Loretta Castelli

Kinetic and functional analysis of transient, persistent and resurgent sodium currents in rat cerebellar granule cells in situ: an electrophysiological and modelling study

Cerebellar neurones show complex and differentiated mechanisms of action potential generation that have been proposed to depend on peculiar properties of their voltage‐dependent Na+ currents. In this study we analysed voltage‐dependent Na+ currents of rat cerebellar granule cells (GCs) by performing whole‐cell, patch‐clamp experiments in acute rat cerebellar slices. A transient Na+ current (INaT) was always present and had the properties of a typical fast‐activating/inactivating Na+ current. In addition to INaT, robust persistent (INaP) and resurgent (INaR) Na+ currents were observed. INaP peaked at ∼−40 mV, showed half‐maximal activation at ∼−55 mV, and its maximal amplitude was about 1.5% of that of INaT. INaR was elicited by repolarizing pulses applied following step depolarizations able to activate/inactivate INaT, and showed voltage‐ and time‐dependent activation and voltage‐dependent decay kinetics. The conductance underlying INaR showed a bell‐shaped voltage dependence, with peak at −35 mV. A significant correlation was found between GC INaR and INaT peak amplitudes; however, GCs expressing INaT of similar size showed marked variability in terms of INaR amplitude, and in a fraction of cells INaR was undetectable. INaT, INaP and INaR could be accounted for by a 13‐state kinetic scheme comprising closed, open, inactivated and blocked states. Current‐clamp experiments carried out to identify possible functional correlates of INaP and/or INaR revealed that in GCs single action potentials were followed by depolarizing afterpotentials (DAPs). In a majority of cells, DAPs showed properties consistent with INaR playing a role in their generation. Computer modelling showed that INaR promotes DAP generation and enhances high‐frequency firing, whereas INaP boosts near‐threshold firing activity. Our findings suggest that special properties of voltage‐dependent Na+ currents provides GCs with mechanisms suitable for shaping activity patterns, with potentially important consequences for cerebellar information transfer and computation.

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.

Cu2+, Co2+, and Mn2+ modify the gating kinetics of high-voltage-activated Ca2+ channels in rat palaeocortical neurons.

The effects of three divalent metal cations (Mn2+, Co2+, and Cu2+) on high-voltage-activated (HVA) Ca2+ currents were studied in acutely dissociated pyramidal neurons of rat piriform cortex using the patch-clamp technique. Cu2+, Mn2+, and Co2+ blocked HVA currents conducted by Ba2+ (IBa) with IC50 of ˜920 nM, ˜58 µM, and ˜65 µM, respectively. Additionally, after application of non-saturating concentrations of the three cations, residual currents activated with substantially slower kinetics than control IBa. As a consequence, the current fraction abolished by the blocking cations typically displayed, in its early phase, an unusually fast-decaying transient. The latter phenomenon turned out to be a subtraction artifact, since none of the pharmacological components (L-, N-, P/Q-, and R-type) that constitute the total HVA currents under study showed a similarly fast early decay: hence, the slow activation kinetics of residual currents was not due to the preferential inhibition of a fast-activating/inactivating component, but rather to a true slowing effect of the blocker cations. The percent IBa-amplitude inhibition caused by Mn2+, Co2+, and Cu2+ was voltage-independent over the whole potential range explored (up to +30 mV), hence the slowing of IBa activation kinetics was not due to a mechanism of voltage- and time-dependent relief from block. Moreover, Mn2+, Co2+, and Cu2+ significantly reduced IBa deactivation speed upon repolarization, which also is not compatible with a depolarization-dependent unblocking mechanism. The above results show that 1) Cu2+ is a particularly potent HVA Ca2+-channel blocker in rat palaeocortical neurons; and 2) Mn2+, Co2+, and Cu2+, besides exerting a blocking action on HVA Ca2+-channels, also modify Ca2+-current activation and deactivation kinetics, most probably by directly interfering with channel-state transitions.

Synthesis and biological evaluation of amidine, guanidine, and thiourea derivatives of 2-amino(6-trifluoromethoxy)benzothiazole as neuroprotective agents potentially useful in brain diseases.

A series of amidine, thiourea, and guanidine derivatives of 2-amino-6-(trifluoromethoxy)benzothiazole termed 2, 3, and 4, respectively, and structurally related to riluzole, a neuroprotective drug in many animal models of brain disease, have been synthesized. The biological activity of compounds 2a-e, 3a-f, and 4a,b was preliminarily tested by means of an in vitro protocol of ischemia/reperfusion injury. The results demonstrated that 2c and 3a-d significantly attenuated neuronal injury. Selected for testing of their antioxidant properties, compounds 3a-d were shown to be endowed with a direct ROS scavenging activity. Compounds 3b and 3d were also evaluated for their activity on voltage-dependent Na(+) and Ca(2+) currents in neurons from rat piriform cortex. At 50 microM, compound 3b inhibited the transient Na(+) current to a much smaller extent than riluzole, whereas 3d was almost completely ineffective.

