Biomedical subjects

I Llano

Publications and source records attributed to I Llano.

33 records · Page 2Linked to original sources

PubMed1994

Prolonged presence of glutamate during excitatory synaptic transmission to cerebellar Purkinje cells.

In the molecular layer of the cerebellar cortex, Purkinje cells and interneurons receive a common excitatory input from parallel fibers. The AMPA/kainate receptor-mediated parallel fiber excitatory postsynaptic current (EPSC) recorded in Purkinje cells decays much more slowly than that recorded in interneurons. We show that this slowness of decay does not result from dendritic filtering and that it is unlikely to reflect the deactivation kinetics of the postsynaptic receptors. Agents blocking glutamate uptake prolong the EPSC in Purkinje cells. We conclude that the slow EPSC decay results from the continued presence of transmitter glutamate. This may be due to retarded transmitter diffusion around spines or to cross-talk between neighboring active synapses.

Animals↗

PubMed1993

Inhibitory synaptic currents in stellate cells of rat cerebellar slices.

1. In thin cerebellar slices of rats aged 14-21 days, voltage-gated currents, synaptic currents and GABA responses were studied with the tight-seal whole-cell recording technique from stellate cells (8-9 micrograms soma diameter) located in the outer two-thirds of the molecular layer. 2. In symmetrical Cl- conditions, stellate cells voltage-clamped at -60 mV showed spontaneous inhibitory postsynaptic currents (IPSCs). As were the GABAA responses of the same cells, the IPSCs were blocked by bicuculline. The frequency of occurrence of IPSCs ranged from 0.2 to 1.9 events per second (21 cells). The mean amplitude of the events ranged from 61 to 226 pA (mean +/- S.E.M.: 132 +/- 11; n = 21). 3. The temporal course of IPSCs was characterized by a rapid rise (mean +/- S.E.M. of the time to peak: 1.1 +/- 0.1 ms, n = 7) and a slow decay. The decay phase was described by a double exponential function with time constants of 8.7 +/- 0.6 ms, and 40.9 +/- 3.7 ms respectively (means +/- S.E.M.; n = 7). 4. A minor fraction (15 to 20%) of the spontaneous synaptic events recorded in control saline had a faster onset than that of the IPSCs and decayed with a rapid mono-exponential decay (time constant of 1.0-1.3 ms). These were excitatory postsynaptic currents (EPSCs) unaffected by bicuculline and blocked by the glutamate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). 5. Bath application of TTX (0.5-1 microM), which blocked voltage-gated Na+ currents in stellate cells, induced a variable decrease in the frequency of IPSCs (mean +/- S.E.M. of the frequency ratio in TTX over control: 0.47 +/- 0.09; n = 12). However, the toxin had no significant effect either on the mean amplitude or on the kinetics of the IPSCs. The mean amplitude of the miniature IPSCs was 141 +/- 13 pA (mean +/- S.E.M.; n = 22). 6. In TTX-containing solutions, the frequency of the IPSCs was unaffected when Ca2+ currents were eliminated either by removal of extracellular Ca2+ and addition of EGTA, or by addition of Cd2+. Miniature IPSCs of 200-300 pA were still observed. 7. In symmetrical Cl- conditions, local application of GABA to stellate cells induced an inward current and an increase in membrane noise. Responses to prolonged applications of GABA showed desensitization in both whole-cell mode and somatic outside-out patches. The chord conductance estimated from recording single GABA channel events in somatic outside-out patches was 28 pS.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗

PubMed1993

Beta-adrenergic enhancement of inhibitory synaptic activity in rat cerebellar stellate and Purkinje cells.

