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Although it is well established that low-voltage activated T-type Ca2+ channels play a key role in many neurophysiological functions and pathological states, the lack of selective and potent antagonists has so far hampered a detailed analysis of the full impact these channels might have on single cell and neuronal network excitability as well as on Ca2+ homeostasis. Recently, a novel series of piperidine-based molecules has been shown to selectively block recombinant T-type but not high-voltage activated (HVA) Ca2+ channels and to affect a number of physiological and pathological T-type channel-dependent behaviours. Here we directly show that one of these compounds, TTA-P2, exerts a specific, potent (IC50: 22nM) and reversible inhibition of T-type Ca2+ currents of thalamocortical and reticular thalamic neurons, without any action on HVA Ca2+ currents, Na+ currents, action potentials, and glutamatergic and GABAergic synaptic currents. Thus, under current clamp conditions the low threshold Ca2+ potential (LTCP)-dependent high frequency burst firing of thalamic neurons is abolished by TTA-P2 whereas tonic firing remains unaltered. Using TTA-P2, we provide the first direct demonstration of the presence of a window component of Ca2+ channels in neurons, and its contribution to the resting membrane potential of thalamic neurons and to the UP state of their intrinsically generated slow (<1Hz) oscillation. Moreover, we demonstrate that activation of only a small fraction of the T-type channel population is required to generate robust LTCPs, suggesting that LTCP driven-bursts of action potentials can be evoked at depolarized potentials where the vast majority of T-type channels are inactivated.
Since their discovery (Carbone and Lux, 1984; Nowycky et al., 1985), T-type Ca2+ channels have been implicated in many physiological and pathological brain states, including sleep (Contreras, 2006; Crunelli et al., 2006), nociception (Todorovic et al., 2001; Kim et al., 2003; Bourinet et al., 2005), and epilepsy (Tsakiridou et al., 1995; Kim et al., 2001; Crunelli and Leresche, 2002a; Vitko et al., 2007; Powell et al., 2009). From a mechanistic perspective, the low threshold Ca2+ potential (LTCP), and associated high frequency burst of action potentials, has become the hallmark for the contribution of T-type Ca2+ channels to single cell and neuronal network excitability in different brain regions, including thalamus (Llinas and Jahnsen, 1982), neocortex (McCormick et al., 1985) and cerebellum (Llinas and Muhlethaler, 1988). However, to limit the function of T-type Ca2+ channels to LTCP generation might lead to underestimating their potential roles in (patho)physiology. Thus, the window component of the T-type Ca2+ current (IT), that relies on the few channels that are constantly opened at ~-60mV, could contribute to the resting membrane potential and has been suggested to be essential for the slow (<1Hz) sleep oscillations of thalamic neurons (Hughes et al., 2002). Moreover, since T-type channels are highly expressed in dendrites (Williams and Stuart, 2000; Joksovic et al., 2005a; McKay et al., 2006; Diana et al., 2007), they could, even in the absence of an LTCP, be involved in signal amplification (Markram and Sakmann, 1994; Magee et al., 1995; Tsay et al., 2007) and synaptic plasticity (Oliet et al., 1997; Aizenman et al., 1998). In addition, the mechanism underlying LTCP-driven action potential bursts during sensory processing in the awake state (i.e. when only a small number of T-type channels is available for activation) is still unclear (Llinas and Steriade, 2006). Conclusive answers to these questions require a potent, specific and reversible block of T-type Ca2+ channels. Whereas antisense strategies (Lambert et al., 1998; Bourinet et al., 2005) and knock-out mice (Kim et al., 2001; Anderson et al., 2005) provide such a specific block, the lack of internal controls, the variable efficiency of the antisense knock-down and the compensatory mechanisms that may develop in knock-out mice compromise the interpretation of the resulting data. Similarly, the usefulness of the available pharmacological approaches (e.g. mibefradil, Ni2+, ethosuximide, etc…) is limited by their poor potency and selectivity (McGivern, 2006).
Here we show that TTA-P2, a novel piperidine-derivative that inhibits recombinant T-type Ca2+ channels (Shipe et al., 2008), induces a potent, specific and reversible block of native IT, LTCPs and their associated high frequency burst firing in thalamic neurons. We also provide the first direct demonstration of the contribution of the window component of IT (ITwindow) to the resting membrane potential and the slow (<1Hz) oscillations of thalamic neurons. Moreover, we demonstrate that activation of only a small fraction of the T-type channel population is required to generate robust LTCPs, suggesting that LTCP driven-bursts of action potentials can be evoked at depolarized potentials where the vast majority of T-type channels are inactivated.