Analysis of resurgent sodium-current expression in rat parahippocampal cortices and hippocampal formation

The resurgent Na(+) current (I(NaR)) is a component of neuronal voltage-dependent Na(+) currents that is activated by repolarization and is believed to result from an atypical path of Na(+)-channel recovery from inactivation. So far, I(NaR) has only been identified in a small number of central neuronal populations in the cerebellum, diencephalon, and brainstem. The possible presence and roles of I(NaR) in neurons of the cerebral cortex and temporal-lobe memory system are still uncharacterized. In this study whole-cell, patch-clamp experiments were carried out in acute rat brain slices to investigate I(NaR) expression and properties in several neuronal populations of the parahippocampal region and hippocampal formation. Specifically, we examined pyramidal neurons of perirhinal cortex areas 36 and 35 (layers II and V); neurons of superficial and deep layers of medial entorhinal cortex (mEC); dentate gyrus (DG) granule cells; and pyramidal cells of the CA3 and CA1 hippocampal fields. I(NaR) was found to be thoroughly expressed in parahippocampal cortices. The most consistent and prominent I(NaR) expression was observed in mEC layer-II cells. A vast majority of areas 36 and 35 neurons (both in layers II and V) and mEC layer-III and -V neurons were also endowed with I(NaR), although at lower amplitude levels. I(NaR) was expressed by approximately 60% of DG granule cells and approximately 35% of CA1 pyramidal cells of the ventral hippocampus, whereas it was never observed in CA3 neurons (both in the ventral and dorsal hippocampus) and CA1 neurons of the dorsal hippocampus. The biophysical properties of I(NaR) were very similar in all of the neuronal types in which the current was observed, with a peak in the current-voltage relationship at -35/-40 mV. Our results show that the parahippocampal region and part of the hippocampal formation are sites of major I(NaR) expression, and provide a new basis for further studies on the molecular correlates of I(NaR).

Resurgent Na+ current in pyramidal neurones of rat perirhinal cortex: axonal location of channels and contribution to depolarizing drive during repetitive firing

The perirhinal cortex (PRC) is a supra‐modal cortical area that collects and integrates information originating from uni‐ and multi‐modal neocortical regions and directed to the hippocampus. The mechanisms that underlie the specific excitable properties of the different PRC neuronal types are still largely unknown, and their elucidation may be important in understanding the integrative functions of PRC. In this study we investigated the expression and properties of resurgent Na+ current (INaR) in pyramidal neurones of rat PRC area 35 (layer II). Patch‐clamp experiments in acute PRC slices were first carried out. A measurable INaR was expressed by a large majority of neurones (31 out of 35 cells). INaR appeared as an inward, slowly decaying current elicited upon step repolarization after depolarizations sufficient to induce nearly complete inactivation of the transient Na+ current (INaT). INaR had a peak amplitude of ∼2.5% that of INaT, and showed the typical biophysical properties also observed in other neuronal types (i.e. cerebellar Purkinje and granule cells), including a bell‐shaped current–voltage relationship with a peak at approximately −40 mV, and a characteristic acceleration of activation and decay speed at potentials negative to −45 mV. Current‐clamp experiments were then carried out in which repetitive action‐potential discharge at various frequencies was induced with depolarizing current injection. The voltage signals thus obtained were then used as command waveforms for voltage‐clamp recordings. These experiments showed that a Na+ current identifiable as INaR activates in the early interspike phase even at relatively high firing frequencies (20 Hz), thereby contributing to the depolarizing drive and possibly enhancing repetitive discharge. In acutely dissociated area 35 layer II neurones, as well as in nucleated patches from the same neurones, INaR was never observed, despite the presence of typical INaTs. Since in both preparations neuronal processes are lost, we carried out experiments of focal tetrodotoxin (TTX) application in slices to verify whether the channels responsible for INaR are located in compartment(s) different from the soma. We found that TTX preferentially inhibited INaR when applied close to the site of axon emergence from soma, whereas application to the apical pole of the soma had a significantly smaller effect on INaR. Our results indicate that in area 35 pyramidal cells INaR is largely generated in the axon initial segment, where it may participate in setting the coding properties of these neurones.

