Biomedical subjects

I Llano

Publications and source records attributed to I Llano.

At least 19 recordsLinked to original sources

PubMed2000

Role of the calcium-binding protein parvalbumin in short-term synaptic plasticity.

GABAergic (GABA = gamma-aminobutyric acid) neurons from different brain regions contain high levels of parvalbumin, both in their soma and in their neurites. Parvalbumin is a slow Ca(2+) buffer that may affect the amplitude and time course of intracellular Ca(2+) transients in terminals after an action potential, and hence may regulate short-term synaptic plasticity. To test this possibility, we have applied paired-pulse stimulations (with 30- to 300-ms intervals) at GABAergic synapses between interneurons and Purkinje cells, both in wild-type (PV+/+) mice and in parvalbumin knockout (PV-/-) mice. We observed paired-pulse depression in PV+/+ mice, but paired-pulse facilitation in PV-/- mice. In paired recordings of connected interneuron-Purkinje cells, dialysis of the presynaptic interneuron with the slow Ca(2+) buffer EGTA (1 mM) rescues paired-pulse depression in PV-/- mice. These data show that parvalbumin potently modulates short-term synaptic plasticity.

2-Amino-5-phosphonovalerate↗

PubMed2000

Action potential-evoked Ca2+ signals and calcium channels in axons of developing rat cerebellar interneurones.

Axonal [Ca2+] transients evoked by action potential (AP) propagation were studied by monitoring the fluorescence of the high-affinity calcium-sensitive dye Oregon Green 488 BAPTA-1, introduced through whole-cell recording pipettes in the molecular layer of interneurones from cerebellar slices of young rats. The spatiotemporal profile of Ca2+-dependent fluorescence changes was analysed in well-focused axonal stretches a few tens of micrometres long. AP-evoked Ca2+ signals were heterogeneously distributed along axons, with the largest and fastest responses appearing in hot spots on average approximately 5 microm apart. The spatial distribution of fluorescence responses was independent of the position of the focal plane, uncorrelated to basal dye fluorescence, and independent of dye concentration. Recordings using the low-affinity dye mag-fura-2 and a Cs+-based intracellular solution revealed a similar pattern of hot spots in response to depolarisation, ruling out measurement artefacts or possible effects of inhomogeneous dye distribution in the generation of hot spots. Fluorescence responses to a short train of APs in hot spots decreased by 41-76 % after bath perfusion of omega-conotoxin MVIIC (5-6 microM), and by 17-65 % after application of omega-agatoxin IVA (500 nM). omega-Conotoxin GVIA (1 microM) had a variable, small effect (0-31 % inhibition), and nimodipine (5 microM) had none. Somatically recorded voltage-gated currents during depolarising pulses were unaffected in all cases. These data indicate that P/Q-type Ca2+ channels, and to a lesser extent N-type channels, are responsible for a large fraction of the [Ca2+] rise in axonalhot spots. [Ca2+] responses never failed during low-frequency (<= 0.5 Hz) stimulation, indicating reliable AP propagation to the imaged sites. Axonal branching points coincided with a hot spot in approximately 50 % of the cases. The spacing of presynaptic varicosities, as determined by a morphological analysis of Neurobiotin-filled axons, was approximately 10 times larger than the one measured for hot spots. The latter is comparable to the spacing reported for varicosities in mature animals. We discuss the nature of hot spots, considering as the most parsimonious explanation that they represent functional clusters of voltage-dependent Ca2+ channels, and possibly other [Ca2+] sources, marking the position of developing presynaptic terminals before the formation of en passant varicosities.

Action Potentials↗

PubMed2000

Presynaptic calcium stores underlie large-amplitude miniature IPSCs and spontaneous calcium transients.

