Vanni Taglietti

Synaptic excitation of individual rat cerebellar granule cells in situ: evidence for the role of NMDA receptors.

1. Current‐clamp recordings were made in whole‐cell patch‐clamp configuration from ninety‐one granule cells in parasagittal cerebellar slices obtained from 21‐ to 31‐day‐old rats. Recordings were performed at 30 degrees C. 2. Resting membrane potential was ‐58 +/‐ 6 mV (n = 43). The membrane voltage response to step current injection showed inward rectification consistent with increasing input resistance during membrane depolarization. Over ‐35 +/‐ 7 mV (n = 14) repetitive firing with little or no adaptation was activated. Spike frequency increased nearly linearly with injected current. 3. Unitary EPSPs obtained by stimulating the mossy fibre bundle had an amplitude of 11.4 +/‐ 2.1 mV (n = 22, holding potential = ‐75 mV). Synchronous activation of greater than one to two mossy fibres was needed to elicit action potentials. Antidromic stimulation elicited antidromic spikes and also EPSPs, presumably through a mossy fibre ‘axon reflex’. 4. EPSPs were brought about by NMDA and non‐NMDA receptor activation, accounting for about 70 and 30%, respectively, of peak amplitude at the holding potential of ‐70 mV. The EPSP decay conformed to passive membrane discharge after blocking the NMDA receptors. 5. No appreciable correlation was found between the time‐to‐peak and decay time constant of the EPSPs, consistent with the compact electrotonic structure of these neurons. 6. During membrane depolarization EPSP amplitude increased transiently, due to both a voltage‐dependent increase of the NMDA component and inward rectification. In addition, EPSPs slowed down due to a slowdown of the NMDA component. 7. Temporal summation during high‐frequency stimulation was sustained by NMDA receptors, whose contribution to depolarization tended to prevail over that of non‐NMDA receptors during the trains. A block of the NMDA receptors resulted in reduced depolarization and output spike frequency. 8. This study, as well as extending previous knowledge to the intracellular level in vivo, provides evidence for a primary role of NMDA receptors in determining mossy fibre excitation of granule cells. It is suggested that the marked voltage dependence of the EPSP time course, which was mainly caused by voltage dependence in NMDA conductance, promotes the NMDA receptor‐dependent enhancement of granule cell coding observed during repetitive mossy fibre activity.

Increased neurotransmitter release during long-term potentiation at mossy fibre–granule cell synapses in rat cerebellum

During long‐term potentiation (LTP) at mossy fibre–granule cell synapses in rat cerebellum synaptic transmission and granule cell intrinsic excitability are enhanced. Although it is clear that changes in granule cell excitability are mediated postsynaptically, there is as yet no direct evidence for the site and mechanism of changes in transmission. To approach this problem, evoked postsynaptic currents (EPSCs) and miniature synaptic currents (mEPSCs) were recorded by patch‐clamp in cerebellar slices obtained from P17–P23 rats. LTP was induced by theta‐burst stimulation paired with depolarization. During LTP, the EPSCs showed a significant decrease in the coefficient of variation (CV; 28.9 ± 5.2%, n= 8; P < 0.002), the number of failures (87.1 ± 41.9%, n= 8; P < 0.04), and the paired‐pulse ratio (PPR; 25.5 ± 4.1%n= 5; P < 0.02). Similar changes were observed by increasing neurotransmitter release (extracellular solutions with high Ca2+/Mg2+ ratio), whereas increases in CV, numbers of failures and PPR occurred when release was decreased (extracellular solutions with low Ca2+/Mg2+ ratio; 10 μm Cl‐adenosine). No changes followed modifications of postsynaptic holding potentials, while CV and failures were reduced when the number of active synapses was increased. LTP was prevented by use of solutions with high Ca2+/Mg2+ ratio. Moreover, LTP and the associated CV decrease were observed in the spillover‐mediated component of AMPA EPSCs and in NMDA EPSCs. During LTP, mEPSCs did not change in amplitude or variability but significantly increased in frequency (47.6 ± 16%, n= 4; P < 0.03). By binomial analysis changes in EPSCs were shown to be due to increased release probability (from 0.6 ± 0.08 to 0.73 ± 0.06, n= 7; P < 0.02) with a constant number of three to four releasing sites. These observations provide evidence for increased neurotransmitter release during LTP at mossy fibre–granule cell synapses.

