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Zn²⁺ Slows Down Ca_V3.3 Gating Kinetics: Implications for Thalamocortical Activity

¹Divisione di Farmacologia, Dipartimento di Neuroscienze, Facoltà di Medicina e Chirurgia, Università di Napoli Federico II, Naples, Italy; ²Montreal Neurological Institute and Department of Neurology and Neurosurgery, McGill University, Montreal, Quebec, Canada; and ³Dipartimento di Medicina Sperimentale, Prima Facoltà di Medicina e Chirurgia, Università di Roma Sapienza, Rome, Italy

Submitted 21 August 2006; accepted in final form 15 August 2007

	ABSTRACT

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
We employed whole cell patch-clamp recordings to establish the effect of Zn²⁺ on the gating the brain specific, T-type channel isoform Ca_V3.3 expressed in HEK-293 cells. Zn²⁺ (300 µM) modified the gating kinetics of this channel without influencing its steady-state properties. When inward Ca²⁺ currents were elicited by step depolarizations at voltages above the threshold for channel opening, current inactivation was significantly slowed down while current activation was moderately affected. In addition, Zn²⁺ slowed down channel deactivation but channel recovery from inactivation was only modestly changed. Zn²⁺ also decreased whole cell Ca²⁺ permeability to 45% of control values. In the presence of Zn²⁺, Ca²⁺ currents evoked by mock action potentials were more persistent than in its absence. Furthermore, computer simulation of action potential generation in thalamic reticular cells performed to model the gating effect of Zn²⁺ on T-type channels (while leaving the kinetic parameters of voltage-gated Na⁺ and K⁺ unchanged) revealed that Zn²⁺ increased the frequency and the duration of burst firing, which is known to depend on T-type channel activity. In line with this finding, we discovered that chelation of endogenous Zn²⁺ decreased the frequency of occurrence of ictal-like epileptiform discharges in rat thalamocortical slices perfused with medium containing the convulsant 4-aminopyridine (50 µM). These data demonstrate that Zn²⁺ modulates Ca_V3.3 channel gating thus leading to increased neuronal excitability. We also propose that endogenous Zn²⁺ may have a role in controlling thalamocortical oscillations.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
T-type channels—which possess peculiar kinetic properties along with differences in ionic permeation when compared with high-voltage activated Ca²⁺ channels (Cataldi et al. 2002; Tsien et al. 1988)—are crucial in regulating the periodic discharge of cells in relay thalamic nuclei and in nucleus reticularis thalami (nRT). These neurons are an integral component of the thalamocortical system that is known to be involved in physiological and pathological conditions such as absence epilepsy (Destexhe and Sejnowski 2003; Perez-Reyes 2003; Steriade 2005; Steriade et al. 1993).

At least three different T-type channel isoforms are expressed in neurons as well as in different compartments of the same neuron (Joksovic et al. 2005a; Talley et al. 1999). These include Ca_V3.1 (Perez-Reyes et al. 1998) and Ca_V3.2 (Cribbs et al. 1998) channels—which carry the classical fast inactivating T-type current (Carbone and Lux 1984; Fox et al. 1987; Kostyuk et al. 1988)—and the Ca_V3.3 channels (Lee et al. 1999), carrying more slowly inactivating currents such as those recorded in nRT cells (Huguenard and Prince 1992). As these different isoforms have specific kinetic properties, neuron behavior will depend on the type of T-current it expresses. Specifically, because slow inactivating Ca_V3.3 channels are required to fire action potentials at a high frequency and for prolonged periods of time (Kozlov et al. 1999), their presence in nRT cells may be essential for periodic spiking (Destexhe and Sejnowski 2003).

Although point mutations affecting channel gating have been identified in absence epilepsy (Khosravani et al. 2004; Vitko et al. 2005), it is unknown how endogenous neurotransmitters or neuromodulators influence T-type-dependent spiking. Here we explore the hypothesis that Ca_V3.3 channel gating could be affected by Zn²⁺, which is stored with glutamate in the terminals of a subset of cells, also called the gluzinergic neurons, and coreleased with this excitatory neurotransmitter (Frederickson et al. 2000, 2005; Howell et al. 1984; Qian and Noebels 2005). That Zn²⁺ may affect T-type channel gating is suggested by the evidence that this metal does affect the gating kinetics of other voltage-gated ion channels (Harrison and Gibbons 1994; Mathie et al. 2006) and greatly increases neuronal excitability in the thalamus, thus suggesting its involvement in absence epilepsy (Gibbs et al. 2000; Wu et al. 2004). Therefore we explored here the hypothesis that Zn²⁺ influences the slowly inactivating T-type current expressed in nRT neurons—which is encoded by the Ca_V3.3 gene (Talley et al. 1999)—by evaluating the effects of exogenous Zn²⁺ on Ca_V3.3 channels heterologously expressed in stably transfected HEK-293 cells as their currents are identical to those recorded in nRT neurons (Joksovic et al. 2005b). This experimental approach allows study of this channel type in isolation without any interference from other ion channels. By doing so, we found that this ion slowed down the gating of Ca_V3.3 channel heterologously expressed in HEK-293 cells. We also discovered that when the changes in gating were translated into simulated nRT neurons by using the NEURON software interface (Hines and Carnevale 1997), the frequency and duration of T-type-dependent action potential trains increased. Finally, we established in rat thalamocortical slices maintained in vitro that chelating endogenous Zn²⁺ decreased the frequency of occurrence of ictal-like epileptiform discharges generated in the presence of the K⁺ channel blocker 4-aminopyridine (4AP).

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
Cell culture

Stably transfected HEK-293 cells (courtesy of Dr. Perez-Reyes), expressing the rat Ca_V3.3a T-type Ca²⁺ channel isoform (Ca_V3.3 cells) (Lee et al. 1999), were cultured in a humidified 5% CO₂ atmosphere using Dulbecco's modified Eagle's medium supplemented with 5% fetal calf serum, 100 IU/ml penicillin, 100 µg/ml streptomycin, and nonessential amino acids; they were kept under constant selection with 1 g/l geneticin. For electrophysiological recordings, cells were plated on poly-L-lysine (30 µg/ml) precoated glass coverslips and used 24–48 h after plating.

Whole cell patch-clamp recordings

Experiments were performed using the whole cell configuration of the patch-clamp technique as previously reported (Cataldi et al. 2004). Briefly, the coverslips used for cell culture were placed into a laminar flow chamber (Warner Instrument, Hamden, CT) mounted on the stage of a Axiovert 25 Zeiss inverted microscope. The cells were continuously superfused by a gravity-fed multilane system controlled by microcomputer-operated electro-valves (Cell Microcontrol, Norfolk, VA) that allowed the rapid exchange of the perfusing solution. Whole cell recordings were performed at room temperature (25–26°C).

Ruptured patches were obtained by suction using fire-polished borosilicate electrodes having a final resistance of 3–5 M and back filled with a CsCl-based internal solution containing (in mM) 110 CsCl, 30 tetraethyl ammonium chloride (TEA-Cl), 10 EGTA, 2 MgCl₂, 10 HEPES, 8 glucose, 15 phosphocreatine di-sodium salt, 5 ATP di-sodium salt, and 1 cAMP sodium salt (pH 7.4 adjusted with CsOH). Unless otherwise specified, the external solution contained (in mM) 125 N-methyl-D-glucamine (NMDG), 2 CaCl₂, 1 MgCl₂, 5 CsCl, 10 glucose, and 10 HEPES (pH 7.4 adjusted with HCl, yielding a final Cl^– concentration of 110 mM, as determined by silver nitrate titration). Under these experimental conditions, Ca²⁺ currents are presumably recorded in isolation because K⁺ currents are blocked by CsCl present in both internal and external solution and by TEA-Cl contained in the internal solution; in addition, no detectable Na⁺ current is expected to flow through the membrane because of the lack of Na⁺ channels in HEK-293 cells and because of the substitution of external Na⁺ with NMDG. The osmolarity of the external solution was adjusted to 300 mosM by adding an appropriate amount of sucrose. All the voltages reported in the text or shown in figures represent the values obtained after correction for junction potential (7 mV in accordance with the estimate provided by the junction potential calculator included in the pClamp8 software) and charge screening (4 mV).