All procedures involving experimental animals were carried out in accordance with the EU Council Directive 86-609, the U.K. Animals (Scientific Procedure Act 1986), and local ethics committee guidelines. All efforts were made to minimize animal suffering and the number of animals used.
Brains were excised from 5-20 day old Wistar rats or 15-30 day old C57Blc6 CaV3.1−/− and wild-type mice (Kim et al., 2001). A block of tissue containing the thalamus was removed and placed in a cold (<4°C) oxygenated (95% O2 : 5% CO2) solution of artificial cerebrospinal fluid (aCSF, in mM): 125 NaCl, 2.5 KCl, 0.4 CaCl2, 1 MgCl2, 1.25 NaH2PO4, 26 NaHCO3, 20 glucose, 5 Na-pyruvate and 1 kynurenic acid (pH 7.3, osmolarity 310mOsm). The block of tissue was glued, ventral surface uppermost, to the stage of a vibroslice (HM650V, Microm), and 200-280μm thick horizontal slices containing the ventrobasal nucleus (VB) and the nucleus reticularis thalami (NRT) were prepared using the internal capsule and the medial lemniscus as landmarks. Slices (three to four per hemisphere) were stored in an oxygenated incubation chamber containing aCSF of the above composition, but without kynurenic acid and with 2mM CaCl2 for at least 1 hour before being transferred to the recording chamber, where they were continuously perfused (1.5ml/min) with oxygenated recording solution of the same composition. Experiments were conducted either at room temperature for voltage-clamp recordings or at 32°C for current-clamp recordings.
Using the patch-clamp technique (Optopatch amplifier, Cairn; Clampex 10, Molecular Devices), whole cell recordings were performed in neurons of the VB or NRT visualized under Nomarski optics (Olympus BX51WI, x60 lens). Current and membrane potential recordings were filtered by a 4-pole Bessel filter set at a corner frequency of 2kHz, digitalized at 10kHz and later analyzed using Igor 6 (WaveMetrics).
Except when specifically mentioned, Ca2+ currents were recorded in 7-10 day old rats. For voltage-clamp experiments electrodes were filled with the following solution (in mM): 110 CsCl, 1 CaCl2, 5 MgCl2, 10 EGTA, 10 HEPES, 4 Na-ATP, 15 phosphocreatine, and 50units/ml creatine phosphokinase (pH 7.3, osmolarity 305mOsm). To isolate Ca2+ currents, the following were added to the extracellular solution: 10mM tetraethylamoniumchloride, 1mM 4-aminopyridine, 0.5μM tetrodotoxin and 2mM CsCl. ITwindow recording was obtained with a similar extracellular solution except for the addition of 1μM nifedipine to suppress the small HVA currents evoked at the end of the slow depolarizing ramp. Leak currents were subtracted off line using templates constructed from the current induced by a 10mV hyperpolarizing step applied from −100mV at the end of each recording. In every experiment, values of access resistance ranged from 4-6 MΩ. At least 70% of the cell capacitance and series resistance were compensated.
Current clamp recordings were performed in 8-20 day old rats and 15-30 day old mice. To record membrane potential, electrodes were filled with the following solution (in mM): 140 potassium-methanesulfonate, 0.1 CaCl2, 5 MgCl2, 1 EGTA, 10 HEPES, and 4 Na-ATP, 15 phosphocreatine and 50units/ml creatine phosphokinase (pH 7.3, osmolarity 290mOsm). Access resistance was monitored by measuring responses to small hyperpolarizing current pulses from resting membrane potential, and only data obtained from pipettes with access resistances of 9-13MΩ with less than 20% change during the experiment were included in this study. When required to analyze the ITwindow contribution to the resting membrane potential, a depolarizing DC current of 75 ± 29pA (n=11) and 85 ± 17pA (n=6) was used to set the membrane potential of thalamocortical and nucleus reticularis neurons to −60mV from their original resting membrane potential of −74 ± 4mV and −85 ± 17mV, respectively.