The Duration and Amplitude of the Plateau Phase of ATP- and ADP-Evoked Ca2+ Signals Are Modulated by Ectonucleotidases in in situ Endothelial Cells of Rat Aorta

ATP has a long-lasting vasodilatory effect, possibly due to its capability to induce a prolonged increase in the intracellular Ca2+ concentration ([Ca2+]i) in endothelial cells (EC) and activate constitutive nitric oxide synthase. However, contradictory data have been reported regarding the time course of ATP-evoked Ca2+ signals in in situ EC. In particular, short-duration Ca2+ signals have been reported, which might be thought to be unable to sustain a prolonged, NO-induced vasodilation. The current experiments were therefore performed in in situ EC of rat aorta in order to more fully define the time course of ATP-evoked Ca2+ signals. 20 µM ATP evoked a short-lasting Ca2+ signal. However, medium stirring, high agonist concentrations, inhibition of ectonucleotidases and application of a poorly hydrolyzable agonist evoked long-lasting Ca2+ signals (up to 20 min at 37°C). These studies suggest that ATP is able to sustain a prolonged [Ca2+]i increase, unless ectonucleotidase activity reduces the agonist concentration near the EC surface to subthreshold values, quickly cutting the Ca2+ signal. Furthermore, the amplitude of the long-lasting phase of the Ca2+ signal depended on the balance between agonist degradation by ectonucleotidases and agonist transport, by diffusion and convection, from bulk solution to the EC surface.

High-voltage-activated Ca2+ currents show similar patterns of expression in stellate and pyramidal cells from rat entorhinal cortex layer II

High-voltage-activated (HVA) Ca2+ currents were studied in acutely isolated neurons from rat entorhinal cortex (EC) layer II. Stellate and pyramidal cells, the two main neuronal types of this structure, were visually identified based on morphological criteria. HVA currents were recorded by applying the whole-cell, patch-clamp technique, using 5-mM Ba2+ as the charge carrier. In both neuronal types, the amplitude of total HVA Ba2+ currents (IBas) showed a significant tendency to increase with postnatal age in the time window considered [postnatal day 15 (P15) to P28-29]. At P20-P29, when IBa expression reached stable levels, IBa density per unit of membrane area was not different in stellate versus pyramidal cells. The same was also observed when Ca2+, instead of Ba2+, was used as the charge carrier. The pharmacological current subtypes composing total HVA currents were characterized using selective blockers. Again, no significant differences were found between stellate and pyramidal cells with respect to the total-current fractions attributable to specific pharmacological Ca2+ channel subtypes. In both cell types, approximately 52-55% of total IBas was abolished by the L-type channel blocker, nifedipine (10 microM), approximately 23-30% by the N-type channel blocker, omega-conotoxin GVIA (1 microM), approximately 22-24% by the P/Q-type channel blocker, omega-agatoxin IVA (100 nM), and approximately 11-13% remained unblocked (R-type current) after simultaneous application of L-, N-, and P/Q-type channel blockers. The Cav 2.3 (alpha1E) channel blocker, SNX-482 (100 nM), abolished approximately 57-62% of total R-type current. We conclude that HVA Ca2+ currents are expressed according to similar patterns in the somata and proximal dendrites of stellate and pyramidal cells of rat EC layer II.

Synthesis and biological evaluation of a new class of benzothiazines as neuroprotective agents

Neurodegenerative diseases are disorders related to the degeneration of central neurons that gradually lead to various, severe alterations of cognitive and/or motor functions. Currently, for no such diseases does any pharmacological treatment exist able to arrest its progression. Riluzole (1) is a small molecule able to interfere with multiple cellular and molecular mechanisms of neurodegeneration, and is the only approved treatment of amyotrophic lateral sclerosis (ALS), the progression of which proved to significantly slow, thus increasing somewhat average survival. Here we report the synthesis of differently functionalized 4H-3,1-benzothiazine (5-6) and 2H-1,4-benzothiazine (7) series as superior homologues of 1. Biological evaluation demonstrated that amidine 4H-3,1-benzothiazine derivatives 5b-d can reduce glutamate and LDH release in the oxygen/glucose deprivation and reperfusion model (OGD/R) applied to brain slices with a higher potency than 1. Moreover the mentioned compounds significantly reduce glutamate- and 6-hydroxydopamine (6-OHDA)-induced cytotoxicity in neuroblastoma cells. In addition, the same compounds limit ROS formation in both neuronal preparations. Finally, 5c proved effective in inhibiting neuronal voltage-dependent Na+ and Ca2+-channels, showing a profile comparable with that of 1.