A Ca2+-independent slow afterhyperpolarization in substantia nigra compacta neurons

Neuroscience 125 (2004) 841– 852

A Ca2ⴙ-INDEPENDENT SLOW AFTERHYPERPOLARIZATION IN SUBSTANTIA NIGRA COMPACTA NEURONS S. NEDERGAARD*

response patterns such as spike frequency adaptation and long-lasting membrane afterhyperpolarization (AHP) following the stimulus. In many neurons the currents that underlie these phenomena have been identified as K⫹ currents activated by Ca2⫹ entry, and are divided into two types, named IAHP (mIAHP) and sIAHP, with distinct pharmacology and kinetics (Sah, 1996; Storm, 1990; Schwindt et al., 1988a). In addition to these Ca2⫹-dependent currents, there is evidence in some neurons that other types of K⫹ currents, such as IM (Madison and Nicoll, 1984; Storm, 1989) and a Na⫹-dependent K⫹ current (Schwindt et al., 1988b,1989; Safronov and Vogel, 1996; Kim and McCormick, 1998; Sanchez-Vives et al., 2000) are implicated in adaptive responses, including slow AHP. In midbrain dopaminergic neurons an AHP component has been identified, which is inhibited by apamin (Shepard and Bunney, 1991; Harris, 1992; Nedergaard et al., 1993), and is underlain by a macroscopic current with kinetics similar to currents ascribed to the SK-type Ca2⫹-activated K⫹ channel in other neurons (mIAHP; Wolfart et al., 2001). The apamin-sensitive current makes a prominent component of the AHP that follows single action potentials (Shepard and Bunney, 1991; Harris, 1992; Nedergaard et al., 1993), and prolonged depolarizing stimuli (Nedergaard et al., 1993; Ping and Shepard, 1999), and has a regulatory influence on the frequency and pattern of spontaneous and evoked discharge (Shepard and Bunney, 1988, 1991; Ping and Shepard, 1996; Wolfart et al., 2001; Wolfart and Roeper, 2002). Nigral SK-type channel activation depends on a rise in intrasomatic/dendritic Ca2⫹ concentration (Wilson and Callaway, 2000) resulting from transmembrane Ca2⫹ influx under resting firing conditions. In addition, this channel has been shown to activate in response to Ca2⫹ release from intracellular stores, either as result of metabotropic glutamate receptor activation (Fiorillo and Williams, 1998), or as a spontaneous event in neonate rats (Seutin et al., 2000). There is also some evidence for the existence of another type of slow inhibitory current in dopaminergic neurons. The response to long-lasting depolarizing current injections involves spike frequency adaptation (Shepard and Bunney, 1991; Nedergaard et al., 1993; Richards et al., 1997) and a prolonged cessation of spontaneous firing after the stimulation (post-stimulus inhibitory period [PSIP]; Shepard and Bunney, 1991). Neither of these responses are sensitive to apamin applied in doses sufficient to inhibit SK-type channels (Shepard and Bunney, 1991; S. Nedergaard, unpublished observations). Furthermore, in a recent whole-cell study, an outward current has been reported (sIAHP), which has a slow decay (seconds) and

Department of Physiology, University of Aarhus, Ole Worms Alle 160, DK-8000 Århus C, Denmark

Abstract—The discharge properties of dopaminergic neurons in substantia nigra are influenced by slow adaptive responses, which have not been fully identified. The present study describes, in a slice preparation from the rat, a complex afterhyperpolarization (AHP), elicited by action potential trains. The AHP could be subdivided into a fast component (AHPf), which was generated near action potential threshold, relaxed within approximately 1 s, and had highest amplitude when evoked by short-lasting (0.1 s) depolarizations, and a slow component (AHPs), which lasted several seconds, was evoked from subthreshold potentials, and required prolonged depolarizing stimuli (>0.1 s). A large proportion of the AHPf was sensitive to (i) 0.1 ␮M apamin, (ii) the Ca2ⴙ antagonists, Cd2ⴙ (0.2 mM) and Ni2ⴙ (0.3 mM), (iii) low (0.2 mM) extracellular Ca2ⴙ concentration, and (iv), Ca2ⴙ chelation with intracellular EGTA. The AHPs was resistant to the above treatments, and it was insensitive to 25 ␮M dantrolene or prolonged exposure to 1 ␮M thapsigargin. The reversal potential of the AHPs (ⴚ97 mV) was close to the Kⴙ equilibrium potential. It was significantly inhibited by 5 mM 4-aminopyridine, 5 ␮M haloperidol, 10 ␮M terfenadine, or high extracellular Mg2ⴙ (10 mM), but not by 30 mM tetraethylammonium chloride, 50 ␮M carbachol, 0.5 ␮M glipizide, 2 ␮M (ⴚ)sulpiride, 100 ␮M N-allyl-normetazocine, or 100 ␮M pentazocine. Haloperidol reduced the poststimulus inhibitory period seen during spontaneous discharge, but had no detectable effect on spike frequency adaptation. It is concluded that the SK-type Ca2ⴙ-activated Kⴙ channels underlies a major component of the AHPf, whereas the AHPs is Ca2ⴙ-independent and relies, in part, on a voltage-dependent Kⴙ current with properties resembling the ether-a-go-go-related gene Kⴙ channel. The latter component exerts a slow, spike-independent, inhibitory influence on repetitive discharge and contributes to the prolonged decrease in excitability following sustained depolarizing stimuli. © 2004 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: Kⴙ channel, ERG, haloperidol, AHP, midbrain, dopamine.

Slow hyperpolarizing currents in central neurons are activated by depolarizing stimuli in the form of single or trains of action potentials, and give rise to characteristic acute *Tel: ⫹45-8942-2800; fax: ⫹45-8612-9065. E-mail address: [email protected] (S. Nedergaard). Abbreviations: AHP, afterhyperpolarization; AHPf, fast afterhyperpolarization; AHPs, slow afterhyperpolarization; EGTA, ethylene glycol-bis(␤-aminoethyl ether)-N,N,N⬘,N⬘-tetraacetic acid; ERG, ether-ago-go-related gene; HERG, human ether-a-go-go-related gene; HEPES, N-(2-hydroxymethyl)piperazine-N⬘-(2-ethanesulfonic acid); PSIP, post-stimulus inhibitory period; (⫹)SKF 10,047, (⫹)-N-allylnormetazocine; TEA, tetraethylammonium chloride; TTX, tetrodotoxin; 4-AP, 4-aminopyridine.