Synaptic responses were induced using a glass pipette (6-8μm tip diameter) filled with the extracellular medium. This stimulating electrode was positioned 25 to 60μm from the recorded neuron to evoke IPSPs/IPSCs or in the medial lemniscus to evoke sensory EPSPs/EPSCs. The stimulus consisted of a 20 to 200μs pulse of 10-40V amplitude. Isolation of GABAA IPSPs/IPSCs and glutamatergic EPSPs/EPSCs was achieved by the addition of 50μM DL-APVplus 10μM CNQX and 1μM SR95531, respectively, to the perfusion medium. Synaptic currents were recorded with electrodes filled with solutions of the following compositions (in mM): for IPSC: 128 potassium-methanesulfonate, 12 CsCl, 0.5 EGTA, 10 Hepes, 3 MgCl2, 0.1 CaCl2, 4 Na-ATP, 0.4 Na-GTP and 15 phosphocreatine; for EPSC: 150 KCl, 1 EGTA, 10 Hepes, 4.6 MgCl2, 0.1 CaCl2, 4 Na-ATP and 0.4 Na-GTP.
Slices of cat thalamus were prepared as previously described (Hughes et al, 2002). Briefly, 6-8 week old cats were deeply anaesthetised with a mixture of O2 and NO2 (2:1) and 5% isoflurane, a wide craniotomy performed and the brain removed. Sagittal slices of the thalamus (300 - 400μm), including the medial geniculate body (MGB), were prepared and incubated at 35°C for 1 hour prior to being maintained at room temperature. For recording, slices were transferred to a submersion chamber on the stage of an Olympus BX61WI microscope and continuously perfused with gassed (95% O2 / 5% CO2) aCSF containing (in mM): 125 NaCl, 2.5 KCl, 2 CaCl2, 1 MgCl2, 1.25 NaH2PO4, 25 NaHCO3, and 25 D-glucose (pH 7.3, 305 mOsm) at 34 ± 0.2°C. Somatic whole-cell patch clamp recordings (3-5MΩ) were performed (Multiclamp 700B, Molecular Devices) on visually identified MGB neurons (IR-Dodt contrast videomicroscopy, Luigs & Neumann) with internal pipette solution containing (in mM): 115 potassium-gluconate, 20 KCl, 2 Mg-ATP, 2 Na-ATP, 0.3 Na-GTP, 10 Na-phosphocreatine, 10 HEPES (pH 7.3, 290mOsm), supplemented with Alexa Fluor 594 (50μM). Slow (<1Hz) sleep oscillations were induced by bath application of 50μM trans-ACPD (Hughes et al., 2002) followed by small steady-state current injection (80-150pA) to bring the neuron into the voltage region of expression of these oscillations. Data were acquired using pClamp 10 software (Molecular Devices), digitized at 20 kHz and filtered at 5kHz. At the end of each experiment a z-series (1μm) of 2 photon excitation fluorescence images (512 × 512 pixels, 400x magnification) was captured using a Prairie Ultima II 2 photon microscope (Prairie Technologies) fitted with a Chameleon Ultra II laser (Coherent) tuned to 810nm (< 4mW power at sample). A maximum intensity z-series projection was then constructed offline using MetaMorph 7.1 (Molecular Devices, USA) to confirm the thalamocortical neuron morphology of the recorded cell.
TTA-P2 (3,5-Dichloro-N-[1-(2,2-dimethyl-tetrahydro-pyran-4-ylmethyl)-4-fluoro-piperidin-4-ylmethyl]-benzamide) (compound (S)-5 in Fig. 3 of Shipe et al., 2008) (Merck & Co., Inc.) was made up as 10mM stock solution in dimethyl sulphoxide (DMSO), kept at −20°C until use and diluted for experiments so that the final concentration of DMSO was ≤0.03%. TTX was obtained from Latoxan, DL-APV from Fluka and CNQX from Tocris. All other chemicals were purchased from Sigma.
Concentration-response curves were generated from the percentage reduction in the peak T-type Ca2+ current amplitude evoked at −50mV. Quantitative comparison of action potential characteristics (threshold, amplitude, half-width) was performed on the first action potential of the spike trains evoked by 200pA depolarizing step from a holding potential of −60mV. Quantitative data in the text and figures are given as mean ± s.d.
