Pyramidal neurones of layer V in rat sensorimotor neocortex are functionally heterogeneous in terms of their firing properties, and can be divided into different subtypes on the basis of their physiological and morphological features (Connors, Gutnick & Prince, 1982; Connors & Gutnick, 1990; Tseng & Prince, 1993). These different intrinsic properties play a crucial role in determining both the subthreshold behaviour and the firing characteristics of the various neuronal subclasses. As in other mammalian and non-mammalian nervous structures, a remarkable subpopulation of layer V pyramidal neurones, currently referred to as intrinsically bursting (IB) cells, is set to discharge with ‘burst’ firing characterized by a short series of action potentials (APs) superimposed on a slow depolarizing potential. It is assumed that the burst discharge generated by neocortical IB neurones significantly contributes to information processing and neocortical rhythm generation (Silva, Amitai & Connors, 1991; Lisman, 1997); moreover in disinhibited neocortical slices, intrinsically bursting neurones have been found to play an important role in epileptic synchronization (Connors, 1984; Chagnac-Amitai & Connors, 1989).
Previous data, obtained from both neocortical slices (Franceschetti, Guatteo, Panzica, Sancini, Wanke & Avanzini, 1995) and acutely dissociated neocortical pyramidal neurones (Guatteo, Franceschetti, Bacci, Avanzini & Wanke, 1996) have demonstrated that neocortical burst generation substantially depends on a tetrodotoxin-sensitive current, and is unaffected by Ca2+ channel blockers. Similar results have been reported for other cortical structures, such as the subiculum (Mattia, Hwa & Avoli, 1993) and CA1 hippocampal region (Azouz, Jensen & Yaari, 1996), in which both the fast spikes and the slow underlying depolarizing potential have been shown to be blocked only by the Na+ channel blocker tetrodotoxin (TTX). On the basis of the results of these experiments, it has been suggested that the persistent fraction of the sodium current (INa,p) (Taylor, 1993; Crill, 1996), which has been shown to be able to sustain plateau potentials (Stafstrom, Schwindt, Chubb & Crill, 1985; Hoehn, Watson & MacVicar, 1993; Fleidervish & Gutnick, 1996), is the best candidate for burst generation in neocortical pyramidal neurones, but no clear proof that it is sufficient for burst generation has been provided. In order to verify this hypothesis we used pharmacological manipulations capable of modifying the kinetics of the Na+ current (INa) to test the consequence of a selective enhancement of INa,p on firing behaviour. The following drugs were employed: anemone toxin II (ATX II), veratridine and iodoacetamide (IAA).
Anemone toxin II (ATX II) was chosen because of its selective inhibition of INa fast inactivation, which has previously been demonstrated in other cell types (Romey, Abita, Schweitz, Wunderer & Lazdunski, 1976; Bergman, Dubois, Rojas & Rathmayer, 1976; Hartung & Rathmayer, 1985; Neumcke, Schwarz & Stämpfli, 1987). ATX II is a small polypeptide produced by Anemonia sulcata that binds to an extracellular site on domain IV of the voltage-dependent Na+ channel α-subunit (Rogers, Qu, Tanada, Scheuer & Catterall, 1996). Since the effects of ATX II on central mammalian neurones have never been studied, a subset of experiments was performed on dissociated pyramidal neurones, designed to confirm the efficacy and selectivity of ATX II on the inactivation properties of the INa in neocortical neurones. In both neocortical slices and dissociated pyramidal neurones, the effects of ATX II were compared with those induced by veratridine, which is known to affect both activation and inactivation of INa (Hille, 1992) and IAA, an alkylating agent potentially active in slowing down INa inactivation (Ulbricht, 1990). The preliminary results have been previously published in abstract form (Mantegazza, Avanzini & Franceschetti, 1996).