The cellular mechanisms responsible for large miniature currents in some brain synapses remain undefined. In Purkinje cells, we found that large-amplitude miniature inhibitory postsynaptic currents (mIPSCs) were inhibited by ryanodine or by long-term removal of extracellular Ca2+. Two-photon Ca2+ imaging revealed random, ryanodine-sensitive intracellular Ca2+ transients, spatially constrained at putative presynaptic terminals. At high concentration, ryanodine decreased action-potential-evoked rises in intracellular Ca2+. Immuno-localization showed ryanodine receptors in these terminals. Our data suggest that large mIPSCs are multivesicular events regulated by Ca2+ release from ryanodine-sensitive presynaptic Ca2+ stores.

Animals↗

PubMed2000

The retrograde inhibition of IPSCs in rat cerebellar purkinje cells is highly sensitive to intracellular Ca2+.

The Ca2+-dependent retrograde inhibition of inhibitory postsynaptic currents (depolarization-induced-suppression of inhibition; DSI) was investigated using fura-2 Ca2+ measurements and whole-cell patch-clamp recordings in rat cerebellar Purkinje cells. DSI was studied in cells loaded with different concentrations of the Ca2+ chelators BAPTA and EGTA. A concentration of 40 mM BAPTA was required to significantly interfere with DSI, whereas 10 mM BAPTA was almost ineffective. 40 mM EGTA reduced DSI, but was less effective than 40 mM BAPTA. Ratiometric Ca2+ measurements indicated that the extent of DSI depended critically on the changes in intracellular calcium ([Ca2+]i). The relationship between DSI and peak Delta[Ca2+]i could be approximated by a hyperbolic function, with apparent half-saturation concentrations of 200 and 40 nM for dendritic and somatic [Ca2+]i, respectively. It is suggested that DSI is due to somatodendritic exocytosis of a retrograde messenger, and that this exocytosis is highly sensitive to [Ca2+]i.

Animals↗

PubMed1999

Fast scanning and efficient photodetection in a simple two-photon microscope.

Two-photon laser scan microscopy carries many advantages for work on brain slices and bulk tissue. However, it has very low signal levels compared to conventional fluorescence microscopy. This is disadvantageous in fast imaging applications when photon shot noise is limiting. Working on brain slices with excitation powers of 8-10 mW at the specimen plane, the resting signal from cerebellar Purkinje cell somas loaded with 10 microM Oregon Green 488 BAPTA-1 averaged 4 detected photons/micros; axons of interneurons loaded with 200 microM of this indicator yielded about 1 photon/micros. To obtain satisfactory images at high time resolution, long pixel dwell times are required and data collection should be restricted to as few pixels as necessary. Furthermore, a large proportion of total measurement time (duty cycle) should be available for data collection. We therefore developed a method for scanning small regions of interest with line repetition rates two to four times higher than conventional ones and a duty cycle of 70%. We also compared the performance of several photodetectors and found the optimum choice to depend strongly on the photon flux during a given application. For fluxes smaller than 5 photons/micros, the photon counting avalanche photodiode shows the best signal to noise ratio. At larger fluxes, photomultipliers or intensified photodiodes are superior.

Animals↗

PubMed1999

Modulation by K+ channels of action potential-evoked intracellular Ca2+ concentration rises in rat cerebellar basket cell axons.

1. Action potential-evoked [Ca2+]i rises in basket cell axons of rat cerebellar slices were studied using two-photon laser scanning microscopy and whole-cell recording, to identify the K+ channels controlling the shape of the axonal action potential. 2. Whole-cell recordings of Purkinje cell IPSCs were used to screen K+ channel subtypes which could contribute to axonal repolarization. alpha-Dendrotoxin, 4-aminopyridine, charybdotoxin and tetraethylammonium chloride increased IPSC rate and/or amplitude, whereas iberiotoxin and apamin failed to affect the IPSCs. 3. The effects of those K+ channel blockers that enhanced transmitter release on the [Ca2+]i rises elicited in basket cell axons by action potentials fell into three groups: 4-aminopyridine strongly increased action potential-evoked [Ca2+]i; tetraethylammonium and charybdotoxin were ineffective alone but augmented the effects of 4-aminopyridine; alpha-dendrotoxin had no effect. 4. We conclude that cerebellar basket cells contain at least three pharmacologically distinct K+ channels, which regulate transmitter release through different mechanisms. 4-Aminopyridine-sensitive, alpha-dendrotoxin-insensitive K+ channels are mainly responsible for repolarization in basket cell presynaptic terminals. K+ channels blocked by charybdotoxin and tetraethylammonium have a minor role in repolarization. alpha-Dendrotoxin-sensitive channels are not involved in shaping the axonal action potential waveform. The two last types of channels must therefore exert control of synaptic activity through a pathway unrelated to axonal action potential broadening.