0306-4522/04$30.00⫹0.00 © 2004 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2004.02.030

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which is unaffected by the removal of extracellular Ca2⫹ (Wolfart et al., 2001). It seems reasonable to assume that phenomena such as the PSIP and frequency adaptation observed in the in vitro slice preparation depend on properties intrinsic to the membrane. However, the nature of these properties and the conditions required for their activation remains to be defined. It is worth noting also, that similar phenomena are observed in connection to a certain state of activity displayed by these neurons in vivo, which involves burst firing (Grace and Bunney, 1984). This type of activity seems to represent a fundamental form of signal processing by which these cells can modify behavioral reactions of the animal (Schultz, 1998). While the expression of bursting behavior essentially depends on intact synaptic connectivity (Overton and Clark, 1997; Kitai et al., 1999; Tepper et al., 1998), it is still not clear how, or to what extent individual intrinsic membrane currents participate in various aspects of this activity. The aim of the present study was to characterize more closely in the in vitro slice preparation, the events following prolonged depolarizations and to examine their possible contribution to the discharge properties.

EXPERIMENTAL PROCEDURES Brain slice preparation and intracellular recording Mesencephalic brain slices were obtained from male Wistar rats (250 –300 g). The animals were deeply anesthetized in an air-tight container with either chloroform or 3.5% isoflurane mixed in air. After decapitation the brain was removed, and a tissue block containing the mesencephalon was isolated for slice preparation. Coronal slices (400 ␮m thick) were cut in a vibrotome and transferred to a N-(2-hydroxymethyl)piperazine-N⬘-(2-ethansulfonic acid) (HEPES)-buffered solution (see below), bubbled with carbogen (95% O2–5% CO2), and stored at room temperature for at least 1 h before use. In the recording chamber, the slice surface was at the interface between a humidified atmosphere of carbogen at 31–33 °C and a standard perfusion solution (see below; flow rate⫽1.5 ml per min). Intracellular recordings were made via borosilicate glass microelectrodes (1.2 mm O.D.) filled with 3 M K-acetate (resistance 50 –90 M⍀) and connected to a highimpedance bridge amplifier (Axoclamp 2A; Axon Instruments, Foster City, CA, USA). Electrodes were lowered into the substantia nigra pars compacta, identified as the area of gray matter extending dorso-lateral to the medial terminal nucleus of the accessory optic tract (Paxinos and Watson, 1998). Recorded signals were digitized and stored on videotape. Data analyses were performed off-line on a PC computer using SIGNAL software (CED, Cambridge, UK). At the end of each experiment, the electrode was retracted a few micrometers from the cell, and the recorded potential was subtracted off-line from the intracellular voltage to obtain the membrane potential.

Materials The HEPES storage solution contained (in mM): 120 NaCl, 2.0 KCl, 1.25 KH2PO4, 2.0 MgSO4, 2.0 CaCl2, 20 NaHCO3, 6.7 HEPES acid, 2.6 HEPES salt, and 10 glucose. The standard perfusion solution contained (in mM): 132 NaCl, 1.8 KCl, 1.25 KH2PO4, 1.3 MgSO4, 2.4 CaCl2, 20 NaHCO3, and 10 glucose. In experiments where CdCl2 and NiCl2 were applied, KH2PO4 and MgSO4 were omitted from the perfusion medium and replaced with equimolar KCl and MgCl2 respectively.

Chemicals were applied to the perfusion medium from premade stocks (500 –1000⫻ final conc.), dissolved in either DMSO (dantrolene, glipizide, haloperidol, terfenadine, thapsigargin), ethanol ((⫺)sulpiride), or water (other chemicals). Applications of light-sensitive compounds were performed in a dark environment. In some experiments, recording electrodes were filled with a solution containing 0.5 M EGTA in 3 mM K-acetate, for chelation of intracellular Ca2⫹. Pharmacological substances were purchased from Sigma, UK, except tetrodotoxin (TTX), thapsigargin (both from Alomone Laboratories, Israel), terfenadine (ICN Biomedicals, USA), and pentazocine (USP, USA).

Data analysis Membrane input resistance was calculated from the slope of the current-voltage curve constructed from measurement of the maximal voltage deflection in response to weak negative current pulses, applied from resting potential. Action potential amplitude was measured from threshold, and the action potential duration from onset to the point where the falling phase crossed the threshold potential. The amplitude of hyperpolarizations following depolarizing current pulses was measured on averaged sweeps (usually three to six) as the height below the average baseline potential recorded before the pulse. The slow component of the AHP (AHPs; see Results) had relatively low amplitude. Therefore, to reduce bias from background noise, the AHPs was taken as mean potential over a 100 ms period (i.e. between 2.0 and 2.1 s after the pulse). In some experiments, the duration of the silent period following an action potential train was compared at different times. This parameter varies with the background firing rate, and to eliminate such influence, the following precautions were taken. First, during the experiment, the spontaneous firing rate was clamped manually as accurately as possible by DC current injection to the cell. Second, in the final analysis, the silent period for each measurement was normalized to the actual firing rate by expressing it as: (t⫺ISI)/ISI; where t⫽time from the pulse offset to the first spontaneous action potential and ISI⫽mean inter-spike interval of at least 10 consecutive action potentials obtained in a period of steady firing prior to pulse injections. Averaged data are presented as the mean⫾S.E.M., unless otherwise stated. For statistical evaluation of drug effects, a paired t-test was used with level of significance set at P⬍0.05.