T-type Ca2+ currents (IT) and HVA Ca2+ currents, similar to those previously described (Guyon and Leresche, 1995), were evoked in thalamocortical (TC) neurons of the ventrobasal (VB) nucleus before, during and after bath application of TTA-P2. As illustrated in Figure 1A and B, application of 1μM and 3μM TTA-P2 resulted in a full block (96 ± 1%) of IT (control: 1135 ± 306pA, TTA-P2: 45 ± 21pA, p<0.001, paired Student t test, n=7), while the compound had no effect on the amplitude of the HVA Ca2+ currents (control: 1182 ± 211pA, TTA-P2: 1072 ± 177pA, p>0.05, paired Student t test, n=7) recorded in the same TC neurons. The maximal effect was achieved within 3-4 minutes from the beginning of TTA-P2 application (Fig. 1A2), and a full recovery of IT could be observed after about 50 min of wash-out (Fig. 1B). The block of IT by TTA-P2 was dose-dependent with an IC50 of 22nM (Fig. 1C). A maximal reduction of IT by TTA-P2 could be induced even in the absence of channel activation (Fig. 1D), indicating the absence of a use-dependent block. I-V relationships were constructed from IT evoked in control conditions and in the presence of 25nM TTA-P2 using slices obtained from very young animals (P5-P6) to optimize the voltage control. TTA-P2 induced a clear decrease in current amplitude with no apparent shift in the voltage-dependence of channel activation and steady-state inactivation (Fig. 1E) (n=5). Therefore, we conclude that the TTA-P2-induced decrease of IT is not due to a significant shift in the activation/inactivation properties of the current.
Precise measurement of the voltage-dependence of Cav3. channel activation and inactivation in recombinant systems demonstrated that in a narrow range of voltages where T-type channels can open, a fraction of the channel population is not inactivated, therefore generating a small tonic current called the window current (ITwindow) (Perez Reyes, 2003). To test the ability of TTA-P2 to suppress ITwindow, experiments were performed on older rats (P19-P20) that express a higher density of T-type channels and therefore display an ITwindow of measurable amplitude. ITwindow was evoked using voltage ramps slow enough to achieve steady state equilibrium between activation and inactivation of the channels during depolarization (Supp. Fig. 1). ITwindow evoked with 10s long ramps from −100mV to −40mV had a mean amplitude of 26 ± 7pA (n=5) and was fully blocked by 1μM TTA-P2 (Fig. 2), similarly to the results obtained for the transient component of IT.
Having established that TTA-P2 potently blocks both the transient and tonic components of IT in TC neurons without affecting the HVA Ca2+ currents, we then investigated its action on neuronal excitability. Under current-clamp conditions, we observed that 1-3μM TTA-P2 produced a 3.1 ± 0.5mV (n=11) hyperpolarization of TC neurons held at −60mV (Fig. 3A1) that could be compensated by injecting a depolarizing DC current of 23 ± 5pA (n=11), a value very similar to the amplitude of ITwindow (26 ± 7pA) directly measured under voltage-clamp conditions (Fig. 2). Such hyperpolarization was not observed when the neurons were held at −70mV (Fig. 3A2), i.e. outside the voltage range of ITwindow (Perez-Reyes, 2003). To confirm that the TTA-P2 induced hyperpolarization resulted from the T-type channel block, and not from unspecific actions on other membrane channels, similar experiments were performed in Cav3.1−/− mice TC neurons which lack IT (Kim et al., 2001). Indeed, no hyperpolarization was observed in Cav3.1−/− TC neurons during wash-in of TTA-P2 (n=6, Fig. 3B1), whereas in TC neurons from wild type mice TTA-P2 induced a membrane hyperpolarization of 3.4 ± 0.4mV (n=5) (Fig. 3B2). As a further control, we tested the effect of TTA-P2 on Cav3.1−/− nucleus reticularis thalami (NRT) neurons, which, due to their selective expression of Cav3.2 and 3.3 channels, show a robust LTCP (see Suppl. Fig.2). As expected, TTA-P2 did hyperpolarize NRT neurons of Cav3.1−/− mice (3.2 ± 0.6 mV; n=4) (Suppl. Fig. 2). Taken together, these data demonstrate that the TTA-P2-induced hyperpolarization is due to the block of ITwindow and indicate a contribution of this current to the resting membrane potential of TC neurons, as previously suggested on the basis of indirect evidence (Crunelli et al., 2005). Therefore, in all following experiments under current clamp condition, the TTA-P2 induced hyperpolarization was compensated by steady-current injection when required.