1. Using the tight-seal whole-cell recording technique, we studied the effects of noradrenaline (NA) on the spontaneous inhibitory synaptic currents (IPSCs) of stellate and Purkinje cells in rat cerebellar slices. 2. In both types of cells, NA (10 microM) induced a marked increase in the frequency of the IPSCs. This effect was observed both in the absence and in the presence of TTX in the saline bathing the cerebellar slices. 3. The NA-induced increase in frequency of IPSCs and miniature IPSCs (mIPSCs) in the two cell types was mimicked by bath applications of isoprenaline (10 microM) and of the adenylyl cyclase activator forskolin (20 microM). Neither phenylephrine nor clonidine changed the frequency of IPSCs in stellate or Purkinje cells. 4. In stellate cells, the beta-agonists and forskolin had variable effects on the amplitudes of both IPSCs and mIPSCs. None of these compounds altered the amplitude of mIPSCs in Purkinje cells. 5. The responses to local applications of GABA to Purkinje cells were unchanged by bath applications of beta-adrenergic agonists or forskolin. A decrease in the response to GABA after treatment with these agents was observed in half the stellate cells examined. 6. We conclude that the major effect of NA on stellate and Purkinje cells is an increase in the frequency of occurrence of spontaneous inhibitory synaptic currents. This action is exerted through the activation of beta-adrenergic receptors and is probably mediated by an intracellular mechanism involving cAMP. The beta-adrenergic modulation of IPSC frequency takes place at the presynaptic level and may involve a change in the process of transmitter release.

Action Potentials↗

PubMed1991

Calcium entry increases the sensitivity of cerebellar Purkinje cells to applied GABA and decreases inhibitory synaptic currents.

The sensitivity to GABA of Purkinje cells in thin cerebellar slices was examined by recording either spontaneous inhibitory synaptic currents or ionic currents elicited by local GABA applications. The effects of Ca2+ entry induced by depolarizing voltage pulses were opposite for the two types of currents. Currents due to exogenous GABA applications were increased by a train of voltage pulses. This potentiation was transient with an average half recovery period of 3.7 min. Spontaneous synaptic currents were reduced by depolarizing voltage pulses, with a half recovery time of about 20 s. The inhibition was largely explained by a decrease of the frequency of synaptic events, suggesting that the primary location of the effect was presynaptic. Thus, a Ca2+ rise increases the sensitivity of Purkinje cells to GABA and induces a retrograde inhibition of presynaptic terminals. The latter effect may be due to a diffusible Ca2(+)-dependent messenger.

Animals↗

PubMed1991

Intradendritic release of calcium induced by glutamate in cerebellar Purkinje cells.

The ability of excitatory amino acids to induce increases in the intracellular Ca2+ concentration ([Ca2+]i) of cerebellar Purkinje cells was examined by digital fluorescence ratio imaging of voltage-clamped Purkinje cells dialyzed with the Ca2+ indicator fura-2. Purkinje cells responded with large inward currents accompanied by increases in dendritic [Ca2+]i when challenged with the excitatory amino acid agonists glutamate and quisqualate. The rise in [Ca2+]i was transient and reached peak values of several hundred nanomolar. The response subsisted in the absence of extracellular Ca2+, a condition that eliminates Ca2+ entry through voltage-gated Ca2+ channels, indicating that Ca2+ arose in large part from an intracellular compartment. In support of this hypothesis, only the first agonist application elicited a [Ca2+]i increase in slices maintained in Ca(2+)-free medium, as expected if the intracellular stores become depleted. These results indicate that metabotropic glutamate receptors are functional in Purkinje cells and point to glutamate as a possible modulator of [Ca2+]i in these neurons.

Animals↗

PubMed1991

Synaptic- and agonist-induced excitatory currents of Purkinje cells in rat cerebellar slices.

1. Postsynaptic currents originating from activation of the two major excitatory inputs to Purkinje cells were studied in thin slices of rat cerebellum, using the tight-seal whole-cell recording technique. Two types of excitatory postsynaptic currents were analysed: those evoked by stimulation of the granule cell-parallel fibre system (PF-EPSC) and those elicited by stimulation of the climbing fibres (CF-EPSC). 2. Both types of postsynaptic currents had a linear current-voltage relation, reversing at membrane potentials close to 0 mV. Their time course of activation was independent of the membrane potential. 3. For both types of postsynaptic currents, the time course of decay was well described by a single exponential function, with a time constant which increased as the membrane potential was made more positive. 4. Postsynaptic currents arising from stimulation of the climbing fibre generally had a slightly faster time course of onset and decay than those associated with stimulation of the granule cell-parallel fibre system. The average values of the 10-90% rise time were 1.8 +/- 0.4 ms (means +/- S.D., n = 7) for PF-EPSCs and 0.8 +/- 0.3 ms (n = 9) for CF-EPSCs. Time constants of decay, at a holding potential of -60 mV, had values of 8.3 +/- 1.6 ms (n = 7) and 6.4 +/- 1.1 ms (n = 9) for PF-EPSCs and CF-EPSCs respectively. 5. CF-EPSCs and PF-EPSCs had the characteristics described above in slices derived from animals aged 9-22 days old and 9-15 days old, respectively. The PF-EPSCs in animals older than 15 days had very slow time courses and positive apparent reversal potentials, suggesting that they originated from distal locations, not under accurate voltage control. 6. In order to assess the quality of the voltage clamp, responses to hyperpolarizing pulses from -70 mV were analysed. The capacitive currents could be fitted by the sum of two exponentials, and were interpreted with an equivalent electrical circuit comprising two main compartments (soma and proximal dendrites on one hand, distal dendrites on the other). Analysis of synaptic currents in terms of this model suggested that the recorded time course of decay was approximately correct. 7. CF-EPSCs as well as PF-EPSCs were insensitive to the NMDA receptor antagonist 3-3(2-carboxypiperazine-4-yl)propyl-1-phosphonate (CPP), but were blocked in a dose-dependent reversible manner by the non-NMDA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX).(ABSTRACT TRUNCATED AT 400 WORDS)