RESULTS Cell characteristics All cells included in the study (n⫽98) were identified as dopaminergic neurons, based on a set of electrophysiological membrane characteristics, shown to be distinctive for cytochemically identified dopamine-containing neurons in this preparation (Grace and Onn, 1989; Richards et al., 1997). Specifically, the following inclusion criteria were applied: (i) action potential duration ⱖ1.5 ms, (ii) the appearance of a prominent sag in the voltage response to hyperpolarizing current pulse injection, and (iii) a delayed return to resting potential after the pulse. Irrespective of these criteria, neurons that were either unable to fire repetitively during a 1 s depolarizing current pulse or had action potential heights of less than 45 mV were discarded. In a representative sample of 60 neurons, the following values were obtained (mean⫾S.D.): membrane input resistance: 133⫾37 M⍀ (range: 70 –262 M⍀), action potential height: 55⫾5 mV (range: 50 – 68 mV), and action potential duration: 2.2⫾0.4 ms (range: 1.6 –3.5 ms).

S. Nedergaard / Neuroscience 125 (2004) 841– 852

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Fig. 1. The PSIP and AHP elicited by action potential trains. (A) High-frequency trains of action potentials evoked by a single depolarizing current pulse (top) and by a 10 Hz train of 5 ms pulses (bottom) during spontaneous pacemaker firing at rest. (B) Responses to stimulation as in (A) from a hyperpolarized membrane potential (⫺55 mV) held by negative DC current injection.

During passive recording, most neurons (45/60) fired spontaneously at a slow, regular rate (mean frequency⫽2.5⫾1.0 Hz). The remaining 15 neurons were either silent or discharged sporadically at a very low rate. The PSIP and AHP evoked by spike trains In regularly firing neurons, a high-frequency train of action potentials induced by injection of positive current for 1 s or more was found to be a reliable stimulus for the PSIP. This response was seen as a period of silence, following the current pulse, which exceeded the normal inter-spike interval (Fig. 1A top). A similar stimulus, delivered from a steady, hyperpolarized membrane potential, was followed by an AHP, consisting of an early peak and a slow repolarization lasting several seconds (Fig. 1B top). By comparison, when the action potential train was elicited by repetitive injection of brief (5 ms) current pulses, the PSIP after the train was short, and often of similar duration to the interval between two consecutive spontaneous spikes (Fig. 1A bottom). The AHP that followed such intermittent stimulation had a fast decay (Fig. 1B bottom). As described elsewhere (Nedergaard, 1999), the dimension (height and duration) of individual action potentials was noted to be considerably reduced when elicited by brief compared with sustained depolarization (not illustrated). Dependency on depolarization length of two AHP components These observations indicated first, that the train-induced silent period is underlain by an AHP, and second, that these responses, rather than being a product of the action potential train per se, depend either on a maintained depolarization between the action potentials, or on the dimension of individual action potentials. To discriminate between these possibilities, further characterization of the AHP was done. In these experiments CsCl (3 mM) was

included in the perfusion medium during the recordings to avoid contribution from the hyperpolarization-activated cation current, Ih. Cesium in itself was noted to reduce the early peak of the AHP, in accordance with previous findings (Ping and Shepard, 1999), but it did not alter the later part of the potential (not illustrated). The dependence of the AHP on the length of the pre-depolarization was examined by injection of depolarizing pulses of fixed amplitude (0.8 –1.0 nA, 0.05 Hz) from holding potentials between ⫺60 and ⫺65 mV. Pulses lasting up to 0.1 s produced a fast AHP (AHPf; Fig. 2A, Ba), which had an average time-to-peak of 46⫾2 ms and relaxed completely within 790⫾90 ms after the pulse (range: 320 –1210 ms; n⫽10). The AHPf, measured at its peak, had maximal amplitude with 0.1 s pulses (mean⫽18.0⫾0.8 mV; n⫽10). Extension of the pulse beyond 0.1 s resulted in the development of another, slow phase of the AHP. This component, henceforth referred to as AHPs, was seen as a slowly decaying potential beginning at the repolarizing phase of the AHPf. To obtain a quantitative estimate of the AHPs, separate from the AHPf, it was chosen to define its amplitude as the membrane-potential undershoot 2 s after the pulse (Fig. 2A). Measurements from 10 neurons showed a gradual amplification of the AHPs with increasing pulse length between 0.1 and 4 s. At 4 s, the mean amplitude was 3.8⫾0.5 mV (n⫽10; Fig. 2A, Bb). Further prolongation of the pulse in three experiments produced additional increase in the amplitude by 40% (10 s) and 43% (20 s), suggesting that full development of the AHPs requires ⬎10 s depolarization. The complete decay time for this component could not be determined precisely. Instead, an estimate of the duration was obtained by measuring the time from pulse offset to half decay. This value was determined in nine cells to be 5.0⫾0.3 s (4 s pulses), 5.1⫾0.3 s (2 s), and 4.6⫾0.2 s (1 s), total range⫽3.6 – 6.7 s. Neither of these mean values varied significantly from the others

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Fig. 2. Characteristics of the AHPf and AHPs. (A) Top panel, responses to square depolarizing current pulses of variable length from ⫺63 mV holding potential. In the bottom panel the AHPs (following termination of 0.1, 1, 2, and 4 s pulses) are shown aligned. Vertical lines indicate where measurement of AHPf and AHPs were taken. (Ba, b) Graphic representation of the mean amplitude (⫾S.E.M., n⫽10) of the two AHP components as function of the pre-pulse duration. In (Bb) the curve marked ‘Tr.’ was obtained with train stimulation (20 pulses/10 Hz), and shows AHPs amplitude versus duration of individual pulses in the train (n⫽5). Plots in (Bc, d) show data obtained before (F) and during (E) perfusion of TTX (0.5–1 ␮M; n⫽4). Values are normalized to the amplitude at 4 s pulse in control medium (⫺100%). All data are with 3 mM Cs⫹ present.