TTA-P2 at concentrations that abolished IT (1 or 3μM) fully blocked the LTCP evoked following a hyperpolarizing step (1.5s long at −100mV, holding potential: −60mV), that de-inactivated the entire T-type channel population (n=11) (Fig. 4A; see also Fig. 3). As shown in Figure 4Ba, TTA-P2 did not affect the depolarizing sag associated with these transient hyperpolarizations, suggesting a lack of effect on the h current.
Except for the small fraction that generate the window current, T-type Ca2+ channels are inactivated at −60mV (Perez-Reyes, 2003). Thus, they should not contribute to the neuronal responses evoked by step depolarization from membrane potentials >-60mV and hence selective block of T-type channels should have no effect under these experimental conditions. Having established that 1μM TTA-P2 in the presence of TTX had no effect on the input resistance of TC neurons when assessed with depolarizing current pulses of increasing intensities (Fig. 4C), the properties of single action potentials and tonic firing were compared in control condition and in the presence of the antagonist. Application of 1μM TTA-P2 had no effect on the threshold (control: −45.9 ± 2.6mV, TTA-P2: −45.8 ± 2.6mV, p>0.05, paired Student t test, n=6), amplitude (control: 74 ± 6.6mV, TTA-P2: 73.7 ± 6.6mV, p>0.05, paired Student t test, n=6), half-width (control: 0.6 ± 0.1ms, TTA-P2: 0.6 ± 0.1ms, p>0.05, paired Student t test, n=6) and afterhyperpolarization (control: 13.1 ± 2.2mV, TTA-P2: 13.3 ± 1.8mV, p>0.05, paired Student t test, n=6) of the action potential (Fig. 4Db), or on the frequency of tonic firing (Fig. 4Da and c). These results are strengthened by the lack of effect of 1μM TTA-P2 on both the fast and persistent Na+ currents underlying tonic action potential firing (Supp. Fig. 3). Taken together, therefore, these data confirm the selectivity of TTA-P2, indicating a lack of action on tonic firing and apparent input resistance, but a potent effect on T-type Ca2+ channel-dependent LTCP-mediated burst firing of TC neurons.
Since TC neurons have been shown to specifically express the Cav3.1 T-type channel isoform while the GABAergic neurons of NRT express both Cav3.2 and 3.3 isoforms (Talley et al., 1999), the effect of TTA-P2 was tested on IT of NRT neurons. Confirming that TTA-P2 potently interacts with all three T-type Ca2+channel isoforms (Shipe et al., 2008), a 97 ± 1% (n=6) decrease in the amplitude of IT recorded in rat NRT neurons was observed upon application of 1μM TTA-P2, whereas the HVA Ca2+ current in the same neurons was unaffected (Fig. 5A).
Current-clamp experiments showed that application of TTA-P2 hyperpolarized NRT neurons held at −60mV by 5 ± 2.2mV (n=6) (Fig. 5B). Such hyperpolarization was compensated by injection of 30 ± 1pA depolarizing DC current (n=6). Similarly to what had been observed in TC neurons, TTA-P2 suppressed the somatically evoked LTCPs (Fig. 5C) but did not affect the tonic firing frequency (Fig. 5D and E) or action potential properties: threshold (control: −47.7 ± 3.6mV, TTA-P2: −48.1 ± 2.5mV, p>0.05, paired Student t test, n=5), amplitude (control: 79.3 ± 5.6mV, TTA-P2: 83.1 ± 4.7mV, p>0.05, paired Student t test, n=5), half-width (control: 0.3 ± 0.1ms, TTA-P2: 0.3 ± 0.1ms, p>0.05, paired Student t test, n=5) and afterhyperpolarization (control: 19.6 ± 3.2mV, TTA-P2: 19.6 ± 2.3mV, p>0.05, paired Student t test, n=5).
