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1Department of Physiology, Feinberg School of Medicine, Northwestern University, Chicago Illinois; and 2Department of Biology, University of Texas at San Antonio, San Antonio, Texas
Submitted 6 September 2007; accepted in final form 13 December 2007
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ABSTRACT |
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subunits because it was attenuated by brief depolarization. D2 and/or D3 dopamine receptors may mediate modulation because a D4-selective agonist was ineffective and mRNA encoding D2 and D3 but not D4 dopamine receptors was detectable. Brain slice recordings confirmed that SKCa channel-mediated action potential afterhyperpolarization was attenuated by D2-like dopamine receptor activation. Together, these data suggest that D2-like dopamine receptors potently modulate the negative feedback control of firing that is mediated by the functional coupling of Cav2.2 and SKCa channels in STN neurons. |
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INTRODUCTION |
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; DeLong 1990; Hornykiewicz 2006; Schultz 2002). The glutamatergic STN drives neuronal activity throughout the basal ganglia including the substantia nigra (Chergui et al. 1994; Kita and Kitai 1987; Kitai and Kita 1987), which in turn directly and positively modulates the excitability of STN neurons through the release of dopamine (Baufreton et al. 2003; Cragg et al. 2004; Mintz et al. 1986; Tofighy et al. 2003; Zhu et al. 2002a). Positive feedback between glutamatergic STN and substantia nigra dopamine neurons may therefore contribute to the activity pattern of STN and dopamine neurons under normal conditions (Schultz 2002; Wichmann et al. 1994) and to excitotoxic degeneration of dopamine neurons in PD (Kress and Reynolds 2005). In PD, degeneration of substantia nigra dopamine neurons, some of which innervate the STN (Cossette et al. 1999; Cragg et al. 2004; François et al. 2000; Hassani et al. 1997), may lead to hypoactivation of STN dopamine receptors, which contributes to the emergence of pathological, correlated, low-frequency, rhythmic, burst activity in the STN and its efferents (Bergman et al. 1994; Filion 1979; Heimer et al. 2002; Levy et al. 2000, 2002). Pathological activity in the STN is associated with the expression of the motor symptoms of PD (Bergman et al. 1994; Levy et al. 2000, 2002). Indeed, "correction" of pathological STN activity by dopamine receptor agonists and/or dopamine replacement therapy and/or direct high-frequency electrical stimulation or ablation of the STN leads to a dramatic amelioration in the symptoms of PD (Alvarez et al. 2005; Benabid 2003; Bergman et al. 1990; Brown et al. 2001; Filion 1979; Hamani et al. 2006; Levy et al. 2001). In addition, hyperactivation of STN dopamine receptors could be involved in the emergence of dyskinesia that invariably accompanies the chronic administration of levodopa or dopamine receptor agonists during the treatment of PD (Fogelson et al. 2005; Lozano et al. 2000). Therefore to understand how dopamine and dopamine depletion influence the activity of the STN it is critical to elucidate precisely the functional impact, signaling elements, and substrates of dopaminergic modulation in the STN.
Although STN neurons express D5 dopamine receptors that potentiate class 1 voltage-dependent Ca2+ (Cav1) channels (Baufreton et al. 2003), and STN afferents express presynaptic D2-like receptors that modulate neurotransmission (Shen and Johnson 2000), the focus of this study is on the action(s) of postsynaptic D2-like dopamine receptors. Activation of postsynaptic D2-like dopamine receptors increases the spontaneous/autonomous activity of STN neurons, apparently through a reduction in the conductance of noninactivating, voltage-independent K+ channels (Zhu et al. 2002a). Our first objective was therefore to further characterize the functional impact of postsynaptic D2-like dopamine receptor modulation on STN neurons. Perforated and whole cell patch-clamp recording of STN neurons in brain slices revealed that D2-like dopamine receptor activation had additional effects that have not been previously described. Thus the precision of autonomous activity was impaired and the characteristic sigmoidal sensitivity of STN neuronal activity in response to depolarizing current injection (Bevan and Wilson 1999; Hallworth et al. 2003; Wilson et al. 2004) was replaced by a relatively linear relationship in which the sensitivity of firing to current injection was enhanced. These effects suggest that voltage/activity-dependent ion channels are additional targets of D2-like dopamine receptor modulation. To further characterize the identity and biophysical properties of modulated channels, recordings were made using acutely isolated STN neurons, in which voltage and recording conditions could be controlled more effectively than in brain slices. Molecular profiling and dopamine receptor-selective drugs were then used to gain insight into the receptor subtypes underlying modulation. Finally, the functional target of D2-like dopamine receptor modulation was verified through brain slice recording.