Different proportions of N-methyl-d-aspartate and non-N-methyl-d-aspartate receptor currents at the mossy fibre-granule cell synapse of developing rat cerebellum

The mossy fibre-granule cell synapse undergoes major developmental changes during the second and third weeks after birth. We investigated synaptic transmission during postnatal days 10-22 by means of whole-cell patch-clamp recordings from granule cells in situ. Parasagittal slices were cut from rat cerebellar vermis, and excitatory postsynaptic currents were evoked in granule cells by mossy fibre stimulation with 1.2 mM Mg++ in the extracellular solution. In the majority of granule cells recorded at postnatal days 16-22, excitatory currents were characterized by a fast initial peak followed by a slower component, while in many of the cells recorded at more immature stages, the fast peak was virtually absent. Pharmacological and kinetic data indicated that the fast and slow components were mediated by non-N-methyl-D-aspartate and N-methyl-D-aspartate receptor activation, respectively. The magnitude of the non-N-methyl-D-aspartate current increased with developmental age, while the magnitude of the NMDA current did not change markedly. The age-dependent change of the non-N-methyl-D-aspartate currents could not be accounted for by changes in recording conditions or granule cell electrotonic properties. Furthermore, from postnatal day 11 to 16 the extent of Mg++ block on the N-methyl-D-aspartate receptor did not change, and could not explain the increasing non-N-methyl-D-aspartate/N-methyl-D-aspartate current ratio. We concluded therefore that the age-dependent increase of the non-N-methyl-D-aspartate current was the main cause of the different postsynaptic current waveforms observed at different ages. The developmental change in the proportion of N-methyl-D-aspartate and non-N-methyl-D-aspartate currents may be relevant to the processes regulating granule cell maturation and excitability.

NMDA receptor 2 (NR2) C-terminal control of NR open probability regulates synaptic transmission and plasticity at a cerebellar synapse

The C-terminal domain of NMDA receptor 2 (NR2) subunits has been proposed to play a critical role in regulating NMDA receptor localization and function in postsynaptic densities. However, the mechanism of this regulation is not completely understood. In this paper we show that C-terminal truncation of NR2A and NR2C subunits in mice (NR2A/CΔC/ΔC) impairs synaptic transmission and plasticity at the cerebellar mossy fiber–granule cell relay. Activation of synaptic NMDA receptors could be distinguished from that of extrasynaptic receptors by using the glutamate scavenger glutamate pyruvate transaminase and the open channel blocker MK801. NR2A/CΔC/ΔC mice exhibited a specific reduction in synaptic NMDA receptor activation attributable to a severalfold decrease in channel open probability but not channel conductance. Immunodetection revealed normal developmental expression of NR subunit proteins. Quantitative immunogold analyses with an antibody to NR1 indicated that the reduction in receptor activation is not attributed to a reduced number of NR1-containing receptors in postsynaptic densities. Thus, NR2A/NR2C subunits and particularly their C termini regulate synaptic NMDA receptor activation and function by enhancing channel open probability, which is critical for long-term potentiation induction.

ST14A cells have properties of a medium-size spiny neuron.

The ST14A cell line was previously derived from embryonic day 14 rat striatal primordia by retroviral transduction of the temperature-sensitive SV40 large T antigen. We showed that cell division and expression of nestin persists at 33 degrees C, the permissive temperature, whereas cell division ceases, nestin expression decreases, and MAP2 expression increases at the nonpermissive temperature of 39 degrees C. In this study, we further characterized the cells and found that they express other general and subtype-specific neuronal characteristics. ST14A cells express enolase and beta III-tubulin. Furthermore, they express the striatal marker DARPP-32, which is up-regulated upon differentiation of the cells by growth in serum-free medium. Stimulation with dopamine, the D2-dopamine receptor agonist quinpirole, or the D1-dopamine receptor agonist SKF82958 results in phosphorylation of CREB. Treatment of the cells with a mixture of reagents which stimulate the MAPK and adenylyl cyclase pathways radically changes the morphology of the ST14A cells. The cells develop numerous neurite-like appearing processes which stain with beta III-tubulin. Moreover, under these conditions, intracellular injection of rectangular depolarizing current stimuli elicits overshooting action potentials with a relatively fast depolarization rate when starting from a strongly hyperpolarized membrane potential. Taken together, these data imply that the ST14A cell line displays some of the characteristics of a medium-size spiny neuron subtype and provides a new tool to elucidate the pathways and molecules involved in medium-size spiny neuron differentiation and disease.