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Wistar rats aged from 9 to 35 days were deeply anaesthetized with ether, killed by decapitation and their brains rapidly removed and placed in cold (4°C) artificial cerebrospinal fluid (ACSF), bubbled with 95 % O2 and 5 % CO2. Coronal slices, with a thickness of 250-350 μm, were prepared from the sensorimotor cortex, using a vibratome (FTB Vibracut 3). For most of the current-clamp experiments, the slices were immediately transferred to an interface recording chamber (Haas type, modified; Haas, Schaerer & Vosmansky, 1979) kept at 35°C and perfused by ACSF. The slices were allowed to equilibrate for 1-1.5 h before recordings were made. The composition of the ACSF was (mM): NaCl, 124; NaHCO3, 26.5; CaCl2, 2; NaH2PO4, 1.25; MgSO4, 2; KCl, 5; glucose, 10; bubbled with 95 % O2 and 5 % CO2. The composition of the low-Ca2+ solution was (mM): NaCl, 120; NaHCO3, 24; CaCl2, 1; NaH2PO4, 1; MgCl2, 4; KCl, 2.5; glucose, 20; bubbled with 95 % O2 and 5 % CO2. Veratridine was added to the superfusion medium. ATX II was dissolved in ACSF and locally applied, using a pipette with a tip diameter of 10-15 μm. Local application of ATX II was used in order to minimize the quantity of perfused drug; local administration of ACSF alone was tested in preliminary control experiments.
In some of the experiments veratridine and IAA were tested using a submersion recording chamber. In that case, slices were maintained in oxygenated low-Ca2+ ACSF until recording, when slices were individually transferred to a submersion recording chamber and perfused with ACSF at a temperature of 30°C.
Kynurenic acid at 2 mM was added to the perfusing solution in most ATX II experiments. Firing behaviour was characterized before and after addition of the excitatory amino acid blocker to the ACSF and postsynaptic potentials were monitored to confirm the block of the excitatory synaptic transmission. Intracellular recordings were made using an IR-283 (Neurodata Inst. Corp.) or Axoclamp-2A (Axon Instruments, Inc.) amplifier. We used voltage follower microelectrode amplifiers, since they have been found to provide more accurate membrane voltage measurements than patch-clamp amplifiers (Magistretti, Mantegazza, Guatteo & Wanke, 1996).
Intracellular recordings were mainly performed using borosilicate glass electrodes pulled with an horizontal puller, and filled with 3 M potassium acetate (resistance, 80-90 MΩ). In some experiments, the electrodes were filled with 1 M IAA and 1 M KCl, in order to inject IAA into the cytoplasm iontophoretically.
Only healthy neurones with a stable resting membrane potential (Vrest) more negative than -60 mV, a stable firing level and overshooting action potentials were selected for the analysis. Voltage and current signals were continuously displayed on a Tektronix storage oscilloscope.
The duration of APs was measured at a level 40 mV depolarized from the Vrest. Membrane input resistance (Rin) was measured at the peak of the voltage deflection of the membrane potential (Vm) induced by the injection of hyperpolarizing 0.4 nA current pulses.
Voltage-clamp experiments were performed by patch clamping acutely dissociated pyramidal neurones prepared from single slices kept for 10-15 min in the following solution (mM): NaCl, 123; NaHCO3, 15; CaCl2, 0.1; EGTA, 0.1; MgCl2, 2; KCl, 3; glucose, 20; kynurenic acid, 1; Hepes-NaOH, 10; pH 7.4. The solution contained 1 mg ml−1 of protease type XIV (Sigma) to digest the extracellular matrix, and was bubbled with 95 % O2 and 5 % CO2 at 35°C. After enzyme treatment, the slices were washed in an enzyme-free solution and kept in a holding chamber as above. One single slice at a time was mechanically dissociated using fire-polished glass pipettes with progressively smaller tips.
The neurones were plated onto Petri dishes (Costar, Cambridge, MA, USA), left 5-10 min to allow attachment and then bath perfused with the following solution (mM): NaCl, 114; NaHCO3, 25; CaCl2, 2; NaH2PO4, 1.25; MgSO4, 2; KCl, 5; Hepes-NaOH, 10; glucose, 10; bubbled with 95 % O2 and 5 % CO2, pH 7.4. Only large neurones showing a pyramidal shape were selected for patch-clamp recordings.