Test pulses were generated and the ensuing currents were collected with an Axon 200 B patch-clamp amplifier (Molecular Devices, Foster City, CA) driven by the pClamp6 software running on a personal computer. Currents were filtered at 2 kHz with the amplifier built-in Bessel filter, and leak currents were subtracted on-line with a P/4 protocol. Using the specific commands of the amplifier, on-line correction of membrane capacitance and series resistance was routinely performed. Data were stored onto the hard disk of the personal computer. Off-line analyses were then performed with the Clampfit 8.0 (Molecular Devices) and Sigmaplot 5.0 (SPSS, Chicago, IL) softwares.

Patch-clamp data analysis and computer modeling

The voltage-dependence of activation constants, V_1/2m (the membrane potential at which the current is half-maximal) and k_m (the voltage required to change the conductance, g, e-fold), were calculated fitting the I-V data to the Goldman and Hodgkin current equation as reported by McCormick and Huguenard (1992)

(1)

where I_Ca is the current carried by Ca²⁺ ions (in A) P is the maximum permeability in cm³/s, z is 2 (the valence of Ca²⁺), V is the applied voltage in V, F is the Faraday constant, R is the gas constant, T is the absolute temperature in Kelvin degrees and represents fractional conductance

(2)

Because

(3)

for fitting purposes, Eq. 2 was expressed in the form

(4)

The values of activation (_m) and inactivation time constants (_h) of the inward Ca²⁺ currents evoked by membrane depolarization to suprathreshold voltages were obtained by fitting current data to the Hodgkin and Huxley equation

(5)

where V is the applied voltage, E_Ca is the reversal potential of Ca²⁺ currents, and the fraction of open activation (m) and inactivation (h) gates are calculated from the relations

(6)

(7)

and

(8)

(9)

where V_1/2m and V_1/2h are respectively the half-voltage of activation and of inactivation and k_m and k_h are the slopes respectively of activation and inactivation.

Data fitting was performed using the NEUROFIT software (developed by Allan Willms, Gregory Ewing, and Howie Kuo at the Department of Mathematics and Statistics of the University of Guelph, Guelph, Canada and freely downloadable at the internet address www.uoguelph.ca/awillms/neurofit/). Tail currents appearing during channel deactivation were fitted to a bi-exponential equation with fast (₁) and slow (₂) time constants. For fitting purposes, the kinetics of tail relaxation was expressed by the single kinetic parameter weighted (_w) that was calculated using the equation

(10)

where A1 and A2 represent the relative amplitude of the fast (A₁) and of the slow (A₂) decay component of the tail current.

Simulations of current-clamp recordings in modeled reticular thalamic cells were performed using the NEURON 5.8 software (Hines and Carnevale 1997), freely downloadable at www.neuron.yale.edu/neuron. The IT2 model file required to run the NEURON simulation was obtained by entering the kinetics parameters—obtained from the experiments described in the results section—into the model file developed by Murbartian et al. (2004), which was kindly provided by Dr. E. Perez-Reyes (Department of Pharmacology, University of Virginia, Charlottesville, VA). This mod file used for Ca_V3.3 cells is a modified version of the model file originally developed by Destexhe et al. (1996) for thalamic reticular cells (downloadable at Dr. Desthexe web site, http://cns.iaf.cnrs-gif.fr/Main.html). The NEURON software extrapolates the changes in membrane voltage evoked by the injection of current into the cells by applying an m² Hogdkin Huxley formalism according to Eq. 5. To get this software running, the fitting parameters needed to calculate m and h, and the kinetic constants _m and _h have to be entered into the program. The values of the kinetic constants of activation (V_1/2m and k_m) were obtained fitting the I-V data to the Goldman and Hodgkin current equation as described in the preceding text, whereas those for inactivation (V_1/2h and k_h) were obtained by fitting the steady-state inactivation data to the Boltzmann equation

(11)

Both the voltage dependence of the activation time constant (_m) and that of the inactivation time constant (_h) were specified by the equation

(12)

where V₁, V₂ and k₁, k₂ represent the half-voltages (V_1/2) and slopes of increasing and decreasing components of the bell shaped curves generated by plotting _m or _h data obtained at the different tested voltages as detailed in RESULTS.

Using Eq. 5, the NEURON software calculates I_Ca as a function of Ca²⁺ conductance g_Ca (5). In our simulation of nRT neurons bathed with 2 mM Ca²⁺ and no Zn²⁺, we used the g_Ca value of 0.0008 (Siemens/cm²) reported in the original nRT model developed by Destexhe et al. (1996) and adapted to HEK cells by Murbartian et al. (2004). Conversely, when the addition of Zn²⁺ was simulated, to reproduce the 55% decrease in Ca²⁺ permeability that we observed in our whole cell recordings, we lowered the above-reported gCa value by introducing a multiplication factor of 0.455, according to the equation

(13)

The entire simulation was run using the option "burst behavior in a three-compartment model" keeping the resting potential at –70 mV, a value close to those observed in thalamic neurons in vivo during sleep (Fuentealba et al. 2005).

Field potential recordings in talamocortical slices

Thalamocortical slices were obtained using the modifications proposed by Biagini et al. (2001) of the original procedures described by Agmon and Connor (1991). Procedures were approved by the Canadian Council of Animal Care. Briefly young male Sprague-Dawley rats (15–28 days old) were deeply anesthesized with isoflurane and killed by decapitation. The skull was quickly opened, and the brain was removed, placed into an ice-cold oxygenated sucrose-containing solution (in mM: 206 sucrose, 3.5 KCl, 1 CaCl₂, 2 MgSO₄, 1.25 NaH₂PO₄, 26 NaHCO₃, 1 MgCl₂, 10 glucose, 0.4 ascorbic acid, and 1 kynurenic acid) and left to refrigerate into this solution for 3–4 min. Then the brain was transferred onto a Plexiglas ramp the surface of which makes a 10° angle with the horizontal plane. Brain stem and cerebellum were removed with a razor blade by a vertical cut. Then the frontal pole was vertically cut along a plane describing a 45° angle with the sagittal plane. During the entire procedure, the brain was repeatedly irrigated with ice-cold cutting solution. The cut surface of the frontal pole was glued onto the chamber of a Leica VT1000S vibratome (Richmond Hill, Ontario, Canada). Then this chamber was filled with an ice-cold cutting solution, which was continuously oxygenated via an oxygen line, and the brain was cut in 450-µm-thick slices. Only the slices clearly showing bundles of fibers connecting cortex and thalamus were chosen for electrophysiological recordings. On average three to four slices per animal showed these characteristics. Chosen slices were transferred onto the surface of a custom-made interface perfusion chamber and let equilibrate for 1–1.5 h while continuously superfused with oxygenated artificial cerebrospinal fluid (ACSF) (containing in mM: 124 NaCl, 2 KCl, 1.25 KH₂PO4, 0.5 MgSO₄, 2 CaCl₂, 26 NaHCO₃, and 10 glucose) at 32°C. At the end of this equilibration period, the K⁺ channel blocker 4AP (50 µM) was added to the medium to induce spontaneous epileptiform activity (Biagini et al. 2001), and the slices were perfused with this 4AP containing ACSF for an additional hour. ACSF-filled borosilicate electrodes (resistance = 10 M) were placed under visual control with the help of a stereomicroscope into the parietal cortex, the ventrobasal nucleus (VB) of the thalamus and the nRT. Field potentials were recorded with preamplifier probes connected with a Cyberamp 300 signal conditioner (Axon Instruments, Molecular Devices), digitized at 5 kHz with a Digidata 1200 A/D converter and stored as pClamp8 files (Axon Instruments, Molecular Devices) for off-line analysis. After recording the baseline activity for >30 min, the cell membrane–permeant Zn²⁺ chelator N,N,N',N-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN) was added to the solution and data were recorded in the presence of this drug for additional 30–45 min. Finally, TPEN was washed out and data were acquired for 30 more min. Data analysis was performed off-line with the Clampfit 8 software (Axon Instruments, Molecular Devices).

