Isabel Llano

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.

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)

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.

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.

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.

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.

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.

Developmental Changes in Parvalbumin Regulate Presynaptic Ca2+ Signaling

Certain interneurons contain large concentrations of specific Ca2+-binding proteins (CBPs), but consequences on presynaptic Ca2+ signaling are poorly understood. Here we show that expression of the slow CBP parvalbumin (PV) in cerebellar interneurons is cell specific and developmentally regulated, leading to characteristic changes in presynaptic Ca2+ dynamics (Cai). Using whole-cell recording and fluorescence imaging, we studied action potential-evoked Cai transients in axons of GABA-releasing interneurons from mouse cerebellum. At early developmental stages [postnatal days 10-12 (P10-P12)], decay kinetics were significantly faster for basket cells than for stellate cells, whereas at P19-P21 both interneurons displayed fast decay kinetics. Biochemical and immunocytochemical analysis showed parallel changes in the expression levels and cellular distribution of PV. By comparing wild-type and PV(-/-) mice, PV was shown to accelerate the initial decay of action potential-evoked Cai signals in single varicosities and to introduce an additional slow phase that summates during bursts of action potentials. The fast initial Cai decay accounts for a previous report that PV elimination favors synaptic facilitation. The slow decay component is responsible for a pronounced, PV-dependent, delayed transmitter release that we describe here at interneuron-interneuron synapses after presynaptic bursts of action potentials. Numerical simulations account for the effect of PV on Cai kinetics, allow estimates for the axonal PV concentration (∼150 μm), and predict the time course of volume-averaged Cai in the absence of exogenous buffer. Overall, PV arises as a major contributor to presynaptic Cai signals and synaptic integration in the cerebellar cortex.

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)

Presynaptic calcium stores and synaptic transmission.

Following the gradual recognition of the importance of intracellular calcium stores for somatodendritic signaling in the mammalian brain, recent reports have also indicated a significant role of presynaptic calcium stores. Ryanodine-sensitive stores generate local, random calcium signals that shape spontaneous transmitter release. They amplify spike-driven calcium signals in presynaptic terminals, and consequently enhance the efficacy of transmitter release. They appear to be recruited by an association with certain types of calcium-permeant ion channels, and they induce specific forms of synaptic plasticity. Recent research also indicates a role of inositoltrisphosphate-sensitive presynaptic calcium stores in synaptic plasticity.