Having established the selectivity and potency of TTA-P2 and its ability to block LTCPs without any effect on single or tonic action potential firing evoked by somatic current injections, we then started to investigate its potential use in network excitability by testing its action on excitatory and inhibitory synaptic currents. As shown in Fig. 6Aa, perfusion of the slice with TTA-P2 (1μM) did not change the amplitude (control: 193 ± 14pA, TTA-P2: 192 ± 6pA, p>0.05, paired Student t test, n=5), rise-time (10-90% rise time control: 1.8 ± 0.2ms, TTA-P2: 1.6 ± 0.1ms, p>0.05, paired Student t test, n=5) and decay (control: τ1 = 8.3 ± 0.8ms, τ2 = 43.5 ± 12.4ms, TTA-P2: τ1 = 7.9 ± 0.7ms, τ2 = 44.6 ± 14.3ms, p>0.05, paired Student t test, n=5) of the GABAA receptor-mediated IPSCs evoked in TC neurons by electrical stimulation of the NRT. Similarly, 1μM TTA-P2 had no effect on the amplitude (control: 415 ± 17pA, TTA-P2: 413 ± 16pA, p>0.05, paired Student t test, n=5), rise-time (10-90% rise time control: 0.6 ± 0.05ms, TTA-P2: 0.5 ± 0.05ms, p>0.05, paired Student t test, n=5) and decay (control: τ1 = 1.7 ± 0.5ms, τ2 = 9.3 ± 2.5ms, TTA-P2: τ1 = 1.9 ± 0.4ms, τ2 = 8.9 ± 1.8ms, p>0.05, paired Student t test, n=5) of the glutamatergic EPSCs evoked by stimulation of the lemniscal afferents to TC neurons (Fig. 6Ab). Since this novel piperidine-derivative had no effect on GABAergic and glutamatergic synaptic currents, we then investigated its action on the characteristic burst firing evoked by these two types of synaptic potentials. As shown in Fig. 6B, application of TTA-P2 at 20nM (IC50) delayed the occurrence of the rebound LTCP evoked by the NRT-elicited IPSP without affecting its associated burst firing (n=5). This result provides the first demonstration that IT participates in the early phase of membrane repolarization following an IPSP, and as a consequence, that the kinetics of this repolarization is highly sensitive to the number of T-type Ca2+ channels available for activation. A full suppression of the rebound LTCP could only be achieved using 50nM TTA-P2 (n=5), a concentration that blocks 70% of IT (see dose-response curve in Fig. 1C). The need to block a large proportion of IT in order to substantially affect synaptically evoked LTCPs was further emphasized by similar experiments on the EPSPs. Indeed, since LTCPs triggered by glutamatergic EPSPs have been shown to occur during tonic or transient hyperpolarization in TC neurons (Steriade et al., 1997) we tested the effect of TTA-P2 on the sensory EPSP evoked by stimulation of the lemniscal fibers in TC neurons maintained at slightly hyperpolarized potentials (−70mV) (Fig. 6C). In this condition, a concentration of TTA-P2 (50nM) that blocks 70% of IT was necessary to decrease the number of action potentials in the LTCP-burst but robust burst firing was still present when only 20% of the T channels were available (i.e. in the presence of 100nM TTA-P2) (Fig. 6C). The full block of the LTCP could only be achieved using 1μM (n=5) (Fig. 6C). Taken together, these results indicate that the number of T-type Ca2+ channels expressed in TC neurons largely exceeds the number of channels required to elicit an LTCP by synaptic potentials.
TC neurons are capable of generating intrinsic membrane potential oscillations that are key to a number of sleep rhythms (Crunelli et al., 2005; Crunelli et al., 2006), including the delta and slow (<1Hz) sleep oscillations. Whereas the former activity mainly requires the interaction of T-type Ca2+ and h currents for its generation, the Up and Down states of the latter oscillation have been suggested to be due to the switching on and off of ITwindow on the basis of dynamic clamp experiments (Hughes et al., 2002; Hughes et al., 2008) and the relatively higher potency of Ni2+ compared to Cd2+ (Williams et al., 1997). The slow oscillation was induced in TC neurons of the MGB in cat thalamic slices by bath application of the metabotropic glutamate receptor agonist t-ACPD, as previously described (Hughes et al., 2002). Application of TTA-P2 (3μM) abolished the slow (<1Hz) oscillation by progressively shortening the duration, and decreasing the amplitude, of the Up state (Fig. 7A and B), such that after a few minutes in the presence of this antagonist the activity expressed by the recorded TC neurons resembled a delta oscillation. This activity too was eventually abolished after an additional few minutes of perfusion with TTA-P2. This pattern of events was observed in all TC neuron tested (n=5), and recovery of the slow and delta oscillations occurred within 25-45 min after beginning of the antagonist washout (Fig. 7C).