Drugs

Geneticin and ATP-sodium salt were obtained from Calbiochem (La Jolla, CA), whereas CsOH was from Aldrich Chemicals (Milan, Italy). DMEM, FCS, penicillin, streptomycin, and nonessential amino acids were from Invitrogen (San Giuliano Milanese, Italy). All the other chemicals were of analytical grade and were from Sigma (Milan, Italy).

Data analysis

All data are reported as means ± SE. When comparing two data sets, the Student's t-test for paired or unpaired data were used as appropriate whereas, repeated-measure ANOVA was used for multiple group comparison. Statistical comparisons were carried out using the Graph-PAD 2.04 software suite (GraphPad Software, San Diego, CA). Curve fitting was performed with the Sigma-plot 5.0 (SPSS) or the N-fit (The University of Texas, Medical Branch at Galveston, Galveston, TX) software.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
Effect of Zn²⁺ on steady-state Ca_V3.3 channel activation and inactivation

To study the effect of Zn²⁺ (300 µM) on the voltage dependence of activation, current versus voltage (I-V) curves were obtained from Ca_V3.3 cells. Briefly, repetitive step depolarizations were delivered at progressively more positive voltages both in the presence and in the absence of this metal (Fig. 1A). The values of half voltage (V_1/2m) and slope (k_m) of the channel activation were extrapolated by fitting the data to the Goldman and Hodgkin current equation reported in METHODS (Fig. 1B). The values obtained with this approach were not significantly different in the two experimental groups neither in the case of V_1/2m (–48.36 ± 0.73 vs. –48.77 ± 1.21 mV, before and after Zn²⁺ addition, respectively) nor in the case of k_m (8.26 ± 0.2 vs. 7.25 ± 0.63, before and after Zn²⁺ addition, respectively). Curve fitting also provided an estimate of the whole cell Ca²⁺ permeability in the presence and in the absence of Zn²⁺, thereby showing that this metal reduces cell permeability by 55.6% (from 0.036 ± 0.004 to 0.0237 ± 0.003 cm³/s, P < 0.001). This reduction is close to that of inward Ca²⁺ current measured either with the protocol shown in Fig. 1, A and B (56.7 ± 7.4%) or by repetitive step depolarization up to –11 mV (56.7 ± 6.9%; Fig. 1C) (cf., Jeong et al. 2003). These findings suggest that even in the presence of high micromolar concentrations of Zn²⁺ Ca_V3.3 channel activity is not totally blocked and, therefore its presence in the extracellular solution does not hamper the flow of Ca²⁺ through Ca_V3.3 channels.

View larger version (20K): [in this window] [in a new window]   FIG. 1. Effects exerted by Zn2+ on voltage dependence of activation and whole cell Ca2+ permeability in CaV3.3 cells. A: representative current traces elicited by consecutive step depolarizations (75-ms duration) to progressively more positive voltages (from –71 to +19 mV in increments of 10 mV) in a CaV3.3 cell perfused with 2 mM Ca2+ before (top) and after (bottom) addition of 300 µM Zn2+ to the perfusion medium. B: mean current-to-voltage plots showing, as a function of step voltage, the means ± SE of the maximal current amplitudes reached in 7 cells before () and after () the addition of 300 µM Zn2+ to the perfusion medium. —, obtained by fitting the data to the Goldman-Hodgkin current equation. *P < 0.05; **P < 0.01. C: current traces evoked by membrane depolarization (up to –11 mV, 75 ms) in a CaV3.3 cell held at –101 mV and perfused with 2 mM Ca2+ before and after Zn2+ addition. In this type of experiment, CaV3.3 cells (n = 5) were continuously perfused with 2 mM Ca2+ and 50 s after the beginning of current recordings, 300 µM Zn2+ was added to the extracellular solution. In A and C, voltage protocols are reported in the insets, where numbers indicate the voltage applied expressed in mV.

View larger version (30K): [in this window] [in a new window]   FIG. 2. CaV3.3 current steady-state inactivation in the presence and absence of Zn2+. A: current traces evoked by repeated membrane depolarizations up to –11 mV (75-ms duration; frequency 0.07 Hz) that were delivered after a conditioning prepulse of 10-s duration in 2 CaV3.3 cells perfused with 2 mM Ca2+ (top) or with 2 mM Ca2++300 µM Zn2+ (bottom); these cells are representative of 2 different groups each of 10 cells. The voltage protocol is reported in the inset where numbers indicate the voltage applied expressed in mV. B: steady-state activation and inactivation curves obtained in cells perfused with 2 mM Ca2+ in the absence (n = 10) and in the presence (n = 10) of 300 µM Zn2+. Steady-state inactivation curves were drawn from the data obtained by applying the protocol shown in A. Each data point represents the means ± SE of the individual values of the maximal inward current amplitude attained in all the cells of the control and Zn2+ groups when the plasmamembrane potential was depolarized up to 0 mV after each prepulse. h curves (—) were obtained by fitting the data to the Boltzman's Eq. 11 as reported in METHODS.

Accordingly to the Hodgkin and Huxley formalism (1952), channel opening and inactivation do not occur instantaneously but take time to appear. Therefore the amount of current that goes through a voltage-gated ion channel at each given time after a depolarization is determined not only by membrane voltage and by the steady-state activation and inactivation of the channels but also by the kinetics of the activation and inactivation gates, which are both intrinsically voltage dependent and can be described with appropriate activation (_m) and inactivation (_h) time constants. This strict voltage dependence of activation and inactivation kinetics is know to apply also to T-type channels as originally described by Coulter et al. (1989) in relay thalamic neurons and may have important functional implications when rapid phenomena such as action potential generation at the postsynaptic membrane of the gluzinergic synapse, occur. Accordingly, we determined the effect of Zn²⁺ on Ca_V3.3 channel activation and inactivation gate kinetics at different membrane voltages. For values of membrane potential more positive than the threshold for Ca_V3.3 channel opening, activation (_m) and inactivation (_h) time constants were determined by fitting the current traces elicited by the step depolarization protocol reported in Fig. 1 to the Hodgkin and Huxley m²h equation described by McCormick and Huguenard (1992). Using this approach we found that Zn²⁺ significantly slowed both the activation time constant (_m) and the inactivation time constant (_h; Fig. 3A). When Ca_V3.3 cells were exposed to 100 µM Zn²⁺ a significant slowing down of current inactivation was observed (_h percent increase 363,6 ± 117,0% of control values, P < 0.05, repeated-measure ANOVA), whereas channel activation was not affected (Fig. 3B). Conversely, Zn²⁺ was ineffective at concentrations of 30 µM (Fig. 3B).