A study of stretch‐activated channels in the membrane of frog oocytes: interactions with Ca2+ ions.

1. We have carried out patch‐clamp measurements on a cationic channel in the plasma membrane of the frog oocyte, which can be specifically activated by membrane stretch. The kinetics of this channel also display a distinct dependence upon membrane potential, the probability of the channel being open increasing with membrane depolarization. 2. When the patch‐clamp pipette filling solution was standard Ringer solution, the single‐channel current‐voltage (I‐V) relationship was linear, the elementary conductance being 38 pS and the reversal potential +7 mV, suggesting very poor selectivity of the channel for the various cations. 3. The I‐V relationship was highly non‐linear having a strong inward‐going rectification when Ca2+‐free solutions were used to fill the patch pipette. These solutions also resulted in a selective, inward cationic permeability through the membrane, with K+ being more permeable than Na+ greater than Li+ greater than Ba2+ greater than Ca2+. 4. Though permeant through the stretch‐activated channel, Ca2+ inhibited in a concentration‐dependent manner the currents carried by other cations. La3+ (0.1 mM) was also an effective channel blocker. 5. The inward current carried by individual cations at a given membrane potential increased with increasing external cation concentration up to a saturating level, this level being maximal for K+ and minimal for Ca2+. Also the half‐saturating concentration was maximal for K+ and minimal for Ca2+ at all membrane potentials. 6. In the presence of a constant Ca2+ concentration (50 microM) increasing [K+] did not change the absolute level at which the current saturated; however the half‐saturating K+ concentration was greatly increased, indicating competitive inhibition between Ca2+ and K+ for the same site. 7. The data are consistent with a model based on Eyring rate theory for current conduction through ionic channels, in which we assume that the ions capable of entering the channel compete for a binding site that they must first occupy before proceeding on. The possible energy profile of the stretch‐activated channel was defined by optimizing the model parameters to obtain the best fit of the experimental data. Ca2+ was found to have a smaller dissociation constant and much longer occupancy time than Na+ or K+, thus accounting for its lower permeability and inhibitory effect on current conduction by other cations through the stretch‐activatable channel.

Age-dependent expression of high-voltage activated calcium currents during cerebellar granule cell development in situ.

Ca2+ currents play a crucial role during neuronal growth. In this paper we describe the development of Ca2+ currents using whole-cell patch-clamp recordings in granule cells of cerebellar slices obtained from 7- to 24day-old rats. Granule cells expressed high-voltage-activated (HVA) Ca2+ currents in different proportions. The percentage of cells with a measurable HVA current, and the size of HVA current increased in parallel with granule cell maturation. At less than 14 days HVA currents consisted of a fast- and slow-inactivating component, while at more than 19 days only the slow-inactivating component remained. The fast-inactivating component had faster activation and inactivation kinetics, a more negative threshold for activation, and steeper steady-state inactivation than the slow-inactivating component. Nifedipine (5 μM) partially blocked both components.ω-Conotoxin (5 μM,ω-CgTx) blocked the slow-inactivating component rather selectively. These results indicate that HVA currents change their gating and pharmacological properties during development. Although the mechanism at the molecular level remains speculative, the developmental changes of the HVA current are relevant to the processes of granule cell maturation and excitability.

Dual effect of Zn2+ on multiple types of voltage-dependent Ca2+ currents in rat palaeocortical neurons.