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METHODS |
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Slice preparation
Electrophysiological recordings were performed using brain slices prepared from 68, 16- to 30-day-old Sprague–Dawley rats (Charles River Laboratories, Wilmington, MA). Animals were deeply anesthetized with a mixture of ketamine [90 mg/kg, administered intraperitoneally (ip)] and xylazine (10 mg/kg, ip) and then perfused transcardially with 20–30 ml of ice-cold modified artificial cerebrospinal fluid (ACSF), which had been bubbled with 95% O2-5% CO2 and contained the following (in mM): 230 sucrose, 26 NaHCO3, 2.5 KCl, 1.25 Na2HPO4, 0.5 CaCl2, 10 MgSO4, and 10 glucose. The brain was then removed, blocked in the sagittal plane, glued to the stage of a vibratome (Vibratome 3000; Vibratome, St. Louis, MO), and submerged in ice-cold modified ACSF. Slices, containing the STN, were cut at a thickness of either 300 µm (slice experiments) or 350 µm (acute isolation experiments) and transferred to a holding chamber at room temperature in "traditional" ACSF that was equilibrated with 95% O2-5% CO2 and contained the following (in mM): 126 NaCl, 26 NaHCO3, 2.5 KCl, 1.25 Na2HPO4, 2 CaCl2, 2 MgSO4, and 10 glucose.
Whole cell and perforated-patch voltage- and current-clamp recordings in brain slices
Slices were transferred to a recording chamber and perfused at 3–5 ml per min with media at 35–37°C that more closely mimicked rodent brain interstitial fluid than "traditional" ACSF (Sanchez-Vives and McCormick 2000). "Synthetic interstitial fluid" was equilibrated with 95% O2-5% CO2 and contained (in mM) 126 NaCl, 26 NaHCO3, 3 KCl, 1.25 Na2HPO4, 1.6 CaCl2, 1.5 MgSO4, and 10 glucose. Recordings were obtained using glass patch pipettes pulled from standard-wall borosilicate glass (Warner Instruments, Hamden, CT) on a P-97 Flaming-Brown micropipette puller (Sutter Instrument, Novato, CA) filled with a solution containing (in mM): 135 K-MeSO4, 3.8 NaCl, 1 MgCl2·6H2O, 10 HEPES, 0.1 Na4EGTA, 0.4 Na3GTP, and 2 Mg1.5ATP, pH adjusted to 7.3 with KOH (290 mOsm). The resistance of filled pipettes ranged between 3 and 6 M
. In the perforated-patch configuration, gramicidin was used as the pore-forming agent (Abe et al. 1994; Kyrozis and Reichling 1995; Myers and Haydon 1972) and was added to the pipette solution at an approximate concentration of 20 µg/ml. A x40 water-immersion objective (Axioskop; Zeiss, Oberkochen, Germany) was used in conjunction with infragradient contrast video microscopy (Infra-patch Workstation; Luigs and Neumann, Ratingen, Germany) to examine the slices. Voltage- and current-clamp recordings were made using an EPC 9/2.C amplifier (Heka, Lambrecht, Germany), which was operated using Pulse 8.5 software (Heka). Signals were low-pass filtered at a frequency (16.67 kHz) that was one third the frequency of digitization (50 kHz). Current-clamp recordings used both the whole cell and perforated-patch configurations, whereas voltage-clamp recordings were made solely using the whole cell configuration. Synaptic transmission at 
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), N-methyl-D-aspartate (NMDA), and both type A and type B -aminobutyric acid (GABAA and GABAB, respectively) receptors was blocked by the continuous application of 20 µM 6,7-dinitroquinoxaline-2,3-dione (DNQX), 50 µM (+)-2-amino-5-phosphonopentanoic acid (APV), 20 µM SR95531 (GABAzine), and 1–2 µM 3-N-[1-(S)-(3,4-dichlorophenyl)ethyl]amino-2-(S)-hydroxypropyl-P-benzyl-phosphinic acid (CGP55845), respectively. In some cases, voltage-dependent Na+ (Nav) channels were blocked with 1 µM tetrodotoxin (TTX) to improve voltage control. Whole cell and perforated patch-clamp recordings were corrected for liquid junction potentials of 9 and 4 mV, respectively (Baufreton et al. 2005).