(P⬎0.05). The relationship between the AHPs and depolarization length was also tested by intermittent stimulation. Injection of a train of 20 pulses at 10 Hz resulted in a response, which was detectable at individual pulse lengths as short as 10 ms (n⫽5; Fig. 2Bb). This result suggested that cumulative activation of the AHPs takes place over intervals of several tens of milliseconds. Effects of TTX Blockade of action potentials by TTX (0.5–1.0 ␮M) gave some reduction of both components of the AHP. As seen in Fig. 2Bc, d, the effect was most prominent on the AHPf elicited by short-lasting depolarizations, whereas the AHPs was much less affected by TTX. The TTX-sensitivity and voltage-dependency of the AHP was studied in more detail by testing the effects of weak pulses applied in small incrementing steps. In three cells examined this way, the threshold of the AHPf, expressed either as applied current (without

TTX) or absolute voltage (with TTX present), was found to lie near the threshold for action potential firing (Fig. 3A, C). In contrast, the AHPs was consistently elicited from membrane potentials well below the firing threshold (10 –15 mV) in the same cells (Fig. 3B, D). Passage of the spike threshold (indicated by arrow in Fig. 3D, left) gave no abrupt shift in AHPs amplitude. Depolarization block of the action potentials, which was usually seen with high-intensity currents, was not associated with reduction of the AHPs (Fig. 3D, left). These data indicated that the action potentials had very little influence on the AHPs. As illustrated in Fig. 3D (left) the TTXinduced inhibition of the AHPs occurred at all current intensities, including those below spike threshold. This latter effect could largely be ascribed to a change in I/V relation during exposure to TTX (same current gave less depolarization with TTX), because no clear effect of TTX was seen at these subthreshold potentials when the data were normalized with respect to absolute voltage (Fig. 3D, right).

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Fig. 3. The influence of membrane voltage and action potential firing on the AHP. (A, B) Averaged responses (n⫽4) to 0.2 s and 2 s square depolarizations to subthreshold potential (⫺51 mV). Holding potential⫽⫺64 mV; 3 mM Cs⫹ present. Same calibration in (A) and (B). Note AHPs in (B) and absence of AHPf in (A). (C, D) Plots from the same experiment of AHPf and AHPs amplitudes versus pulse intensity (left) and absolute potential reached during the pulse (right; only data without action potential firing included). Symbols refer to control data (F) and 1 ␮M TTX (E). White arrows indicate transition from sub- to supra-threshold pulse intensity. Black arrows indicate action potential threshold (⫺44 mV). Histogram in D (left) shows average number of action potentials per pulse without TTX. The decrease in number at high current intensities reflects depolarization block.

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The specific antagonist of SK-type Ca -activated K channels, apamin (0.1 ␮M), when applied to the bath in the presence of Cs⫹ (3 mM) and TTX (0.5–1 ␮M), gave variable inhibition of the AHPf depending on the pre-pulse duration. Thus, when this response was evoked by short (ⱕ0.2 s) pulses, it was completely abolished by apamin and reversed into a depolarizing afterpotential (Fig. 4A, B). The AHPf evoked by pulses longer than 0.2 s was only partly inhibited by apamin, and the effect was inversely correlated to pulse length (only 34% block was obtained with 4 s pulses, n⫽5). In the same cells, apamin had no significant effect on the AHPs (mean amplitude⫽107⫾10% of control; Fig. 4A, B). In one experiment, the apamin concentration was increased from 0.1–1 ␮M. This resulted in a slight further reduction of the AHPf and still no effect on the AHPs (not illustrated). These data suggested that the AHPs is distinct from the SK-type K⫹ channel. Reversal of the AHPs near EK To observe a possible reversal potential of the AHPs, voltage deflections obtained during negative current steps in the presence and absence of a depolarizing pre-pulse were subtracted (Fig. 5). Averaged data from eight cells showed reversal of the AHPs at ⫺97⫾1 mV. The proximity of this value to the estimated EK (⫺100 mV) indicated that K⫹ is the main charge carrier for the current underlying the AHPs. Role of calcium for the AHPs The purpose of the following experiments was to examine the possible calcium-dependency of the AHPs and to

further characterize the pharmacological profile of this response. In these experiments, the amplitude of the pre-pulse was adjusted when needed to obtain a constant depolarization before and after application of test solution. In one series of recordings, the Ca2⫹ concentration in the perfusion medium was lowered from 2.4 to 0.2 mM in combination with raised concentration of Mg2⫹. This protocol was chosen because it was associated with no detectable instability of membrane properties, unlike when nominally Ca2⫹-free media were used. After blockade of action potentials (0.5–1 ␮M TTX) and in the continued presence of 3 mM Cs⫹, perfusion with 0.2 mM Ca2⫹/ 3.5 mM Mg2⫹ for 15–35 min caused a marked reduction of the AHPf (Fig. 6A). The effect varied from a complete block with 0.1 s pulses to 44% reduction with 4 s pulses (Fig. 6B). The amplitude of the remaining component of the AHPf was directly proportional to the pulse length. The AHPs, on the other hand, was unaffected by this treatment (amplitude 97⫾2% of control; n⫽4). In another set of experiments, the low Ca2⫹ (0.2 mM) was combined with increased Mg2⫹ concentration to 10 mM. Here, the AHPs was clearly diminished (amplitude 53⫾13%; n⫽4). The possibility that this latter effect was due to the high Mg2⫹ rather than the low Ca2⫹ concentration was tested further by application of 10 mM Mg2⫹ with normal extracellular Ca2⫹. This treatment also gave reduction of the AHPs (n⫽2); subsequent lowering of Ca2⫹ to 0.2 mM in these experiments did not further reduce the AHPs. Apamin (0.1 ␮M), when added in the presence of low Ca2⫹, had only little effect on the remaining AHPf (Fig. 6A; two of two cells tested), indicating that block of SK-type K⫹ channel was nearly complete at 0.2 mM Ca2⫹. The effects of the

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inorganic Ca2⫹ channel antagonists Cd2⫹ (0.2– 0.4 mM) and Ni2⫹ (0.3 mM) were examined with normal external Ca2⫹ concentration. Responses evoked after 12–25 min application showed a large reduction of the AHPf (amplitude 33⫾8% of control; pulse length 2– 4 s; n⫽6) but no overall effect on the AHPs (101⫾14%; n⫽6; Fig. 6C, D). In separate experiments intracellular loading of the calcium chelating agent, EGTA (0.5 M in recording electrode), was performed in normal external medium. The AHP recorded a few minutes after stabilization from impalement was taken as control. After a total recording period of 40 – 60 min the AHPf was reduced in all of four cells tested. In two experiments, an afterdepolarization developed in place of the AHPf (Fig. 6C). The AHPs, however, was not affected in either of the cells, even after prolonged (up to 2 h) exposure to EGTA (Fig. 6C, D). In another four cells, bath application of the intracellular Ca2⫹ store reuptake inhibitor, thapsigargin (1 ␮M) for 40 –120 min failed to reduce the AHPs (amplitude 107⫾5%). Finally, dantrolene (25 ␮M, 35–90 min), an inhibitor of intracellular Ca2⫹ release, was also found to be ineffective (AHPs amplitude 103⫾10% of control, n⫽4).