The effects of Zn(2+) were evaluated on high-voltage-activated Ca(2+) currents expressed by pyramidal neurons acutely dissociated from rat piriform cortex. Whole-cell, patch-clamp experiments were carried out using Ba(2+) (5 mM) as the charge carrier. Zn(2+) blocked total high-voltage-activated Ba(2+) currents with an IC(50) of approximately 21 microM. In addition, after application of non-saturating Zn(2+) concentrations, residual currents activated with substantially slower kinetics than control Ba(2+) currents. Both of the above-mentioned effects of Zn(2+) were also observed in high-voltage-activated currents recorded in the presence of nearly-physiological concentrations of extracellular Ca(2+) (1 and 2 mM) rather than Ba(2+). Under the latter conditions, 30 microM Zn(2+) inhibited high-voltage-activated currents somewhat less than observed in extracellular Ba(2+) (approximately 47% and approximately 41%, respectively, vs. approximately 59%), but slowed Ca(2+)-current activation to very similar degrees. All of the pharmacological components in which Ba(2+) currents could be dissected (L-, N-, P/Q-, and R-type) were inhibited by Zn(2+), the percentage of current blocked by 30 microM Zn(2+) ranging from 34 to 57%. Moreover, the activation kinetics of all pharmacological Ba(2+) current components were slowed by Zn(2+). Hence, the lower activation speed observed in residual Ba(2+) currents after Zn(2+) block is due to a true slowing of macroscopic Ca(2+)-current activation kinetics and not to the preferential inhibition of a fast-activating current component. The inhibitory effect of Zn(2+) on Ba(2+) current amplitude was voltage-independent over the whole voltage range explored (-60 to +30 mV), hence the Zn(2+)-dependent decrease of Ba(2+) current activation speed is not the consequence of a voltage- and time-dependent relief from block. Zn(2+) also caused a slight, but significant, reduction of Ba(2+) current deactivation speed upon repolarization, which is further evidence against a depolarization-dependent unblocking mechanism. Finally, the slowing effect of Zn(2+) on Ca(2+)-channel activation kinetics was found to result in a significant, extra reduction of Ba(2+) current amplitude when action-potential-like waveforms, rather than step pulses, were used as depolarizing stimuli. We conclude that Zn(2+) exerts a dual action on multiple types of voltage-gated Ca(2+) channels, causing a blocking effect and altering the speed at which channels are delivered to conducting states, with mechanism(s) that could be distinct.

Epidermal growth factor induces intracellular Ca2+ oscillations in microvascular endothelial cells

An increase in intracellular Ca2+ concentration ([Ca2+]i) may play a role in the proliferative effect of several growth factors. In this study, the changes in [Ca2+]i elicited by epidermal growth factor (EGF) in rat cardiac microvascular endothelial cells (CMEC) have been investigated by using fura‐2 conventional and confocal microscopy. A large heterogeneity in the latency and in the pattern of the Ca2+ response was found at each dose of EGF (2.5–100 ng/ml), whereas some cells displayed a non‐oscillatory behavior and others exhibited a variable number of Ca2+ oscillations. On average, the fraction of responsive cells, the total number of oscillations and the duration of the Ca2+ signal were higher at around 10 ng/ml EGF, while there was no dose‐dependence in the lag time and in the amplitude of the [Ca2+]i increase. EGF‐induced Ca2+ spikes were abolished by the tyrosine kinase inhibitor genistein, but not by its inactive analogue daidzein, and by the phospholipase C blocker NCDC. Only 1–2 transients could be elicited in Ca2+‐free solution, while re‐addition of extracellular Ca2+ recovered the spiking activity. The oscillatory signal was prevented by the SERCA inhibitor thapsigargin and abolished by the calcium entry blockers Ni2+ and La3+. Moreover, EGF‐induced Ca2+ transients were abolished by the InsP3 receptor blocker caffeine, while ryanodine was without effect. Confocal imaging microscopy showed that the Ca2+ response to EGF was localized both in the cytoplasm and in the nucleus. We suggest that EGF‐induced [Ca2+]i increase may play a role in the proliferative action of EGF on endothelial cells.

Voltage‐dependent Kinetics of N‐Methyl‐d‐aspartate Synaptic Currents in Rat Cerebellar Granule Cells

Decay kinetics of N‐methyl‐d‐aspartate excitatory postsynaptic currents (NMDA‐EPSCs) have been voltage‐dependent in some, but not all neurons studied so far, and almost no information has been available on the voltage‐dependence of the rising phase. In this work we investigated the effect of membrane potential on rising and decay kinetics of the NMDA‐EPSC in cerebellar granule cells using the tight‐seal whole‐cell recording technique. NMDA‐EPSCs were evoked by electrical mossy fibre stimulation in the presence of 10 μM 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione, 1.2 mM Mg2+ and 5 μM glycine. The rate of rise of NMDA‐EPSCs remained substantially unchanged when the cell was depolarized, indicating that the limiting step of channel opening was voltage‐insensitive. The NMDA‐EPSC, however, flattened around the peak and the time‐to‐peak increased. This observation was explained by the influence of decay. Decay was biphasic and slowed down with membrane depolarization. Moreover, the fast component of decay increased less than the slow component. This complex voltage‐dependence may extend the integrative role of the NMDA current during synaptic transmission.