View larger version (25K): [in this window] [in a new window]   FIG. 3. Effect of different Zn2+ concentrations on CaV3.3 current kinetics. A: slowing of CaV3.3 current kinetics on exposure to 300 µM Zn2+. Current traces were recorded in the same CaV3.3 cell in response to consecutive step depolarizations delivered at progressively more positive voltages. Cells were held at –90 mV, and step voltage was increased in 10-mV increments with an interepisode interval of 10 s. Long depolarizations (300 ms) were applied to resolve the slow inactivation of current traces at the more negative voltages. B: current traces recorded in three different CaV3.3 cells before and after the addition to the extracellular solution of 10, 30, and 100 µM Zn2+ (from the top to the bottom). Cells were held at –91 mV and step depolarized up to –11 mV. Long depolarizations (300 ms) were applied to resolve the slow inactivation of current traces at the more negative voltages. Each trace is from a different cell representative of a group of 5. In A and B, voltage protocols are reported in the insets, where numbers indicate the voltage applied expressed in mV.

View larger version (25K): [in this window] [in a new window]   FIG. 4. - CaV3.3 current deactivation in the presence and in the absence of Zn2+. A: tail current traces evoked by membrane repolarization in 2 different cells, one perfused with 2 mM Ca2+ without Zn2+ and the other with 2 mM Ca2+ plus 300 µM Zn2+. Tail currents were evoked by membrane repolarization at –111 mV as indicated in the inset. B: tail current traces evoked by membrane repolarization at different voltages in 2 CaV3.3 cells exposed to 2 mM Ca2+ with or without 300 µM Zn2+. For sake of clarity, only the tails are shown in A and B. C: mean 1 (left) and 2 (right) values as a function of the repolarization voltage obtained by the biexponential fitting of tail current decay. Each data point is the means ± SE of the values calculated by the biexponential fitting of the current traces recorded at each Zn2+ concentration in 6 cells. *P < 0.05; **P < 0.01.

View larger version (26K): [in this window] [in a new window]   FIG. 5. CaV3.3 current recovery from inactivation in the presence and absence of Zn2+. Left: families of current traces elicited by repeated 80-ms step depolarizations up to –11 mV, delivered at progressively longer time intervals after repolarizing the plasma-membrane to –101 (A) or –71 (B) mV after a 10-s inactivating prepulse to –51 mV. Both in A and B, 2 different CaV3.3 cells, one perfused with 2 mM Ca2+ (top) and the other with 2 mM Ca2+ plus 300 µM Zn2+ (bottom), are shown. Right: mean recovery from inactivation curves obtained by averaging the data obtained in CaV3.3 cells repolarized to –101 mV (A) or –71 mV (B) perfused with 2 mM Ca2+ in the presence and in the absence of 300 µM Zn2+. Each data point represents the means ± SE of all the maximal amplitude of the currents elicited after the inactivating prepulse in each cell under control and Zn2+ at the time indicated in the graph, expressed as percentages of the maximal inward current recorded during the entire protocol, which was conventionally set as the 100%. The cells depicted in A (right) are representative of a group of 9 (2 mM Ca2+ group) and of 11 cells (Zn2+ group), whereas the cells depicted in B (right) are representative of a group of 4 cells both in the case of the control and of the Zn2+ exposed cells. In A and B, voltage protocols are reported in the insets where numbers indicate the voltage applied expressed in mV.

View larger version (17K): [in this window] [in a new window]   FIG. 6. Voltage-dependence curves of the activation and inactivation time constants of CaV3.3 channels in the presence and in the absence of Zn2+. A: voltage-dependence curve of the time constant of the activation gate. For voltages more negative than –61 mV, each data point represents the means ± SE of the values of weighted deactivation obtained by applying the protocol described in Fig. 5A at the corresponding repolarization voltage. For voltages equal to or more positive than –61 mV, the data points represent the values of activation obtained by fitting the current traces elicited by the step protocol reported in Fig. 1A to Eq. 5. B: voltage-dependence curve of the time constant of the inactivation gate. For voltages more negative than –61 mV, each data point represents the means ± SE of the time constants of recovery from inactivation obtained by applying the protocol described in Fig. 4A at the corresponding repolarization voltage. For voltages equal to or more positive than –61 mV, the data points represent the values of the time constants of inactivation obtained by fitting to Eq. 5 the current traces elicited by step depolarization according to the protocol reported in Fig. 1A. Both in A and in B, —, obtained by fitting the data to Eq. 12. *P < 0.05; **P < 0.01.

To explore how Zn²⁺-induced modifications in Ca_V3.3 channel gating could affect the ability of Ca_V3.3 channels to generate action potentials, we measured the Ca²⁺ currents elicited by mock action potentials delivered to Ca_V3.3 cells through the patch pipette (Kozlov et al. 2005). Cells were first depolarized by delivering an ascending ramp from –101 up to +39 mV in 3 ms and, then, immediately repolarized by a second descending ramp bringing the potentials down to –101 mV in 6 ms. This protocol elicited large inward currents in cells perfused with 2 mM Ca²⁺ (Fig. 7). When the same cells were perfused with 2 mM Ca²⁺ +300 µM Zn²⁺, the currents were significantly smaller, peaked significantly later, and persisted for a significantly longer period of time because their relaxation kinetics was slowed (Fig. 7). These data indicate that, in the presence of Zn²⁺, Ca²⁺ influx through Ca_V3.3 channels is maintained for a significantly longer time interval than under control condition, thus suggesting that Zn²⁺ can influence excitability in cells expressing Ca_V3.3 channels.

View larger version (11K): [in this window] [in a new window]   FIG. 7. Ca2+ currents evoked by mock action potentials in the presence and in the absence of Zn2+. Current traces elicited by a train of 3 mock action potentials generated by the patch amplifier according to the protocol reported in the inset where the numbers indicate the applied voltage expressed in mV. The traces shown were obtained from a single CaV3.3 cell representative of a group of 7 before and after the addition of Zn2+ (300 µM) to the bathing 2 mM Ca2+ solution.

To establish whether Zn²⁺ modifies the frequency and/or the duration of neuronal action potential trains, we inserted Zn²⁺-induced changes in Ca_V3.3 channel gating parameters into the NEURON simulated model developed by Destexhe et al. (1996). This model reproduces the electrophysiological behavior of nRT neurons, a cell type where T-type channels participate in depolarization-induced action potentials and are required for triggering rebound burst firing. To run the nRT simulation, we introduced the kinetic parameters obtained from Ca_V3.3 cells exposed or not to Zn²⁺ into the IT2 model file, which provides to the NEURON program the parameters needed to build up the simulated T-type current (see METHODS). Conversely, the parameters describing the contribution of the other ion channels, i.e., voltage-gated Na⁺ and K⁺ channels, included in the NEURON model were not modified.

When the injection of a 200-ms hyperpolarizing current (0.3 nA) pulse was simulated, we observed under control conditions the appearance of a burst firing with the crescendo-decrescendo pattern typical of nRT cells; in contrast, this protocol was characterized by a dramatic increase in burst firing frequency in the presence of Zn²⁺ (Fig. 8A). It is worth noticing that the burst firing onset in the presence of Zn²⁺ was significantly delayed and that the simulated neuron returned to baseline condition in 300 ms from the beginning of burst firing. Similarly, when we simulated the injection of a 200-ms depolarizing current (0.3 nA) pulse, we observed an increase in action potential frequency in the presence of Zn²⁺ (Fig. 8B). To reproduce the Zn²⁺-induced blockade of Ca_V3.3 channels, both the simulation involving the injection of a negative current pulse and that involving the injection of a positive current pulse were run introducing into the model a value for Ca²⁺ whole cell conductance 55.5% lower that that used in control cell recording. Therefore these data suggest that, despite the reduction in the amplitude of the currents flowing through Ca_V3.3 channels by Zn²⁺, this metal caused a substantial increase in neuronal excitability.