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed1990

Synaptic currents in cerebellar Purkinje cells.

Cerebellar Purkinje cells are known to receive strong excitatory input from two major pathways originating outside the cerebellum and inhibitory input from two types of neurons in the cerebellar cortex. The functions and synaptic strengths of these pathways are only partially known. We have used the patch-clamp technique applied to Purkinje cells in thin slices of rat cerebellum to measure directly the postsynaptic currents arising from the two major excitatory pathways and one of the inhibitory inputs. Inhibitory synaptic currents occur spontaneously with high frequency and are variable in amplitude, ranging, in our recording conditions with high internal Cl-, from less than 100 pA to more than 1 nA. These currents are blocked by the gamma-aminobutyrate type A antagonist bicuculline. One of the excitatory inputs is all or none. For threshold stimulation, the synaptic current is either full amplitude, when the presynaptic fiber is successfully stimulated, or completely absent. This synaptic current is often larger than 1 nA and is virtually eliminated by 2 microM 6-cyano-7-nitroquinoxaline-2,3-dione, a blocking agent thought to be specific for glutamate receptors that are not of the N-methyl-D-aspartate type. Its all-or-none character identifies it as arising from a climbing-fiber synapse. The other excitatory input produces a synaptic current that is smoothly graded as a function of stimulus intensity. This response we believe arises from the stimulation of mossy fibers or granule cells. The synaptic current associated with this input is also largely eliminated by 2 microM 6-cyano-7-nitroquinoxaline-2,3-dione.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed1989

Sodium and potassium conductances in somatic membranes of rat Purkinje cells from organotypic cerebellar cultures.

1. The somatic voltage-gated conductances of Purkinje cells in organotypic cultures (Gähwiler, 1981) were studied using the outside-out patch recording configuration of the patch-clamp technique (Hamill, Marty, Neher, Sakmann & Sigworth, 1981). 2. When activated by step depolarizations, the tetrodotoxin-sensitive voltage-dependent Na+ current presented two distinct phases: an initial surge of inward current fluctuations which activates rapidly upon pulse onset and decays within 20-40 ms, and a later phase in which discrete bursts of single-channel activity are interspersed with silent periods. 3. Ensemble fluctuation analysis of the current fluctuations during the early phase of the Na+ current and measurements of single channels during both early and late phases indicate that a single type of Na+ channel can account for both phases of the Na+ current. This channel has an elementary current amplitude of -2 pA at -40 mV. This amplitude did not vary significantly between -60 and -20 mV. The mean open time depended on membrane potential, increasing by a factor of three between -60 and -20 mV. 4. The early component of the Na+ current activated at a threshold of -60 mV and reached its maximum amplitude at -20, mid-point for the activation curve being -40 mV. Times-to-peak current decreased with membrane potential, from 3.5 ms at -60 mV to 0.3 ms at 0 mV. The decay phase of the current presented two exponential components, with time constants of 1.5 and 10 ms at -40 mV. The steady-state inactivation curve had a mid-point at -75 mV. 5. The late component of the Na+ current was observed in the voltage range from -60 to -20 mV, with a maximum at -40 mV. Its maximum amplitude corresponded to approximately 1.7% of the peak amplitude of the early component. 6. Macroscopic potassium currents were observed upon step depolarizations above a threshold of -30 mV. The currents activated in a voltage-dependent fashion, times-to-peak decreasing with depolarization, and partially inactivated during 40 ms depolarizing steps. Peak current amplitudes at any given membrane potential were decreased by depolarizing the holding potential. The macroscopic properties of the K+ current varied from patch to patch. 7. Two types of single-channel K+ currents were observed during steady-state depolarizations. The unitary current amplitudes were 2.7 and 10.4 pA at 30 mV, corresponding to chord conductances of 28 and 90 pS respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1988