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Fig. 5. Reversal potential of the AHPs. (A) Superimposed voltage records from a series of step hyperpolarizations following 2 s depolarizing current pulses (black traces). Control responses in the absence of pre-pulse are shown as gray. The curves were subtracted to obtain AHPs amplitude at each membrane potential. Arrow indicates time of measurement. Cs⫹ (3 mM) was present. (B) Plot of AHPs amplitude versus membrane potential from the same experiment. Each point represents the average of three to five measurements. The reversal potential was identified as the cross-point of the linear regression line on the voltage axis (⫺99 mV).

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Fig. 4. Effect of apamin on the AHP components. (A) Responses to depolarizing current pulses (top: 200 ms; bottom: 4 s) before and during exposure to 0.1 ␮M apamin. Note in top panel the block of the AHPf. (B) Plots of normalized AHPf and AHPs amplitudes versus pulse length (n⫽5). F, control; E, 0.1 ␮M apamin. Cs⫹ (3 mM) and TTX (0.5–1 ␮M) were present throughout.

The above results indicated that the AHPs is mediated by a Ca2⫹-insensitive, voltage-dependent K⫹ current. The effects of the conventionally used K⫹ channel antagonists, 4-aminopyridine (4-AP) and tetraethylammonium chloride (TEA), were therefore examined. Control data were collected in the presence of Cs⫹ and TTX together with the calcium blockers Cd2⫹ and Ni2⫹ (to avoid generation of Ca2⫹-spikes). Perfusion with 5 mM 4-AP resulted in a significant reduction of the AHPs to 49⫾11% of control amplitude (Fig. 7A, B; n⫽6). The AHPf (component persisting after Ca2⫹ channel blockade) was also significantly diminished in the presence of 4-AP (amplitude: 57⫾7% of control). In two cells tested, partial recovery of the 4-AP effect was observed after 30 min washout. No effect was seen with low doses (0.1– 0.5 mM) of 4-AP (n⫽4; not illustrated). In contrast, TEA applied in high doses (10 – 30 mM) had no overall effect on the AHPs (amplitude: 95⫾5% in 30 mM TEA, n⫽7). However, the AHPf was markedly affected by TEA at these concentrations (Fig. 7); with 30 mM this component was reduced approximately to

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Fig. 6. Calcium-dependency of the AHP. (A) Superimposed records of depolarizations (4 s) evoked in normal Ca2⫹ (Ca 2.4), after 35 min perfusion with medium containing 0.2 mM Ca2⫹/3.5 mM Mg2⫹, and after subsequent addition of 0.1 ␮M apamin (3 mM Cs⫹ and 1 ␮M TTX present throughout). The current pulse intensity (not shown) was adjusted to give similar depolarization in each condition. Baseline potential⫽⫺60 mV. (B) Plots of the normalized AHP amplitudes versus pulse duration from four cells in 2.4 mM Ca2⫹ (F) and 0.2 mM Ca2⫹/3.5 mM Mg2⫹ (E). (C) Upper panel: AHP responses elicited in a control period and after 22 min perfusion with 0.4 mM Cd2⫹/0.3 mM Ni2⫹ (3 mM Cs⫹ and 1 ␮M TTX included; baseline potential⫽⫺60 mV). Lower panel: from another cell impaled in normal medium with an electrode containing 0.5 mM EGTA. The traces were obtained in the beginning of the recording (control) and after 50 min (EGTA). Periods of current injection are truncated for clarity. (D) Summary of the effects of calcium antagonism. The bars indicate average amplitudes (n⫽4 – 6 cells in each group) of AHPf and AHPs normalized to control values (⫽100%). * P⬍0.05.

zero (n⫽3), or reversed into a depolarizing afterpotential (n⫽4). In addition to 4-AP and TEA, the sensitivity of the AHPs to glipizide (0.5 ␮M, n⫽4) and carbachol (50 ␮M, n⫽3) were tested. No effects were seen with either substance, which indicated that neither the ATP-dependent K⫹ current nor the M-type K⫹ current participated in the response. Effects of haloperidol and related substances The slow properties of the AHPs seem not immediately compatible with either of the classical transient or delayed rectifier K⫹ channel subtypes. In this respect, another class of K⫹ channels would seem more likely to underlie this conductance: the ether-a-go-go-related gene (ERG) K⫹ channel. Members of this group of channels have been demonstrated in the midbrain. It was therefore tested if an antagonist of this channel type, the antipsychotic drug haloperidol (Suessbrich et al., 1997), could affect the response. These experiments showed a marked inhibitory effect of haloperidol on the AHP. Thus, a mean reduction to 45⫾10% (range 12– 68%) of the AHPs amplitude was found in six cells after 15–25 min exposure to 5 ␮M haloperidol (Fig. 7A, B). The AHPf was also significantly inhibited by haloperidol (Fig. 7A), but to a lesser extent (to 83⫾18% of the control amplitude). Apart from the block of ERG channels, other mechanisms could, in theory, explain this effect, since haloperidol binds to several types of receptors expressed by dopaminergic neurons. One of these is the dopamine D2 autoreceptor, which is antagonised by haloperidol. A specific antagonist of this receptor,