View larger version (21K): [in this window] [in a new window]   FIG. 8. Computer simulation of CaV3.3-dependent nRT burst firing in the presence and in the absence of Zn2+. A: burst firing elicited by the injection of a pulse of hyperpolarizing current (–0.3 nA) in the absence (top) and in the presence (bottom) of 300 µM Zn2+ using the NEURON nucleus reticularis thalami (nRT) cell model simulation as detailed in METHODS. B: trains of simulated action potentials elicited by the injection of a 200-ms depolarizing current pulse (0.3 nA) in the absence (top) and in the presence (bottom) of 300 µM Zn2+ in the same model nRT cell as in A. Both in A and in B, voltage traces were generated using the option "burst behavior in a 3-compartment model" of the NEURON software with the resting potential set at –70 mV and the gating kinetic constants obtained by applying the experimental protocols reported in Figs. 1–6 and described throughout the text. For more details, see METHODS.

Overall, the data obtained in vitro from HEK Ca_V3.3 cells and from the nRT computer model indicate that Zn²⁺ may exert an excitatory effect on thalamic activity. To further test this hypothesis, we explored the consequences of Zn²⁺ chelation in an acute talamocortical slice preparation. Spontaneous field activity was induced by perfusing these slices with 50 µM 4AP (Biagini et al. 2001). As illustrated in Fig. 9A (control panel), two different types of epileptiform field activity were observed: prolonged (>8 s) ictal-like discharges and short (0.5–4 s) interictal-like events. As expected with preserved thalamocortical connectivity, the majority of discharges occurred synchronously in the cortex, VB and nRT, with a few tens of ms delay between these structures (cf., Biagini et al. 2001). After a 15- to 30-min exposure to the Zn²⁺ chelator TPEN (300 µM), ictal discharges significantly decreased in frequency (Fig. 9A, TPEN panel) in all structures while interictal discharges remain unchanged. These data are summarized in Fig. 9, B and C. It should be emphasized that TPEN did not induce any significant change in the mean amplitude and duration of either ictal or interictal discharges and that TPEN-induced effect on ictal discharge occurrence was not reversible on drug washout.

View larger version (20K): [in this window] [in a new window]   FIG. 9. Effects of Zn2+ chelation on spontaneous epileptiform discharges induced by 4-aminopyridine in acute thalamocortical slices. A: extracellular field potentials recorded before, during and after exposure to the Zn2+ chelator N,N,N',N-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN, 300 µM). The recording electrodes were positioned in the parietal cortex (CTX) in the more dorsal portion of nRT and ventrobasal nucleus (VB) as illustrated in the inset. B: effects induced by TPEN on the frequency of ictal and interictal discharges in 5 thalamocortical slices. *P value <0.05 as evaluated with repeated-measure ANOVA followed by the Neuman-Keuls test.

	DISCUSSION

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Surface charge screening by Zn²⁺ represents an obvious possible explanation for the effects induced by this ion on channel gating. In line with this view, it has been reported that like other metal ions, Zn²⁺ effectively binds to and neutralizes negative surface charges on the membrane, thus modifying the local electrical field around ion channels (Hille 1992). Even though the shielding effect of high concentrations of Zn²⁺ may have major consequences on channel gating, this mechanism does appear to be relevant when Ca_V3.3 cells were exposed to 300 µM Zn²⁺. In fact, while the Gouy-Chapman theory (Hille 1992) predicts that the half voltages for channel activation and inactivation and the _m and _h curves would be shifted rightward owing to surface charge screening, we did not find any significant shift suggesting that the concentration used in this study (i.e., 300 µM) was too low to significantly shield surface charges. Interestingly, it has been proposed that metal ions, such as Ni²⁺ or La³⁺—which are known to bind to ion channels—affect channel gating in a way that cannot be explained merely on the basis of the Gouy-Chapman theory of surface screening (Armstrong and Cota 1990; Elinder et al. 1996; Hille 1992). This could also be the case of Zn²⁺ that binds to the channel pore of voltage-dependent Ca²⁺ channels. In this context, it is tempting to speculate that Zn²⁺, while standing into the pore of Ca_V3.3 channels, may cause structural rearrangements of this channel region that contribute to the gating process. In line with this view, a crucial role in the gating process has been demonstrated for the pore region of T-type channels, the gating of which has been shown to be influenced by changes in the nature of the permeant ion (Shuba et al. 1991) or by point mutations in the selectivity filter of the channel pore (Talavera et al. 2003).

As the presence of Zn²⁺ dramatically slowed down Ca_V3.3 channel inactivation and deactivation kinetics, we believe that exposure of Ca_V3.3 to Zn²⁺ alters the function of this class of T-type channels during physiological processes that are characterized by rapid changes in membrane voltage. This is the case, for instance, of action potential generation and propagation. Therefore we hypothesize that Zn²⁺ would modify the response of Ca_V3.3 channels to incoming action potentials and/or their ability to generate hyperpolarization-induced low-threshold spikes, which entrain Na⁺-dependent action potential bursts (Perez-Reyes 2003). Our data presented support this idea. Indeed by exploring the ability of Ca_V3.3 channels to open in response to mock action potential, we found that the shape of Ca²⁺ currents was significantly different during Zn²⁺ application. Thus in the presence of this ion, the currents elicited by mock action potential were smaller but more persistent than under control conditions, suggesting that when Zn²⁺ is released in the extracellular compartment, Ca²⁺ influx through T-type channels lasts longer than under control conditions. Accordingly, using the NEURON computer simulation of nRT neurons (Dexthexe et al. 1996; Murbartian et al. 2004), we found that Zn²⁺ increases the frequency of burst firing. This type of computer simulation has already been adapted to Ca_V3.3 cells (Murbartian et al. 2004), and simulations in HEK-293 cells have been used to infer conclusions on neuronal T-type channel activity in physiological (Kozlov et al. 1999) and pathophysiological conditions (Vitko et al. 2005).

Assuming that our simulation data can be extrapolated to real nRT cells, the ability of Zn²⁺ to affect Ca_V3.3-dependent burst firing may have relevant implications in pathophysiological conditions in which the extracellular concentration of Zn²⁺ released from the terminals of the gluzinergic fibers, which impinge onto nRT neurons (Mengual et al. 2001), reaches values close to what used in our experiments (300 µM). The extracellular concentrations of Zn²⁺ have not been clearly established to date (Frederickson et al. 2006a; Kay 2003). It is, however, believed that Zn²⁺ release is negligible under basal conditions (Frederickson et al. 2006b), whereas it can increase up to 30–100 µM during ischemia or seizures (Thompson et al. 2002; Ueno et al. 2002). In addition, much higher concentrations of this ion are presumably reached in restricted areas around the site of release of Zn²⁺-containing vescicles. It should be emphasized that we observed relevant kinetic effects also in cells exposed to 100 µM Zn²⁺. In addition, it should also be noticed that, concomitantly with intense synaptic stimulation, as in epilepsy (Heinemann and Louvel 1983; Heinemann et al. 1977) or ischemia (Harris et al. 1981), extracellular Ca²⁺ concentration is expected to drop as a consequence of neuronal sink action. This could significantly shift rightward the Zn²⁺ effect-concentration curve assuming a binding competition between Ca²⁺ and Zn²⁺ as should happen if, as we hypothesized, the pore of Ca_V3.3 could represent the site for Zn²⁺ action on Ca_V3.3 channel kinetics.

