic response. 6. To test whether interneurones could be coupled by electrical synapses, paired recordings were performed from neighbouring interneurones. No correlation was found between the firing patterns of such paired recordings, indicating that electrical coupling among presynaptic neurones is not responsible for large IPSC fluctuations as recorded in Purkinje cells. 7. Finally, IPSC fluctuations were investigated in paired recordings from two Purkinje cells. IPSCs corresponding to the activity of common interneurones were identified on the basis of temporal correlation. By plotting the amplitudes of such common IPSCs in one cell against those obtained simultaneously in the other cell, the pattern of IPSCs due to a single presynaptic neurone could be identified. These results show that fluctuations of IPSCs due to the same interneurone in one postsynaptic Purkinje cell are independent of those occurring in another Purkinje cell. 8. The results indicate that the major source of fluctuations is localized within the axonal arborization of presynaptic interneurones. The results with presynaptic Cs+ require that the fluctuations involve the concerted release of several presynaptic vesicles. Two possible mechanisms for such multiquantal events are discussed: fluctuations in presynaptic depolarization, and fluctuations in a regenerative Ca2+ amplification mechanism.

Animals↗
PubMed1996

Cyclic AMP-regulated GABA release at inhibitory synapses in rat cerebellar slices.

We have investigated the effects of agents interfering with the cAMP pathway on the rate of miniature IPSCs in cerebellar slices. Noradrenaline and group II glutamate metabotropic receptor agonists respectively enhance and reduce the rate of miniature IPSCs, presumably because they respectively increase and decrease the presynaptic concentration of cAMP.

Adenine↗
PubMed1996

HOE 694 affords protection versus veratrine contractures in rat atria by Na+ channel blockade.

We examined the effects of the benzoylguanidine derivative HOE 694, an inhibitor of Na(+)-H+ exchange, against veratrine-induced diastolic contractures and action potentials recorded in rat isolated left atria. Concentration-dependent protective effects against veratrine-contractures, in the absence of negative inotropic responses, were observed with HOE 694 (IC50 = 20.1(7.6-27.0) microM, n = 24) and with the chemically related amiloride derivatives DMA, EIPA, HMA and MIA, but not with amiloride itself. Concomitant Na(+)-H+ exchange blockade by a high concentration of amiloride (100 microM) failed to significantly modify the protective effects of HOE 694. HOE 694 decreased Vmax significantly at 10 microM (166.7 +/- 21 vs 154.7 +/- 20 V/s, P < 0.05, n = 6) without any effect on resting potential or action potential duration. High concentrations (100 microM) of HOE 694 further decreased Vmax and increased action potential duration. The protective effects of HOE 694 were compared with three of the class 1 antiarrhythmic agents, quinidine, lidocaine and flecainide against veratrine contracture. These Na+ channel blockers exerted protective effects in the same range of concentrations as HOE 694. Our findings demonstrate that HOE 694 prevents veratrine contractures at concentrations which presumably affect Na(+)-H+ exchange. However, the mechanism by which HOE 694 affords protection is apparently mediated by class 1-type Na+ channel blockade.

Action Potentials↗
PubMed1995

Presynaptic metabotropic glutamatergic regulation of inhibitory synapses in rat cerebellar slices.

1. The effects of the metabotropic glutamate agonist trans-ACPD (t-ACPD) were investigated in various locations of the inhibitory network made by GABAergic interneurones in the molecular layer of rat cerebellar slices. 2. t-ACPD exerted complex effects on spontaneous IPSCs in Purkinje cells. IPSC frequency was transiently inhibited during short (< 1 min) applications, and enhanced upon washing. During prolonged exposure to t-ACPD, IPSCs became organized in high-frequency bursts interspersed with periods of deep inhibition. 3. In interneurones, the frequency of spontaneous IPSCs was enhanced by t-ACPD. As in Purkinje cells, spontaneous IPSCs had a tendency to cluster in bursts in the presence of t-ACPD. 4. Evoked IPSCs recorded either in interneurones or in Purkinje cells upon stimulation of presynaptic interneurones were reversibly inhibited by t-ACPD. 5. Miniature IPSCs (mIPSCs) were recorded in the presence of tetrodotoxin both in Purkinje cells and in interneurones. t-ACPD did not alter the mean amplitude of mIPSCs in either cell type. It reduced the frequency of mIPSCs in Purkinje cells, but did not alter their rate in interneurones. 6. In cell-attached recordings on interneurone somata, t-ACPD was found to induce clustering of action potentials and to enhance the mean rate of firing. These results apply whether t-ACPD was tested in normal saline, in the presence of glutamatergic ionotrophic blockers, or in the presence of a mixture of glutamatergic and GABAergic blockers. 7. The results suggest that t-ACPD has at least two different modes of action. One effect is to alter the intrinsic firing rate of interneurones, presumably through an action on somatic conductance mechanisms. The other is to decrease the efficacy of the interneurone-interneurone and interneurone-Purkinje cell synapses, presumably through an action on axonal conductance systems and/or vesicle release mechanisms.

Action Potentials↗
PubMed1995

Modulation of inhibitory synapses in the mammalian brain.

Recent studies have identified new forms of short-term and long-term modulation at inhibitory synapses. In cerebellum and hippocampus, a transient inhibition of synaptic efficacy has been found to involve a Ca(2+)-dependent retrograde messenger. Exciting results from work in the visual cortex suggest that forms of both long-term potentiation and long-term depression occur in cortical inhibitory synapses.

Animals↗
PubMed1995

Ketanserin inhibits the transient outward current in rabbit ventricular myocytes.

The effects of ketanserin on the calcium-independent component of the transient outward potassium current (Ito) was investigated in isolated rabbit ventricular myocytes using the whole-cell patch-clamp technique. Ketanserin concentration-dependently (0.1-100 microM) reduced Ito (IC50 2.25 +/- 2.2 microM; Hill coefficient, nH 0.91), and decreased both the fast and slow Ito inactivation time constants so that the decay of the peak outward current was significantly accelerated. Our results indicate that, in addition to its antagonist activity on 5HT2A/C receptors, ketanserin reduces the calcium-independent component of Ito. Blockade of Ito could explain the proarrhythmic properties of ketanserin in vivo.

Animals↗