4-Aminopyridine↗

PubMed1999

Leishmania amazonensis infection induces changes in the electrophysiological properties of macrophage-like cells.

Whole cell patch-clamp recordings were used to study the electrical properties of the macrophage-like cell line J774.1, after infection with Leishmania amazonensis. Infection induced a significant increase in cell size and membrane capacitance, suggesting that parasite invasion leads to the addition of plasma membrane to the host cell. By 24 hr after infection, the host cell membrane potential was significantly more hyperpolarized than control cells, and this difference remained for the subsequent 72 hr post-infection. The hyperpolarization was paralleled by an increase in the density of inward rectifying K(+) currents. The shape of the conductance vs. voltage curve, the kinetic properties and the pharmacological profile of these currents were not significantly altered by infection. These results suggest that infection by L. amazonensis causes an increase in the number of functional inward rectifying K(+) channels, leading to hyperpolarization of the host cell membrane.

Animals↗

PubMed1998

Intracellular calcium clearance in Purkinje cell somata from rat cerebellar slices.

1. The mechanisms governing the return of intracellular calcium (Cai2+) to baseline levels following depolarization-evoked [Ca2+]i rises were investigated in Purkinje cell somata using tight-seal whole-cell recordings and fura-2 microfluorometry, for peak [Ca2+]i ranging from 50 nm to 2 microM. 2. Cai2+ decay was well fitted by a double exponential with time constants of O.6 and 3 s. Both time constants were independent of peak [Ca2+]i but the contribution of the faster component increased with [Ca2+]i. 3. Thapsigargin (10 microM) and cyclopiazonic acid (50 microM) prolonged Cai2+ decay indicating that sarco-endoplasmic reticulum Ca2+ (SERCA) pumps contribute to Purkinje cell Cai2+ clearance. 4. A modest participation in clearance was found for the plasma membrane Ca2+ (PMCA) pumps using 5,6-succinimidyl carboxyeosin (40 microM). 5. The Na(+)-Ca2+ exchanger also contributed to the clearance process, since replacement of extracellular Na+ by Li+ slowed Cai2+ decay. 6. Carbonyl cyanide m-chlorophenylhydrazone (CCCP, 2 microM) and rotenone (10 microM) increased [Ca2+]i and elicited large inward currents at -60 mV. Both effects were also obtained with CCCP in the absence of external Ca2+, suggesting that mitochondrial Ca2+ uptake uncouplers release Ca2+ from intracellular stores and may alter the membrane permeability to Ca2+. These effects were irreversible and impeded tests on the role of mitochondria in Cai2+ clearance. 7. The relative contribution of the clearance systems characterized in this study varied as a function of [Ca2+]i. At 0.5 microM Cai2+, SERCA pumps and the Na(+)-Ca2+ exchanger contribute equally to removal and account for 78% of the process. Only 45% of the removal at 2 microM Cai2+ can be explained by these systems. In this high [Ca2+]i range the major contribution is that of SERCA pumps (21%) and of the Na(+)-Ca2+ exchanger (18%), whereas the contribution of PMCA pumps is only 6%.

Animals↗

PubMed1998

Calcium currents and calcium signaling in rod bipolar cells of rat retinal slices.