The use of a potent, selective T-type Ca2+ channel antagonist has enabled the discovery of novel roles for these channels in the modulation of single neuron excitability. In particular, the present results provide the first direct demonstration of the contribution of ITwindow to the resting membrane potential of thalamic neurons and to the UP state of their intrinsic slow (<1Hz) sleep oscillation, and show that only a small fraction of the T-type Ca2+ channel population is sufficient to generate robust LTCPs in these neurons.
Our experiments show that TTA-P2 fully blocks IT in TC and NRT neurons. Since TC neurons specifically express the Cav3.1 isoform while NRT neurons express Cav3.3 and 3.2 (Talley et al., 1999), these results are in agreement with previous data on recombinant Cav3.1, 3.2 and 3.3 channels that showed an identical potency of TTA-P2 on all three isoforms (Shipe et al., 2008). The similarity of the TTA-P2 IC50 for native T-type channels in TC neurons (22nM) and recombinant channels expressed in HEK cells (93nM for Cav3.1) (Shipe et al., 2008) demonstrates that the potency of TTA-P2 does not depend on potential splice variant difference between native and heterologous systems and/or the presence of still unknown auxiliary subunits in TC neurons (see Perez-Reyes, 2003). Our results also demonstrate that the effects of TTA-P2 on the intrinsic and synaptic excitability of TC and NRT neurons specifically involve T-type Ca2+ channel activation, since this antagonist does not modify the input resistance, the depolarizing sag present in the voltage response to negative current pulses, the transient and persistent Na+ currents, the action potential properties and tonic firing.
The pharmacological profile of TTA-P2, therefore, is superior to existing putative T-type Ca2+ channel blockers. One of the oldest and possibly best established blockers of T-type Ca2+ channels is the divalent cation Ni2+ (Fox et al., 1987) that was shown to be selective for low-voltage over HVA Ca2+ channels in cells expressing the Cav3.2 isoform (Lee et al., 1999). However, for a substantial block of the other two Cav3 isoforms a concentration of Ni2+ that already affects HVA currents is required (Zamponi et al., 1996). The antihypertensive mibefradil and the antiepileptics based on succinimides have also been extensively used in recombinant and native preparations, but the potency and selectivity of these drugs are not high and vary according to cell type and recording conditions (McGivern, 2006). With an IC50 in the micromolar range, mibefradil is an effective T-type Ca2+ channel blocker, but also inhibits HVA Ca2+ and Na+ channels, as well as voltage-gated and ATP-sensitive K+ channels (Mishra and Hermsmeyer, 1994; Bezprozvanny and Tsien, 1995; Nilius et al., 1997; Viana et al., 1997; Gomora et al., 1999; Liu et al., 1999). The use of ethosuximide and α-methyl-α-phenylsuccinimide have provided contrasting results, from a maximum 50% block of IT when used in millimolar concentrations (Coulter et al., 1989; Gomora et al., 2001) to no effect but with inhibition of the persistent Na+ and Ca2+-activated K+ currents (Leresche et al., 1998; Crunelli and Leresche, 2002b). Finally, U-92032 (Porcello et al., 2003), some anesthetic and antipsychotic drugs (Herrington et al., 1991; Takenoshita and Steinbach, 1991; Todorovic et al., 2000; Joksovic et al., 2005b; Orestes et al., 2008) also block IT but concurrently affect many other targets. Thus, the poor specificity and/or potency of these antagonists have so far hampered the interpretation of studies on neuronal excitability that have used these compounds. Instead, the use of the selective and potent TTA-P2 opens a novel avenue to investigate the role of T-type Ca2+ currents in single cell excitability, synaptic physiology and neuronal network activities. In particular, it should allow precise dissection of physiological mechanisms such as the amplification of synaptic inputs by T-type Ca2+ channels (Markram and Sakmann, 1994; Magee et al., 1995; Tsay et al., 2007), the IT-dependent component of synaptic plasticity (Oliet et al., 1997; Aizenman et al., 1998; Uebele et al., 2009), and the relative contribution of cortical and thalamic T-type Ca2+ channels to the pathophysiology of absence seizures (Vitko et al., 2007; Ernst et al., 2009; Powell et al., 2009).
By using the novel compound TTA-P2, the present results have already allowed us to consolidate and enlarge our current understanding of the contribution of T-type Ca2+ channels to single neuron excitability.