Patch-clamp recording of amino acid-activated responses in "organotypic" slice cultures.

Patch-clamp recording techniques were used to study the properties of amino acid-activated channels in cultured "organotypic" slices from rat cerebellum and hippocampus. Hippocampal pyramidal cells responded to the three main glutamatergic agonists, N-methyl-D-aspartate (N-Me-D-Asp), quisqualate, and kainate, whereas Purkinje cells responded only to quisqualate and kainate. Analysis of single-channel events recorded in outside-out patches from hippocampal neurons showed large conductance events (50 pS), which occurred more frequently in the presence of glycine. These events could be produced by N-Me-D-Asp and also, at low frequency, by quisqualate. On the other hand, 50-pS events were never observed in Purkinje neurons. This supports the hypothesis that N-Me-D-Asp and "non-N-Me-D-Asp" receptors are distinct molecular entities. Comparison of whole-cell and outside-out patch recordings from Purkinje cells revealed a clear spatial segregation of gamma-aminobutyric acid (GABA) and glutamate receptors: although GABA receptors are found at high density in somatic membrane, quisqualate and kainate receptors are mostly extrasomatic. The results show that organotypic slice cultures are amenable to patch-clamp methods. They also show that, in these cultures, amino acids receptors have specific distribution patterns according to cell type and to region within a cell.

Action Potentials↗

PubMed1988

Potassium conductance of the squid giant axon. Single-channel studies.

The patch-clamp technique was implemented in the cut-open squid giant axon and used to record single K channels. We present evidence for the existence of three distinct types of channel activities. In patches that contained three to eight channels, ensemble fluctuation analysis was performed to obtain an estimate of 17.4 pS for the single-channel conductance. Averaged currents obtained from these multichannel patches had a time course of activation similar to that of macroscopic K currents recorded from perfused squid giant axons. In patches where single events could be recorded, it was possible to find channels with conductances of 10, 20, and 40 pS. The channel most frequently encountered was the 20-pS channel; for a pulse to 50 mV, this channel had a probability of being open of 0.9. In other single-channel patches, a channel with a conductance of 40 pS was present. The activity of this channel varied from patch to patch. In some patches, it showed a very low probability of being open (0.16 for a pulse to 50 mV) and had a pronounced lag in its activation time course. In other patches, the 40-pS channel had a much higher probability of being open (0.75 at a holding potential of 50 mV). The 40-pS channel was found to be quite selective for K over Na. In some experiments, the cut-open axon was exposed to a solution containing no K for several minutes. A channel with a conductance of 10 pS was more frequently observed after this treatment. Our study shows that the macroscopic K conductance is a composite of several K channel types, but the relative contribution of each type is not yet clear. The time course of activation of the 20-pS channel and the ability to render it refractory to activation only by holding the membrane potential at a positive potential for several seconds makes it likely that it is the predominant channel contributing to the delayed rectifier conductance.

Animals↗

PubMed1987

Dependence of intracellular effects of GTP gamma S and inositoltrisphosphate on cell membrane potential and on external Ca ions.

Single cells from rat lacrimal glands were studied with the tight-seal whole-cell recording method. Ca-dependent K and Cl currents were measured in response to dialysis with inositoltrisphosphate or GTP gamma S, two compounds known for elevating internal Ca2+ concentration. The activation of the Ca-dependent currents elicited by either compound was partially inhibited by sustained depolarization or by removal of external Ca2+. Conversely, hyperpolarization or removal of external Mg2+ led to augmentations of the Ca-dependent currents. These effects became apparent about 1 min after initiation of cell dialysis with inositoltrisphosphate- or GTP gamma S-containing solutions, and they further developed during the ensuing 10 min. Holding potential and external divalent cations did not affect the Ca-dependent currents elicited by dialysing the cells with strongly buffered solutions containing 0.5 microM free Ca2+. In Ca2+-free external saline, cell currents were independent of the holding potential. It is suggested that InsP3 augments intracellular Ca2+ levels not only by releasing Ca2+ from internal stores but also by slowly increasing the Ca permeability of the plasma membrane. The results indicate that Ca2+ entry through the plasma membrane increases with hyperpolarization. The similarity of the effects seen in InsP3- and GTP gamma S-dialysed cells gives support to the hypothesis that production of the former depends on a GTP-binding protein.