In order to isolate the Na+ currents, the following solution (physiological Na+ solution), was locally perfused (mM): NaCl, 120; CaCl2, 1.3; MgCl2, 2; CdCl2, 0.4; NiCl2, 0.3; TEACl, 20; Hepes-NaOH, 10; glucose, 10; pH 7.4. In the experiments aimed at evaluating the properties of the fast Na+ current (see Results), NaCl was partially replaced with choline chloride (low-Na+ solution: NaCl, 10 mM; choline chloride, 110 mM) in order to reduce voltage-clamp errors. The drugs (ATX II, TTX, veratridine) were dissolved in physiological Na+ or low-Na+ solutions and locally applied. The borosilicate glass capillary recording pipettes were pulled using a horizontal puller and filled with the following solution (mM): CsF, 120; MgCl2, 1; EGTA-CsOH, 10; Na2ATP, 2; Hepes-CsOH 10; phosphocreatine-diTris, 10; with 20 units ml−1 creatine phosphokinase; pH 7.2. With this solution the resistance of the electrodes was 2-3 MΩ. In some experiments 1 mM IAA was added to the internal solution.
In order to avoid contaminating currents, the traces recorded in the presence of both 1 μM TTX and the test drug were subtracted from those recorded in the presence of the drug alone. When INa was measured using a step voltage stimulus, the INa,p amplitude was determined as the mean current between 100 and 200 ms after the beginning of the stimulus.
In order to record both voltage-activated and Ca2+-activated K+ currents the following external solution was locally perfused (mM): NaCl, 140; CaCl2, 2; MgCl2, 2; KCl, 3; Hepes-NaOH, 10; glucose, 10; pH 7.4. TTX was added at 1 μM to block INa, and ATX II was dissolved in that solution. Recording pipettes were filled with (mM): potassium gluconate, 119; KCl, 13; MgCl2, 2; EGTA-KOH, 5; Na2ATP, 2; Hepes-KOH, 10; NaGTP, 0.2; phosphocreatine-diTris, 10; with 20 units ml−1 creatine phosphokinase; pH 7.2.
The following external solution was used to isolate Ca2+ currents (mM): choline chloride, 80; CaCl2, 2; MgCl2, 2; KCl, 3; Hepes-NaOH, 10; TEA-Cl, 40; 4AP, 5; glucose, 10; pH 7.4. ATX II was dissolved in this solution. The recording pipettes were filled with (mM): CsCl, 78; TEACl, 40; EGTA-CsOH, 10; MgATP, 2; Hepes-CsOH, 10; GTP-Na, 0.2; cAMP, 1; phosphocreatine-diTris, 10; with 20 units ml−1 creatine phosphokinase; pH 7.2.
The voltage-clamp recordings were made at room temperature (21-24°C), using a Biologic RK-400 patch-clamp amplifier; junction potential was not corrected. Capacitative currents were minimized by means of the amplifier circuitry, and 70-80 % series resistance compensation was routinely used. The remaining transients and leakage currents were eliminated using P/4 subtraction.
The pCLAMP 6.0.3 software and a Digidata 1200 interface (Axon Instruments) were used to generate the stimulus protocols and to acquire the signals; the voltage signals were filtered at 3 kHz, and the current signals at 5 kHz (voltage steps) or 0.5 kHz (voltage ramps). The data were analysed using pCLAMP and Origin 4.0 (Microcal Inc.) software on a Pentium 166 PC. Voltage dependency was fitted to Boltzmann relationships in the form:
y= ((A1 - A2)/(1 + exp(V - V½)/k)) +A2,
using the Levenberg-Marquardt algorithm, where V½ is membrane potential at half-maximal inactivation. The statistical results are given as means ± standard error of the mean (s.e.m.); statistical significance was evaluated using the two-tailed Student's test for paired data (only P values < 0.01 were accepted).
ATX II was purchased from Calbiochem-Novabiochem Corp. (La Jolla, CA, USA); veratridine, IAA, TTX, kynurenic acid, phosphocreatine, creatine phosphokinase, GTP and cAMP were purchased from Sigma.