Combined electrophysiological and imaging techniques were used to study calcium currents (ICa) and their sites of origin at rod bipolar cells in rat retinal slices. We report here for the first time the successful whole-cell patch-clamp recording from presynaptic boutons that were compared with somatic recordings. TTX-resistant inward currents were elicited in response to depolarization. The kinetic and pharmacological properties of ICa were very similar for recordings obtained from the soma and the presynaptic terminals. ICa activated maximally between -30 and -20 mV was enhanced by Bay K 8644 and was blocked by isradipine and nifedipine. Peak amplitude and time to peak were -31.3 +/- 1.2 pA and 3.2 +/- 0.2 msec with somatic recordings (n = 54), whereas the corresponding values were -31.6 +/- 6.1 pA and 3.2 +/- 0.7 msec in recordings obtained directly from terminals (n = 6). ICa showed little inactivation during sustained depolarizations. No T-type ICa was observed with depolarizations from -90 mV. Concomitant with Ca2+ entry, depolarization induced the appearance of transient outward currents that resembled IPSCs and were blocked by GABA and glycine receptor antagonists, suggesting that they arise from activation of amacrine feedback synapses. Upon depolarization, intracellular Ca2+ ([Ca2+]i) rises were restricted to the presynaptic terminals with no somatic or axonal changes and were linearly dependent on pulse duration when using a low-affinity Ca2+ indicator. In cone bipolar cells, ICa inactivated markedly, and [Ca2+]i rises occurred in the axon, as well as in the presynaptic terminals.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1997

Two different conductances contribute to the anion currents in Coffea arabica protoplasts.

The anion conductance of the plasma membrane of Coffea arabica protoplasts was isolated and characterized using the whole-cell patch clamp technique. Voltage pulse protocols revealed two components: a voltage-gated conductance (Gs) and a voltage-independent one (Gl). Gs is activated upon depolarization (e-fold activation every +36 mV) with time constants of 1 sec and 5 sec at all potentials. Gl and Gs also differ by their kinetic and biophysical properties. In bi-ionic conditions the current associated with Gs shows strong outward rectification and its permeability sequence is F- > NO3- > Cl-. In the same conditions the current associated with Gl does not rectify and its permeability sequence is F- > > NO3- = Cl-. Furthermore, at potentials over +50 mV Gs, but not Gl, increases with a time constant of several minutes. Finally the gating of Gs is affected by stretch of the membrane, which leads to an increased activation and a reduced voltage sensitivity. Anion conductances similar to the ones described here have been found in many plant preparations but Gl-type components have been generally interpreted as the background activation of the slow voltage-gated channels (corresponding to Gs). We show that in coffee protoplasts Gl and Gs are kinetically and biophysically distinct, suggesting that they correspond to two different molecular entities.

Anions↗

PubMed1997

GABAergic and glycinergic IPSCs in ganglion cells of rat retinal slices.

GABAergic and glycinergic IPSCs were studied in identified retinal ganglion cells (RGCs) of light-adapted rat retinal slices, using whole-cell recording techniques. GABAergic IPSCs were blocked specifically by SR95531 (3 microM) and bicuculline (3 microM) and glycinergic IPSCs by strychnine (0.3 microM). From 37 RGCs studied, 25 showed exclusively GABAergic IPSCs, 6 presented only glycinergic IPSCs, and 6 showed both. This distribution may result from differences in amacrine cells input rather than from receptor heterogeneity, because both GABA and glycine elicited Cl--selective currents in all RGCs tested. TTX markedly reduced GABAergic IPSCs frequency, whereas glycinergic IPSCs were unaffected. Ca2+-free media, with or without high Mg2+, blocked TTX-resistant GABAergic and glycinergic IPSCs. These results suggest that GABAergic IPSCs in RGCs can be elicited either by Na+-dependent action potentials or by local Ca2+ influx in medium or large dendritic field GABAergic amacrine cells, whereas glycinergic IPSCs are generated by action potential-independent Ca2+ influx in narrow field glycinergic amacrine cells. Both types of IPSCs had fast rise times and biexponential decays, but glycinergic IPSC decay was significantly slower than that of GABAergic IPSCs. An elementary conductance of 54 pS for the glycine-gated channels was estimated from single-channel events, clearly detected in the falling phase of glycinergic IPSCs, and from responses to exogenous glycine.