On the basis of these considerations, we believe that our data have implications in brain ischemia or in epilepsy, two neurological conditions in which extracellular Zn²⁺ concentrations reach high micromolar values. This hypothesis is supported by the results obtained from acute thalamocortical slices exposed to the convulsant drug 4AP, which represents an in vitro model of epileptic discharge (Biagini et al. 2002; D'Arcangelo et al. 2002). We found that exposure to the Zn²⁺ chelator TPEN decreased the occurrence of 4AP-induced ictal-like discharges in nRT and cortex. TPEN presumably chelated the intracellular releasable Zn²⁺ as suggested by the ability of this drug to deplete brain terminals from Zn²⁺ (Cuajungco and Lees 1996). Despite its ability to chelate also iron and copper TPEN is usually considered a selective Zn²⁺ chelator, and it has been used to assess the role of this ion in neurotransmission in the hippocampus (Matias et al. 2006).

The ability of exogenous Zn²⁺ to increase the frequency of thalamocortical discharges in brain slices has been reported by Gibbs et al. (2000). However, no information is available on the physiological meaning of this Zn²⁺ action in the thalamocortical system; in addition, it was unknown whether endogenous Zn²⁺ could exert similar effects. At the best of our knowledge, our data represent the first evidence in favor of the idea that endogenous Zn²⁺ plays a role in thalamocortical excitability as shown by the dramatic consequences of its chelation. This raises the question of what are the sources of the endogenous Zn²⁺ affecting thalamocortical discharges. The rat cortex is rich in Zn²⁺, which mostly originates into the cortex itself as cortico-cortical connections are enriched in synaptic Zn²⁺ (Casanovas-Aguilar et al. 1998, 2002). In contrast, projecting thalamocortical fibers connecting the VB with cortex are not zincergic (Brown and Dyck 2005). However, dense zincergic terminals are concentrated in specific thalamic regions such as the dorsal portion of nRT, where we positioned our recording electrode; in addition, both anterodorsal and lateral dorsal thalamic nuclei display intense Timm staining (Mengual et al. 2002). Finally, zincergic terminals into the thalamus appear to have a cortical origin, thus identifying the zincergic thalamic afferents as a subset of glutamatergic corticothalamic fibers. As the ventrobasal thalamus is not significantly innervated by zincergic fibers and exogenous Zn²⁺ does suppress the activity of thalamic relay neurons (Noh and Chung 2003), it is unlikely that the decrease in VB ictal firing induced by Zn²⁺ chelation could have been determined by a direct effect on thalamic relay neurons. Conversely, the most likely explanation for TPEN effect is a decrease in the excitatory drive by afferent fibers of nRT and/or cortical origin. In line with this hypothesis comes a study, which was published while the present paper was under review, showing that Zn²⁺ does affect the gating of the nRT-specific T-type channel isoform Ca_V3.3 but not that of the T-type channel isoform Ca_V3.1 that is preferentially expressed in thalamic relay neurons (Traboulsie et al. 2007).

A limitation of our study is that we did not include in our computer simulation the effect of Zn²⁺ on other ion channels such as voltage-gated Na⁺, I_h, and type A K⁺ channels or ligand-gated GABA_A channels, which are expressed in nRT neurons and have a role in determining their electrical properties (Abbass 2006; Destexhe and Sejnowski 2003; Yue and Huguenard 2001). Evidence reported in the literature suggests that some of these mechanisms could cooperate in a determining the pro-excitatory effect of this metal ion. For instance, it has been reported that Zn²⁺ is ineffective on voltage-gated Na⁺ channels or induce only a marginal decrease in Na⁺ currents (Harrison and Gibbons 1994; Horning and Trombley 2001), whereas the effect on type A K⁺ channels is strictly voltage dependent as these currents are enhanced by Zn²⁺ at membrane potentials more positive than –50 mV and inhibited at more negative potentials, such as those applied by us in the simulation experiments (Horning and Trombley 2001). As far as I_h channels are concerned, very few data are available on Zn²⁺ effect on these channel type, which is inhibited only by millimolar Zn²⁺ concentrations in neuroendocrine cells (Raymond and Lapied 1999). Finally, GABA_A receptor blockade in the nRT has been reported to contribute to the pro-excitatory action of exogenous Zn²⁺ (Gibbs et al. 2000). However, it is unlikely that this can be the only factor because we have found that exogenously added Zn²⁺ increases the frequency of thalamocortical epileptiform discharges in the presence of saturating concentrations of the GABA_A receptor antagonist picrotoxin (Cataldi and Avoli, unpublished data).

In conclusion, endogenous Zn²⁺ controls thalamocortical firing, and this effect can be at least in part determined by its ability to slow down the gating of Ca_V3.3 channes, the major T-type channel isoform expressed in nRT neurons.

	GRANTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
This study was supported by the following grants: Programma Speciale art. 12bis comma 6, D. Lgs. 229/99, Special Project "Alzheimer 2001/2004" to L. Annunziato from the Italian Ministry of Health and Regione Campania; COFIN 2004 to M. Cataldi and L. Annunziato from the Italian Ministry of Education, University and Research; PNR-FIRBRBNE01E7YX_007 2001 to L. Annunziato from the Italian Ministry of Education, University and Research; Ricerca Finalizzata, legge 502/92 "Geni di vulnerabilità e di riparazione DNA" to L. Annunziato from the Italian Ministry of Health; Legge regionale 28/5/02, Finanziamento 2003 to L. Annunziato from Regione Campania; Programma Operativo Regionale (POR), Centro regionale di competenza GEAR to L. Annunziato from Regione Campania; 12th Italian-Chinese Executive program for scientific and technological Cooperation for the period 2006–2009 to L. Annunziato from the Italian Foreign Ministry; the CIHR (MOP 8109) and the Savoy Foundation to M. Avoli. V. Lariccia was supported by a fellowship from the Centro Regionale di Competenza di Genomica Funzionale. M. Cataldi was supported by a travel grant from the Federico II University of Naples, " Programma di scambi internazionali con universitá ed istituti di ricerca stranieri per la mobiltá di breve durata di docenti, ricercatori e studiosi."

	ACKNOWLEDGMENTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
The authors are indebted to Dr. E. Perez-Reyes for kindly providing the stably transfected HEK-293, to Dr. G. Panuccio for friendly help, to V. Grillo and C. Capitale for technical support, and to Dr. P. Merolla for editorial help.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Address for reprint requests and other correspondence: L. Annunziato, Div. di Farmacologia, Dipt. di Neuroscienze, Via Pansini 5, 80131 Napoli, Italy (E-mail: lannunzi{at}unina.it)

	REFERENCES

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
Abbas SY, Ying SW, Goldstein PA. Compartmental distribution of hyperpolarization-activated cyclic-nucleotide-gated channel 2 and hyperpolarization-activated cyclic-nucleotide-gated channel 4 in thalamic reticular and thalamocortical relay neurons. Neuroscience 141: 1811–1825, 2006.[CrossRef][Web of Science][Medline]

Agmon A, Connors BW. Thalamocortical responses of mouse somatosensory (barrel) cortex in vitro. Neuroscience 41: 365–379, 1991.[CrossRef][Web of Science][Medline]

Armstrong CM, Cota G. Modification of sodium channel gating by lanthanum. Some effects that cannot be explained by surface charge theory. J Gen Physiol 96: 1129–1140, 1990.[Abstract/Free Full Text]

Biagini G, D'Antuono M, Tancredi V, Motalli R, Louvel J, D'Arcangelo G, Pumain R, Warren RA, Avoli M. Thalamocortical connectivity in a rat brain slice preparation: participation of the ventrobasal complex to synchronous activities. Thalamus Related Syst 310: 169–179, 2001.