Firstly, we have directly demonstrated that there are sufficient numbers of T-type Ca2+ channels expressed in thalamic neurons to provide a robust redundancy mechanism for LTCPs. Previous studies in thalamic slices had suggested that a full block of LTCPs requires a marked reduction of the number of available T channels. However difficulties with the potency and specificity of the various antagonists used in these studies (i.e. U-92032, ethosuximide, methylphenylsuccinimide, mibefradil and valproate) did preclude a direct measurement of the minimal number of T channels required to evoke an LTCP in various physiological situations (Porcello et al., 2003; Broicher et al., 2007), while a computational model indicated that LTCPs could still be evoked when the T conductance was decreased by 50% (Broicher et., 2007). Our data directly show that LTCPs and their associated burst firing could still be readily evoked when up to 70% of the channels are blocked. Indeed, application of increasing concentrations of TTA-P2 demonstrates that only when less than 30% of the T-type channel population is available for activation, the GABAergic inputs occurring at depolarized potentials are unable to generate rebound LTCPs. Similarly, in physiological conditions more favorable to T-type channel de-inactivation (i.e. slightly hyperpolarized potentials), lemniscal EPSP triggers a strong LTCP and associated high-frequency firing when only 20% of the channels are available. These data, together with the demonstration of the physiological impact of the potentiation of IT that occurs at depolarized potentials in TC neurons (Bessaih et al., 2008) explain why high frequency bursts of action potentials can be recorded in awake animals when TC neurons are depolarized and most of the T-type Ca2+ channels are inactivated (Llinas and Steriade, 2006).
Secondly, we have directly demonstrated the presence of a window component of a Ca2+ current in neurons and its contribution to their resting membrane potential, a role that was proposed more than a decade ago on the basis of indirect in vitro experiments (Williams et al., 1997) using the dynamic-clamp technique (Hughes et al., 1999b) in thalamic neurons. As the window component is an inherent biophysical property of all three Cav3 isoforms, this result suggests a similar control by T-type Ca2+ channels of the resting membrane potential of other neuronal types that possess this class of membrane channels (Perez-Reyes, 2003). Although it is at present difficult to precisely ascertain the extent of this contribution to the ‘resting’ membrane potential in vivo, and in particular in non-anesthetized freely moving animals, its role under conditions of progressively increasing membrane hyperpolarization, such as it occurs in thalamic and cortical neurons during the behavioural shift from an attentive waking state to relaxed wakefulness and then to early sleep stages (Crunelli et al., 2006), should not be underestimated. A similar contribution of ITwindow to the membrane potential may also be of significance in pathological states, such as absence seizures, where neuronal hyperpolarization has been highlighted as one of the important underlying cellular abnormalities (Polack et al., 2007) together with an increase in IT (Tsakiridou et al., 1995; Ernst et al., 2009).
Thirdly, we have shown that the Up state of the slow (<1Hz) oscillation of thalamic neurons depends on IT. The biophysical mechanism of this slow oscillation in TC (and NRT) neurons requires the membrane potential bistability that is brought about by the interaction of ITwindow and the leak current (Hughes et al., 2002; Crunelli et al., 2005; Zhu et al., 2006), such that the Up and Down states are defined by ITwindow being switched on and off, respectively. The Ca2+ entry via the T-type channels activates ICAN which in turn determines the duration of the UP state. As predicted by previous dynamic clamp experiments (Hughes et al., 1999a; Crunelli et al., 2005), the selective block of IT by TTA-P2 shortens the duration and decreases the amplitude of the Up state, eventually leading to a delta oscillation, that is still evoked by the interaction between the remaining vast majority of T-type Ca2+ and h channels. Thus, the present experiments provide the first direct demonstration of a role for the window component of a Ca2+ current in a physiologically relevant brain activity, the slow (<1Hz) oscillation.
In conclusion, our results clearly demonstrate that TTA-P2 is a potent and selective T-type Ca2+ channel blocker that should prove a valuable tool for identifying the precise contribution of these channels to the excitability of single cells, as we have shown here for thalamic neurons, and to the dynamical output of neuronal networks in physiological and pathological conditions. Since TTA-P2 easily crosses the blood-brain barrier after oral dosing (Shipe et al., 2008), its use would not be restricted to in vitro analysis but could be easily extended to behavioural studies in freely moving animals.
This work was supported by the ANR-06-Neuro, CNRS LEA 528 (Thalamic function in health and disease states) and the Wellcome Trust (grant 71436).
Conflict of interest. JJR and VNU are employees of Merck and Co., Inc. (USA) and potentially own stock and/or stock options in the company.