(⫺)sulpiride was therefore employed. No effect of 2 ␮M (⫺)sulpiride was seen, however, in any of five cells tested (AHPs amplitude⫽105⫾3% of control; Fig. 7B). The ␴ receptor ligands (⫹)-N-allylnormetazocine ((⫹)SKF 10,047) and pentazocine were also tested. Neither of these substances had statistically significant effect on the AHPs: amplitude⫽111⫾6% (n⫽6) and 105⫾3% (n⫽5) in the presence of either 100 ␮M (⫹)SKF 10,047 (Fig. 7B) or 100 ␮M pentazocine (Fig. 7A, B) respectively. These findings therefore indicated that the observed effect of haloperidol was not via dopamine or ␴ receptors. In an attempt to further clarify if ERG channels could be involved in the AHPs, the effect of another antagonist of this channel, terfenadine, was tested. As illustrated in Fig. 7A, terfenadine (10 ␮M) inhibited the AHP in a manner similar to haloperidol. Specifically, in six cells tested with 10 ␮M terfenadine, the mean amplitude of AHPs and AHPf was reduced to 49⫾9% (Fig. 7B) and 76⫾3% respectively. To test whether the haloperidol-sensitive component could have functional impact on the discharge properties, seven neurons were examined, which showed regular firing under control conditions. Application of 5 ␮M haloperidol gave an increase in the resting firing frequency in all of the cells, on average from 2.9⫾0.4 Hz to 3.5⫾0.4 Hz (P⬍0.05). The number of action potentials evoked by depolarizing current pulses of similar amplitude was generally increased by haloperidol. To make a reliable comparison of the PSIP before and after haloperidol, the above effects were compensated as closely as possible by adjustments of current injection. Under these conditions, the

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Fig. 7. Sensitivity of the AHPs to channel and receptor antagonists. (A) Voltage trajectories following depolarizing current pulses in different cells before and during perfusion with 5 mM 4-AP, 30 mM TEA, 5 ␮M haloperidol, 100 ␮M pentazocine, or 10 ␮M terfenadine. Unmarked traces are controls. Pulse lengths: 2– 4 s; baseline potentials: 55– 64 mV. In all experiments, TTX (0.5 ␮M), Cs⫹ (3 mM), Cd2⫹ (0.2 mM), and Ni2⫹ (0.3 mM) were present throughout. (B) Histogram of the AHPs amplitude recorded in the presence of the substances indicated. Bars indicate mean values (n⫽5–7 cells in each group) normalized to control (⫽100%). * P⬍0.05.

directly measured PSIP following depolarizing current pulses (2– 4 s duration; 0.05 Hz) was reduced in all cells during exposure to haloperidol (example in Fig. 8A). After normalization to the actual background inter-spike interval (see Experimental Procedures), the average reduction of the PSIP in response to haloperidol amounted to 28⫾5%. (Fig. 8B, n⫽7, P⬍0.05). The effect reversed partially after washout of haloperidol for 30 min (three of three cells). As described previously (Shepard and Bunney, 1991; Richards et al., 1997), the response to depolarizing stimuli is characterized by adaptation of the firing frequency, which is most pronounced between the first and second interspike interval. The frequency of the following spikes shows a much slower accommodation. This can be visualized by plotting the instantaneous frequency against spike interval position in the train, as in Fig. 8C. Overlay of the frequency plots obtained in control medium and in the presence of haloperidol showed similar pattern of frequency adaptation in individual neurons (Fig. 8C). An estimate of the overall degree of adaptation was obtained by calculating the ratio between the first and last inter-spike interval. The mean of this value was not significantly altered by haloperidol (Fig. 8D; P⬎0.05). Haloperidol had minor and inconsistent effects on the membrane input resistance: in three cells, the input resistance increased by 3–7%, in one cell a 9% decrease was seen, and in the remaining cells there was no detectable change. The possible contribution from a local anesthetic effect of haloperidol was examined in each cell by comparing the amplitude, time-to-peak, and duration of spontaneous action potentials sampled before and during the application. At 5 ␮M concentration, none of these parameters were

affected. This finding indicated that a local anesthetic effect is unlikely to explain the effects of haloperidol at this dose, in agreement with the conclusions reached by Pucak and Grace (1996). An increase in the dose to 50 ␮M, however, gave a clear broadening of the action potential and a decrease in its peak amplitude (n⫽1; not illustrated).

DISCUSSION The present findings suggest that the PSIP following action potential trains in dopaminergic neurons of the substantia nigra is due to generation of slow AHP. Activation of this response depends mainly on a sustained subthreshold membrane depolarization during the train and less so on the action potentials. The support for this conclusion is first, that intermittent depolarizations were ineffective in producing the response (Fig. 1), and second, that the major part of the AHPs was insensitive to action potential blockade by TTX (Fig. 2). The latter observation excludes furthermore that AHPs is simply a function of the action potential dimension, shown to depend on the stimulation parameters used (Nedergaard, 1999). The importance of a maintained depolarization is further supported by the very close correlation between the AHPs and depolarization length, and by the observed voltage-range for activation of the AHPs, which was negative to action potential threshold (Fig. 3). Part of the TTX-effect on the AHPs could be ascribed to a reduction in depolarization induced by the current pulse. This effect is likely to reflect a block of the TTX-sensitive persistent sodium conductance, previously shown to exist at subthreshold membrane potentials in these cells (Grace and Onn, 1989; Kang and Kitai, 1993a).

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Interval position Fig. 8. Effects of haloperidol on PSIP and spike-frequency adaptation. (A) Responses to a ⫹0.3 nA current pulse were obtained from a control period (3 mM Cs⫹ present) and after 15 min perfusion with 5 ␮M haloperidol. A weak hyperpolarizing holding current was injected in the presence of haloperidol to compensate for an increased background firing rate. Note that the PSIP following the current pulse is reduced after addition of haloperidol. (B) Summary of the effect of haloperidol on the PSIP, normalized to the background inter-spike interval. Data pairs represent values obtained from individual cells, before (CON) and during application of 5 ␮M haloperidol (HALO). (C) From the same experiment as in (A) is shown the mean instantaneous firing frequency as function of the position of the inter-spike interval during the current pulse (0.3 nA, 4s). F, control, E, haloperidol. (D) The degree of frequency adaptation represented as the first divided by the last inter-spike interval. Averaged values from six experiments are shown from control periods (CON) and in the presence 5 ␮M haloperidol (HALO).