Brown CE, Dyck RH. Retrograde tracing of the subset of afferent connections in mouse barrel cortex provided by zincergic neurons. J Comp Neurol 486: 48–60, 2005.[CrossRef][Web of Science][Medline]

Carbone E, Lux HD. A low voltage-activated, fully inactivating Ca²⁺ channel in vertebrate sensory neurons. Nature 310: 501–502, 1984.[Medline]

Casanovas-Aguilar C, Miro-Bernie N, Perez-Clausell J. Zinc-rich neurons in the rat visual cortex give rise to two laminar segregated systems of connections. Neuroscience. 110: 445–458, 2002.[CrossRef][Web of Science][Medline]

Casanovas-Aguilar C, Reblet C, Pérez-Clausell J, Bueno-López JL. Zinc-rich afferents to the rat neocortex: projections to the visual cortex traced with intracerebral selenite injections. J Chem Neuroanat 15: 97–109, 1998.[CrossRef][Web of Science][Medline]

Cataldi M, Gaudino A, Lariccia V, Russo M, Amoroso S, di Renzo G, Annunziato L. Imatinib-mesylate blocks recombinant T-type calcium channels expressed in human embryonic kidney-293 cells by a protein tyrosine kinase-independent mechanism. J Pharmacol Exp Ther 309: 208–215, 2004.[Abstract/Free Full Text]

Cataldi M, Perez-Reyes E, Tsien RW. Differences in apparent pore sizes of low- and high-voltage-activated Ca²⁺ channels. J Biol Chem 277: 45969–45976, 2002.[Abstract/Free Full Text]

Coulter DA, Huguenard JR, Prince DA. Calcium currents in rat thalamocortical relay neurons: kinetic properties of the transient, low-threshold current. J Physiol 414: 587–604, 1989.[Abstract/Free Full Text]

Cribbs LL, Lee JH, Yang J, Satin J, Zhang Y, Daud A, Barclay J, Williamson MP, Fox M, Rees M, Perez-Reyes E. Cloning and characterization of alpha1H from human heart, a member of the T-type Ca²⁺ channel gene family. Circ Res 83: 103–109, 1998.[Abstract/Free Full Text]

Cuajungco MP, Lees GJ. Prevention of zinc neurotoxicity in vivo by N,N,N', N'-tetrakis (2-pyridylmethyl) ethylene-diamine (TPEN). Neuroreport 7: 1301–1304, 1996.[Web of Science][Medline]

D'Arcangelo G, D'Antuono M, Biagini G, Warren R, Tancredi V, Avoli M. Thalamocortical oscillations in a genetic model of absence seizures. Eur J Neurosci 16: 2383–2393, 2002.[CrossRef][Web of Science][Medline]

Destexhe A, Contreras D, Steriade M, Sejnowski TJ, Huguenard JR. In vivo, in vitro, and computational analysis of dendritic calcium currents in thalamic reticular neurons. J Neurosci 16: 169–185, 1996.[Abstract/Free Full Text]

Destexhe A, Sejnowski TJ. Interactions between membrane conductances underlying thalamocortical slow-wave oscillations. Physiol Rev 83: 1401–1453, 2003.[Abstract/Free Full Text]

Elinder F, Madeja M, Arhem P. Surface charges of K channels. Effects of strontium on five cloned channels expressed in Xenopus oocytes. J Gen Physiol 108: 325–332, 1996.[Abstract/Free Full Text]

Fox AP, Nowycky MC, Tsien RW. Single-channel recordings of three types of calcium channels in chick sensory neurones. J Physiol 394: 173–200, 1987.[Abstract/Free Full Text]

Frederickson CJ, Giblin LJ 3rd, Rengarajan B, Masalha R, Frederickson CJ, Zeng Y, Lopez EV, Koh JY, Chorin U, Besser L, Hershfinkel M, Li Y, Thompson RB, Krezel A. Synaptic release of zinc from brain slices: factors governing release, imaging, and accurate calculation of concentration. J Neurosci Methods 154: 19–29, 2006a.[CrossRef][Web of Science][Medline]

Frederickson CJ, Giblin LJ, Krezel A, McAdoo DJ, Muelle RN, Zeng Y, Balaji RV, Masalha R, Thompson RB, Fierke CA, Sarvey JM, de Valdenebro M, Prough DS, Zornow MH. Concentrations of extracellular free zinc (pZn)(e) in the central nervous system during simple anesthetization, ischemia and reperfusion. Exp Neurol 198: 285–293, 2006b.[CrossRef][Web of Science][Medline]

Frederickson CJ, Koh JY, Bush AI. The neurobiology of zinc in health and disease. Nat Rev Neurosci 6: 449–462, 2005.[CrossRef][Web of Science][Medline]

Frederickson CJ, Suh SW, Silva D, Frederickson CJ, Thompson RB. Importance of zinc in the central nervous system: the zinc-containing neuron. J Nutr 130: 1471S–1483S, 2000.[Abstract/Free Full Text]

Fuentealba P, Timofeev I, Bazhenov M, Sejnowski TJ, Steriade M. Membrane bistability in thalamic reticular neurons during spindle oscillations. J Neurophysiol 93: 294–304, 2005.[Abstract/Free Full Text]

Gibbs JW 3rd, Zhang YF, Shumate MD, Coulter DA. Regionally selective blockade of GABAergic inhibition by zinc in the thalamocortical system: functional significance. J Neurophysiol 83: 1510–1521, 2000.[Abstract/Free Full Text]

Harris RJ, Symon L, Branston NM, Bayhan M. Changes in extracellular calcium activity in cerebral ischemia. J Cereb Blood Flow Metab. 1: 203–9, 1981.[Web of Science][Medline]

Harrison NL, Gibbons SJ. Zn²⁺: an endogenous modulator of ligand- and voltage-gated ion channels. Neuropharmacology 33: 935–952, 1994.[CrossRef][Web of Science][Medline]

Heinemann U, Louvel J. Changes in [Ca²⁺]_o and [K⁺]_o during repetitive electrical stimulation and during pentetrazol induced seizure activity in the sensorimotor cortex of cats. Pfluegers 398: 310–317, 1983.[CrossRef]

Heinemann U, Lux HD, Gutnick MJ. Extracellular free calcium and potassium during paroxsmal activity in the cerebral cortex of the cat. Exp Brain Res 27: 237–243, 1977.[Web of Science][Medline]

Hille B. Ionic Channels of Excitable Membranes. Sunderland, MA: Sinauer, 1992.