Adaptation, Ocular↗

PubMed1997

Spatial heterogeneity of intracellular Ca2+ signals in axons of basket cells from rat cerebellar slices.

1. Using tight-seal whole-cell recording and digital fluorescence imaging, we studied intracellular calcium (Ca2+i) dynamics in cerebellar basket cells, whose dendrites, axon and presynaptic terminals are coplanar, an optimal configuration for simultaneous optical measurements of all functional domains. 2. In Cs(+)-loaded neurones, depolarizing pulses induced large Ca2+i transients in single axonal varicosities and synaptic terminals, contrasting with much weaker signals between varicosities or in the somato-dendritic domain. 3. Axonal branch points consistently displayed [Ca2+]i rises of similar magnitude and time course to those in axonal terminals and varicosities. 4. In biocytin-filled basket cells, varicosity-like swellings were present along the axon including its branch points. Thus, axonal enlargements are not due to fluorescence-induced cell damage. 5. The spatial heterogeneity of Ca2+i signals was also observed in K(+)-loaded cells upon depolarizing trains, suggesting that this behaviour is an intrinsic property of Ca2+i homeostasis in basket cells. 6. We conclude that depolarization of basket cell axons evokes high local Ca2+i signals in synaptic terminals, en passant varicosities and branch points. While high [Ca2+]i in presynaptic structures presumably triggers transmitter release, Ca2+i transients at branch points may control signal transmission in the axonal arborization.

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

High endogenous calcium buffering in Purkinje cells from rat cerebellar slices.

1. The ability of Purkinje cells to rapidly buffer depolarization-evoked intracellular calcium changes (delta [Ca2+]i) was estimated by titrating the endogenous buffer against incremental concentrations of the Ca(2+)-sensitive dye fura-2. 2. In cells from 15-day-old rats, pulse-evoked delta [Ca2+]i were stable during the loading with 0.5 mM fura-2 through the patch pipette. In cells from 6-day-old rats, delta [Ca2+]i decreased by approximately 50% during equivalent experiments. This decrease was not related to changes in Ca2+ influx, since the integral of the Ca2+ currents remained constant throughout the recording. 3. Experiments with high fura-2 concentrations (1.75-3.5 mM) were performed in order to obtain for each cell the curve relating delta [Ca2+]i to fura-2 concentration. From this relationship, values for the Ca2+ binding ratio (the ratio of buffer-bound Ca2+ changes over free Ca2+ changes) were calculated. 4. In Purkinje cells from 15-day-old rats, the Ca2+ binding ratio was approximately 2000, an order of magnitude larger than that of other neurones and neuroendocrine cells studied to date. This Ca2+ binding ratio was significantly smaller (approximately 900) in Purkinje cells from 6-day-old rats. 5. We propose that the large Ca2+ binding ratio of Purkinje cells is related to the presence of large concentrations of Ca2+ binding proteins and that these cells regulate their ability to handle Ca2+ loads during development through changes in the concentration of Ca2+ binding proteins.

Aging↗

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↗

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↗

PubMed1994

Calcium-induced calcium release in cerebellar Purkinje cells.

Depolarization-induced intracellular Ca2+ rises were measured in fura-2-loaded, voltage-clamped Purkinje cells. The peak Ca2+ rise increased more than linearly with voltage step duration, suggesting the presence of Ca(2+)-induced Ca2+ release. In cells from young animals, in which Ca2+ currents could be satisfactorily recorded, a supralinear relation was also found between peak Ca2+ rise and Ca2+ current integral. Responses to long pulses were inhibited in cells dialyzed with 20 microM ruthenium red and potentiated in cells bathed in the presence of 20 microM ryanodine. Upon repetitive depolarization, increasing Ca2+ rises were elicited by successive voltage pulses, probably because of a potentiating effect of residual Ca2+. Altogether, the results indicate an important contribution of Ca(2+)-induced Ca2+ release to Ca2+ signals of Purkinje cells.

Aging↗