The opening of persistent sodium channels may therefore, indirectly, contribute to activation of the AHPs. The data here do not support, however, that Na⫹ entry via these channels is acting as a direct trigger for the AHP, as described in other cell types (Schwindt et al., 1989; Kim and McCormick, 1998; Sanchez-Vives et al., 2000). Characteristics of the AHP Two distinct components of the AHP could be separated in terms of kinetics, voltage-dependency, and pharmacology. The AHPf could be activated by short-lasting (⬎0.1 s) depolarizations, had a threshold close to that of the action potential, and it was sensitive to i) blockade of low- and high-voltage-activated Ca2⫹ channels by the inorganic Ca2⫹ antagonists, Cd2⫹ and Ni2⫹, ii) reduction of the extracellular Ca2⫹ concentration, iii) chelation of intracellular Ca2⫹ with EGTA, and iv) apamin. The data suggest that Ca2⫹-dependent currents, in particular the apaminsensitive SK-type K⫹-current, are the main contributors to the AHPf, which agrees with earlier findings in these cells (Shepard and Bunney, 1991; Nedergaard et al., 1993; Ping

and Shepard, 1999; Wolfart and Roeper, 2002). The degree of block induced by apamin or low Ca2⫹ media was inversely related to the length of pre-depolarization, which indicated that the Ca2⫹-dependent AHP declined with time. This observation could be consistent with the recent demonstration that SK-type K⫹ channels are functionally linked to the T-type Ca2⫹ channel, and that use-dependent block of IAHP occurs in parallel with cumulative T channel inactivation during repetitive depolarizations (Wolfart and Roeper, 2002). However, this explanation only seems to cover the present data in part, since an appreciable apamin-sensitive component was seen to exist even after ⬎1 s depolarizations (at which time the T-channel would be inactivated; Kang and Kitai, 1993b). Under such conditions, other sources of Ca2⫹ for the SK-type channel may prevail, as for example the N-type channel (Wolfart and Roeper, 2002), that underlies a major component of highvoltage activated Ca2⫹ current isolated by prolonged depolarization, and which undergoes slow inactivation (Kang and Kitai, 1993a). The data here also showed that a Ca2⫹independent part of the AHPf developed in parallel with the

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decline of the Ca2⫹-dependent part. Although the composition of this potential remains to be resolved, it seems likely that currents underlying the AHPs contribute, since it was partly sensitive to antagonists of the AHPs. In addition, a large, TEA-sensitive component appears to be involved. The AHPs was distinguished by a decay time lasting several seconds, and a slow activation, which involved an apparent delay of 0.1– 0.2 s followed by gradual increase over a period of at least 10 s. The conductance underlying this component is presumably a K⫹ conductance (reversal at ⫺97 mV), which is activated in a voltage-dependent manner from membrane potentials beginning at least 10 –15 mV below firing threshold. No evidence could be found that intracellular Ca2⫹ elevation is a necessary trigger for the AHPs. Thus, attempts to reduce Ca2⫹ activity, either by reduced influx from extracellular space, or by chelation of intracellular Ca2⫹, or by pharmacological inhibition of release from intracellular stores, were all without effect (except when low extracellular Ca2⫹ was used in combination with 10 mM Mg2⫹; see below). Which channels are involved in the AHPs? The observations outlined above seem in favor of the proposal that a voltage-dependent K⫹ current is responsible for the AHPs. The sensitivity of the AHPs to 4-AP gives further support for this view. However, a more specific identification of the channel(s) cannot be established on the basis of this observation. The transient outward (A-type) potassium current can, however, be excluded (even though it is sensitive to millimolar concentrations of 4-AP and insensitive to TEA) because its inactivation property clearly does not match the persisting nature of the AHPs (rebound activation of transient outward currents is also unlikely to contribute significantly to the AHPs, as judged from the lack of unlinearity in the voltage-response relationship; Fig. 5). Indeed, the current underlying this potential would appear to have much slower activation and deactivation kinetics than any known member of the Kv class (Shaker-related) K⫹ channels (see Coetzee et al., 1999). The lack of effect of 50 ␮M carbachol (or 30 mM TEA), which is sufficient to block the M-current in other central neurons (Halliwell and Adams, 1982; Storm, 1989), suggests that this current makes no major contribution to the AHPs. The AHPs was also sensitive to haloperidol. This effect could not be attributed to binding of haloperidol to either dopamine or ␴ receptors. The haloperidol effect was, however, reproduced by the second generation H1 receptor antagonist, terfenadine, which, along with haloperidol, is a blocker of a sub-class of channels (ERG), belonging to the ether-a-go-go family of K⫹ channels (Suessbrich et al., 1996, 1997; Secondo et al., 2000). The three known members of the ERG K⫹ channel (ERG1-3) are all represented in the rat brain (Shi et al., 1997; Saganich et al., 2001), and ERG1 channel transcripts have been demonstrated in mesencephalic nuclei containing dopaminergic neurons (substantia nigra compacta and ventral tegmental area; Saganich et al., 2001). The kinetic properties of ERG channels are complex and have mostly been studied in non-neuronal preparations. In brief, depolarization leads to