Hines ML, Carnevale NT. The NEURON simulation environment. Neural Comput 9: 1179–1209, 1997.[CrossRef][Web of Science][Medline]

Hodgkin AL, HuxleyAF. A quantitative description of membrane current and its application to conduction and excitation in nerve. J Physiol 117: 500–544, 1952.[Free Full Text]

Horning MS, Trombley PQ. Zinc and copper influence excitability of rat olfactory bulb neurons by multiple mechanisms. J Neurophysiol 86: 1652–1660, 2001.[Abstract/Free Full Text]

Howell GA, Welch MG, Frederickson CJ. Stimulation-induced uptake and release of zinc in hippocampal slices. Nature 308: 736–738, 1984.[CrossRef][Medline]

Huguenard JR, Prince DA. A novel T-type current underlies prolonged Ca²⁺-dependent burst firing in GABAergic neurons of rat thalamic reticular nucleus. J Neurosci 12: 3804–3817, 1992.[Abstract]

Jeong SW, Park BG, Park JY, Lee JW, Lee JH. Divalent metals differentially block cloned T-type calcium channels. Neuroreport 14: 1537–1540, 2003.[CrossRef][Web of Science][Medline]

Joksovic PM, Bayliss DA, Todorovic SM. Different kinetic properties of two T-type Ca²⁺ currents of rat reticular thalamic neurons and their modulation by enflurane. J Physiol 566: 125–142, 2005a.[Abstract/Free Full Text]

Joksovic PM, Brimelow BC, Murbartián J, Perez-Reyes E, Todorovic SM. Contrasting anesthetic sensitivities of T-type Ca²⁺ channels of reticular thalamic neurons and recombinant Ca(v) 3.3 channels. Br J Pharmacol 144: 59–70, 2005b.[CrossRef][Web of Science][Medline]

Kay AR. Evidence for chelatable zinc in the extracellular space of the hippocampus but little evidence for synaptic release of Zn. J Neurosci 23: 6847–6855, 2003.[Abstract/Free Full Text]

Khosravani H, Altier C, Simms B, Hamming KS, Snutch TP, Mezeyova J, McRory JE, Zamponi GW. Gating effects of mutations in the Cav3.2 T-type calcium channel associated with childhood absence epilepsy. J Biol Chem 279: 9681–9684, 2004.[Abstract/Free Full Text]

Kostyuk PG, Shuba Ya M, Savchenko AN. Three types of calcium channels in the membrane of mouse sensory neurons. Pfluegers 411: 661–669, 1988.[CrossRef]

Kozlov AS, McKenna F, Lee JH, Cribbs LL, Perez-Reyes E, Feltz A, Lambert RC. Distinct kinetics of cloned T-type Ca²⁺ channels lead to differential Ca²⁺ entry and frequency dependence during mock action potentials. Eur J Neurosci 11: 4149–4158, 1999.[CrossRef][Web of Science][Medline]

Lee JH, Daud AN, Cribbs LL, Lacerda AE, Pereverzev A, Klockner U, Schneider T, Perez-Reyes E. Cloning and expression of a novel member of the low voltage-activated T-type calcium channel family. J Neurosci 19: 1912–1921, 1999.[Abstract/Free Full Text]

Mathie A, Sutton GL, Clarke CE, Veale EL. Zinc and copper: pharmacological probes and endogenous modulators of neuronal excitability. Pharmacol Ther 111: 567–583, 2006.[CrossRef][Web of Science][Medline]

Matias CM, Matos NC, Arif M, Dionisio JC, Quinta-Ferreira ME. Effect of the zinc chelator N,N,N',N'-tetrakis (2-pyridylmethyl)ethylenediamine (TPEN) on hippocampal mossy fiber calcium signals and on synaptic transmission. Biol. Res. 39: 521–530, 2006.[Web of Science][Medline]

McCormick DA, Huguenard JR. A model of the electrophysiological properties of thalamocortical relay neurons. J Neurophysiol 68: 1384–1400, 1992.[Abstract/Free Full Text]

Mengual E, Casanovas-Aguilar C, Perez-Clausell J, Gimenez-Amaya JM. Thalamic distribution of zinc-rich terminal fields and neurons of origin in the rat. Neuroscience 102: 863–884, 2001.[CrossRef][Web of Science][Medline]

Murbartian J, Arias JM, Perez-Reyes E. Functional impact of alternative splicing of human T-type Cav3.3 calcium channels. J Neurophysiol 92: 3399–3407, 2004.[Abstract/Free Full Text]

Noh JH, Chung JM. Zinc reduces low-threshold Ca²⁺ currents of rat thalamic relay neurons. Neurosci Res 47: 261–5, 2003.[CrossRef][Web of Science][Medline]

Perez-Reyes E. Molecular physiology of low-voltage-activated t-type calcium channels. Physiol Rev 83: 117–161, 2003.[Abstract/Free Full Text]

Perez-Reyes E, Cribbs LL, Daud A, Lacerda AE, Barclay J, Williamson MP, Fox M, Rees M, Lee JH. Molecular characterization of a neuronal low-voltage-activated T-type calcium channel. Nature 391: 896–900, 1998.[CrossRef][Medline]

Qian J, Noebels JL. Visualization of transmitter release with zinc fluorescence detection at the mouse hippocampal mossy fiber synapse. J Physiol 566: 747–758, 2005.[Abstract/Free Full Text]

Raymond V, Lapied B. Hyperpolarization-activated inward potassium and calcium-sensitive chloride currents in beating pacemaker insect neurosecretory cells (dorsal unpaired median neurons). Neuroscience 93: 1207–1218, 1999.[CrossRef][Web of Science][Medline]

Shuba YM, Teslenko VI, Savchenko AN, Pogorelaya NH. The effect of permeant ions on single calcium channel activation in mouse neuroblastoma cells: ion-channel interaction. J Physiol 443: 25–44, 1991.[Abstract/Free Full Text]

Steriade M. Sleep, epilepsy and thalamic reticular inhibitory neurons. Trends Neurosci 28: 317–24, 2005.[CrossRef][Web of Science][Medline]

Steriade M, McCormick DA, Sejnowski TJ. Thalamocortical oscillations in the sleeping and aroused brain. Science 262: 679–85, 1993.[Abstract/Free Full Text]

Talavera K, Janssens A, Klugbauer N, Droogmans G, Nilius B. Pore structure influences gating properties of the T-type Ca²⁺ channel alpha1G. J Gen Physiol 121: 529–540, 2003.[Abstract/Free Full Text]

Talley EM, Cribbs LL, Lee JH, Daud A, Perez-Reyes E, Bayliss DA. Differential distribution of three members of a gene family encoding low voltage-activated (T-type) calcium channels. J Neurosci 19: 1895–1911, 1999.[Abstract/Free Full Text]

Thompson RB, Whetsell WO, Jr, Maliwal BP, Fierke CA, Frederickson CJ. Fluorescence microscopy of stimulated Zn(II) release from organotypic cultures of mammalian hippo-campus using a carbonic anhydrase-based biosensor system. J Neurosci Methods 96: 35–45, 2000.[CrossRef][Web of Science][Medline]

Traboulsie A, Chemin J, Chevalier M, Quignard JF, Nargeot J, Lory P. Subunit-specific modulation of T-type calcium channels by zinc. J Physiol 578: 159–171, 2007.[Abstract/Free Full Text]

Tsien RW, Lipscombe D, Madison DV, Bley KR, Fox AP. Multiple types of neuronal calcium channels and their selective modulation. Trends Neurosci 11: 431–438, 1988.[CrossRef][Web of Science][Medline]

Ueno S, Tsukamoto M, Hirano T, Kikuchi K, Yamada MK, Nishiyama N, Nagano T, Matsuki N, Ikegaya Y. Mossy fiber Zn²⁺ spillover modulates heterosynaptic N-methyl-D-aspartate receptor activity in hippocampal CA3 circuits. J Cell Biol 158: 215–220, 2002.[Abstract/Free Full Text]

Vitko I, Chen Y, Arias JM, Shen Y, Wu XR, Perez-Reyes E. Functional characterization and neuronal modeling of the effects of childhood absence epilepsy variants of CACNA1H, a T-type calcium channel. J Neurosci 25: 4844–4855, 2005.[Abstract/Free Full Text]

Wu J, Ellsworth K, Ellsworth M, Schroeder KM, Smith K, Fisher RS. Abnormal benzodi-azepine and zinc modulation of GABA_A receptors in an acquired absence epilepsy model. Brain Res 1013: 230–240, 2004.[CrossRef][Web of Science][Medline]

Yue BW, Huguenard JR. The role of H-current in regulating strength and frequency of thalamic network oscillations. Thalamus Related Syst 1: 95–103, 2001.

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