Animals↗

PubMed1987

Protein kinase C activators inhibit the inositol trisphosphate-mediated muscarinic current responses in rat lacrimal cells.

1. Pre-incubation (1-5 min) with 12-O-tetradecanoylphorbol-13-acetate (TPA, 8-16 nM), a tumour-promoting phorbol ester known to activate protein kinase C, was found to inhibit acetylcholine-induced Ca2+-dependent K+ and Cl- currents in cells isolated from rat lacrimal glands. 2. Previous work showed that the same currents may be elicited by dialysing cells with a high-Ca2+ (1.0 microM) solution, with inositol trisphosphate (InsP3), or with guanosine 5'-[gamma-thio]triphosphate (GTP-gamma-S). 3. After TPA incubation both types of currents could be elicited by dialysis with elevated Ca2+ solutions, although the magnitude of the K+ current was slightly reduced in comparison with control cells. 4. Responses to intracellular dialysis with InsP3 (20 microM) were similar to those in untreated cells, indicating that the Ca2+ release process was unaffected. 5. However, the response to GTP-gamma-S (0.2-0.5 mM) dialysis was strongly inhibited in TPA-treated cells. 6. These results suggest that protein kinase C exerts an inhibitory action on the pathway leading from receptor activation to inositol trisphosphate production.

Acetylcholine↗

PubMed1986

Ionic conductances of squid giant fiber lobe neurons.

The cell bodies of the neurons in the giant fiber lobe (GFL) of the squid stellate ganglion give rise to axons that fuse and thereby form the third-order giant axon, whose initial portion functions as the postsynaptic element of the squid giant synapse. We have developed a preparation of dissociated, cultured cells from this lobe and have studied the voltage-dependent conductances using patch-clamp techniques. This system offers a unique opportunity for comparing the properties and regional differentiation of ionic channels in somatic and axonal membranes within the same cell. Some of these cells contain a small inward Na current which resembles that found in axon with respect to tetrodotoxin sensitivity, voltage dependence, and inactivation. More prominent is a macroscopic inward current, carried by Ca2+, which is likely to be the result of at least two kinetically distinct types of channels. These Ca channels differ in their closing kinetics, voltage range and time course of activation, and the extent to which their conductance inactivates. The dominant current in these GFL neurons is outward and is carried by K+. It can be accounted for by a single type of voltage-dependent channel. This conductance resembles the K conductance of the axon, except that it partially inactivates during relatively short depolarizations. Ensemble fluctuation analysis of K currents obtained from excised outside-out patches is consistent with a single type of K channel and yields estimates for the single channel conductance of approximately 13 pS, independently of membrane potential. A preliminary analysis of single channel data supports the conclusion that there is a single type of voltage-dependent, inactivating K channel in the GFL neurons.

Animals↗

PubMed1984

Analysis of sodium current fluctuations in the cut-open squid giant axon.

Patch pipettes were used to record the current arising from small populations of sodium channels in voltage-clamped cut-open squid axons. The current fluctuations associated with the time-variant sodium conductance were analyzed with nonstationary statistical techniques in order to obtain an estimate for the conductance of a single sodium channel. The results presented support the notion that the open sodium channel in the squid axon has only one value of conductance, 3.5 pS.

Animals↗

PubMed1980

Current recorded from a cut-open giant axon under voltage clamp.

Squid giant axons were cut open and the resulting membrane sheet was positioned in a chamber separating two compartments. Under voltage clamp, normal sodium, potassium, and gating currents could be recorded for several hours. This preparation allows direct access to the internal side of the axonal membrane and should prove useful for recording of ionic currents, gating currents, and channel-induced current fluctuations in the same membrane sheet.

Animals↗