slow activation of the channel followed by fast, voltagedependent, inactivation. During membrane repolarization the channel quickly returns to the activated state, from which deactivation occurs at a slow rate. Activation begins at rather negative membrane potentials (⫺60 to ⫺50 mV for ERG1; Saganich et al., 2001) and the rate of activation depends on the voltage. With moderate depolarization around V ⁄ (⫺20 mV), the time of depolarization needed for full steady-state activation of ERG channels has been estimated to 15 s or more, and accumulation of the current is evident during train activation (10 Hz), even with pulses as short as 5 ms (Scho¨nherr et al., 1999). The deactivation time constant is in the range of seconds between ⫺60 and ⫺80 mV (Scho¨nherr et al., 1999; Saganich et al., 2001). The exact values obtained for the individual processes show some variability depending on the preparation used. Nevertheless, by comparison, these reported kinetics of the ERG channel seem to correlate rather well with characteristics of the AHPs observed here. Haloperidol and terfenadine gave a similar, but only partial, inhibition of the AHPs, even though the concentrations used were sufficient to block ERG channels in other systems (Suessbrich et al., 1996). A low efficacy may be explained by the voltage- and use-dependency of drug binding, shown for both haloperidol and terfenadine (Suessbrich et al., 1996, 1997), or by limited diffusion into the tissue. On the other hand, a newly discovered ERG current in cerebellar Purkinje cells from slices could be reduced by 78% in the presence of 2 ␮M haloperidol (Sacco et al., 2003). Alternatively, the haloperidol-resistant part of the AHPs could be mediated by a different type of conductance. If so, it seems possible that 4-AP acted on a different conductance. This issue was not further pursued here, however, it has recently been reported that 4-AP indeed is a blocker of the human ERG (HERG) channel in cardiac cells (IC50⫽4.4 mM; Ridley et al., 2003). It was also observed that the AHPs amplitude decreased during exposure to low extracellular Ca2⫹ and high (10 mM) Mg2⫹, and this effect was attributable to the high Mg2⫹ concentration. This finding could be analogous to the one described by Faravelli et al. (1996), that a rise in external Mg2⫹ reduces the peak current through HERG channels (see also Ho et al., 1998), and thus give additional support for the idea that an ERG channel is involved. On the other hand, an isolated lowering of the external Ca2⫹ concentration is shown to give a marked negative shift in the voltage-dependence of activation of IERG (Johnson et al., 2001), and should oppose the effect of increased Mg2⫹. It is unclear if these effects could cancel each other. The present study showed no net effect of equimolar substitution of Ca2⫹ with Mg2⫹ on the AHPs. The IKr from cardiac myocytes, which is encoded by ERG (Sanguinetti et al., 1995), is subject to complex modulation by divalent cations, including Cd2⫹ and Ni2⫹ (Follmer et al., 1992; Paquette et al., 1998). The effect consists of an increase in Imax, a positive shift in the activation curve, and accelerated rate of decay. Hence, depending on the voltage, Cd2⫹ and Ni2⫹ should be expected to enhance or inhibit the AHPs, provided it was mediated via ERG channels. However, the present study showed no effect of these cations 12

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on the AHPs. On the other hand, because the depolarizations used here were unclamped and in a narrow range, it is difficult to establish if these apparent discrepancies are real. Taken as a whole, the properties of the AHPs observed so far correspond, in many respects, to those described for the ERG K⫹ channel. However, there are some apparent inconsistencies, as outlined above, which will need a more extensive biophysical and pharmacological analysis to resolve. Functional importance of the AHPs The properties of the AHPs indicate that the underlying current will be active under steady-state firing conditions. The pacemaker potential traverses the region of activation of this current, which, in turn, would oppose the depolarization and increase the interval to the next action potential. Because of its slow decay, the AHPs current would only partly deactivate during the hyperpolarized state between action potentials, resulting in temporal summation of the current at normal resting firing rates. Such influence is supported by the observed acceleratory effect of haloperidol on the firing rate. It should be noted that similar effect of haloperidol (10 ␮M) has been reported previously (Pucak and Grace, 1996), and ascribed to dopamine receptor blockade. The present study does not eliminate this as a possible mechanism for this particular effect. However, with the lower dose of haloperidol used here, an input resistance increase was not observed in all cells, which seems compatible with a primary effect on a voltagedependent- rather than a tonically active current. It appears that a prominent role of the AHPs is to give a prolonged period of inhibition triggered by a preceding sustained excitatory stimulus. Interestingly, a correlate of such slow excitation-inhibition sequence is exhibited by these neurons in vivo during periods of burst firing. A burst episode typically consists of a train of two to 12 action potentials generated on a slow depolarizing wave and is followed by a prolonged silent period (Grace and Bunney, 1984). Since a slow wave is an ideal trigger for the AHPs, the latter is likely to be activated by the burst and could contribute to the inter-burst silent period. Although afferent synaptic inputs are essential for the expression of the bursting state, intrinsic membrane currents could actively influence this type of firing (Overton and Clark, 1997; Kitai et al., 1999). Evidence from intracellular recordings in vivo has prompted the suggestion that termination of a burst is due to membrane repolarization mediated by a K⫹ current, likely to be triggered by accumulation of intracellular Ca2⫹ (Grace and Bunney, 1984). While such Ca2⫹-dependent current would clearly differ from the AHPs, no available evidence rules out the possibility that an additional, purely voltage-dependent K⫹ current contributes to the inhibitory state between bursts. This possibility will, however, need a direct verification, since data from the slice preparation may not be immediately translatable into the in vivo situation. For example, spikefrequency adaptation and spike-dependent summation of the Ca2⫹-dependent AHP are reported to be more pronounced in dopamine neurons recorded in vivo (Grace, 1991). Although the reason for this discrepancy remains

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unknown, it raises the possibility that the relative contribution from Ca2⫹-dependent and Ca2⫹-independent processes to the AHP could also be different in vivo compared with the brain slice. In any event, the present data indicate that the AHPs may not have a significant role in active repolarization, in particular since no effect on spike-frequency adaptation was detected with haloperidol. This result was unexpected, since the AHPs, given its slow activation, would seem suitable as a mechanism behind the slow phase of the accommodation. In addition, evidence has recently been found that the ERG channel enhances frequency adaptation in cerebellar Purkinje neurons (Sacco et al., 2003; see also Chiesa et al., 1997; Scho¨nherr et al., 1999). The present results with haloperidol could indicate that, although the AHPs is strongly activated by and clearly expressed after a depolarization, other processes are responsible for the slow adaptive responses during the depolarization. The possibility remains, of course, that frequency adaptation is highly unlinearly related to the magnitude of the AHPs, so that the partial blockade obtained with haloperidol is insufficient to affect adaptation. In conclusion, the AHPs described here shares some of its features with slow hyperpolarizations in other central neurons. The very slow activation and decay are also characteristic for the slow AHP (sIAHP) represented in a number of cell types (Sah, 1996). The AHPs is however, distinguished by its independence from intracellular Ca2⫹ and its low activation threshold, which provides a mechanism for slow, spike-independent regulation of excitability. In this respect, the function of the AHPs seems very similar to the one served by the Na⫹-dependent K⫹ channel in some neurons (Schwindt et al., 1989). However, the present study suggests that in substantia nigra neurons, a voltage-dependent K⫹ channel type is involved, which has many characteristics in common with the ERG K⫹ channel. Future studies should be undertaken to give a full account of the elements underlying slow adaptive responses in these neurons, and to investigate the susceptibility of individual components to regulation by neurotransmitters.

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(Accepted 26 February 2004)