Acute isolation and whole cell voltage-clamp recordings
Single slices were removed from the holding chamber and placed in "background" solution containing the following (in mM): 140 NaCl, 2 KCl, 2 MgCl2, 1 CaCl2, 23 glucose, and 15 HEPES, pH adjusted to 7.2 with NaOH (300–310 mOsm). The STN was then carefully dissected under microscopic guidance. Care was taken to make excisions from entirely within the clearly visible boundaries of the nucleus to minimize contamination by surrounding structures. The dissected STN was transferred to a chamber containing oxygenated dissociation solution (in mM: 82 Na2SO4, 30 K2SO4, 5 MgCl2, 10 HEPES, 10 glucose) and 3 mg/ml protease XXIII (Sigma–Aldrich, St. Louis, MO), pH 7.4 with NaOH, for 30 min at 30–32°C. The tissue was then washed for 15 min in warmed, oxygenated dissociation solution containing 1 mg/ml bovine serum albumin (Sigma–Aldrich) and 1 mg/ml trypsin inhibitor (Sigma–Aldrich). The tissue was then washed several times in Tyrode's solution containing (in mM): 150 NaCl, 4 KCl, 2 CaCl2, 2 MgCl2, 10 HEPES, and 10 glucose, pH 7.4 with NaOH and mechanically dissociated using a graded series of fire-polished glass pipettes. The resulting suspension of neurons was then allowed to settle in a cell culture dish mounted to the stage of an inverted microscope (Olympus IX70, Olympus America, Center Valley, PA) before perfusion with background solution. Whole cell voltage-clamp recordings were obtained at room temperature using glass patch pipettes. For the majority of experiments, pipettes were filled with methylsulfate-based pipette solution (as described for slice experiments) and recordings were made in the presence of background solution. For experiments in which currents flowing through Cav channels were isolated, the pipette solution consisted of (in mM): 170 N-methyl-D-glucamine (NMG), 40 HEPES, 4 MgCl2, 0.1 Na4EGTA, 0.4 Na3GTP, and 2 Mg1.5ATP, pH 7.2–3 with H2SO4, 265–270 mOsm/l and the external solution consisted of (in mM) 127 NaCl, 10 glucose, 10 HEPES, 1 MgCl2, 5 BaCl2, and 0.5 CsCl, pH adjusted to 7.2 with NaOH (300–310 mOsm). All experiments in acutely isolated neurons were conducted in the presence of 1 µM TTX in the external solution to improve voltage control. Data were recorded using an Axopatch 200B amplifier controlled by Clampex 9.0 (Molecular Devices, Sunnyvale, CA). Data were filtered at 2–10 kHz and digitized at 10–50 kHz, respectively. Series resistances of 7–25 M were electrically compensated by 70–85%, and a junction potential of 9 mV (16 mV in isolated Cav channel current experiments) was accounted for in all of the voltage-clamp waveforms that were applied.
Drugs
APV, CGP55845, DNQX, GABAzine, isradipine, quinpirole hydrochloride, R-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5- tetrahydro-1H-3-benzazepine (SCH23390) hydrochloride, and sulpiride were purchased from Tocris-Cookson (Ellisville, MO). Apamin, iberiotoxin, 
-conotoxin GVIA, [(4-phenylpiperazinyl)-methyl]benzamide (PD168077) maleate salt, 3,4-dihydroxyphenethylamine hydrochloride (dopamine), and N-ethylmaleimide (NEM) were obtained from Sigma–Aldrich. All drugs with the exception of dopamine and NEM were prepared as stock solutions and stored at –20°C. Stock solutions of dopamine and NEM were made fresh on the day of the experiment and kept on ice until use. Final dilutions of -conotoxin GVIA and apamin were made in background/external solution containing 0.01% cytochrome C to minimize nonspecific binding of toxin. Sodium metabisulfite (50 µM) (Sigma–Aldrich) was added as an antioxidant to all dopamine receptor agonists and antagonists and respective control solutions (Sigma–Aldrich). Drugs were applied using a gravity-fed "rapid solution changer" system (RSC-160; Biologic, Claix, France). The capillary through which each drug was delivered was placed about 1 mm from the cell under study.
Data analysis
Data were analyzed using Origin 7.5 (Microcal, Northampton, MA) and Igor Pro 6.0 (WaveMetrics, Lake Oswego, OR). The threshold for action potentials was determined as described previously (Baufreton et al. 2005). Currents recorded in acutely isolated neurons were normalized to cell capacitance. Numerical data are presented as means ± SD. Distributions of data are stated numerically as ranges and/or represented graphically with box plots (when sample sizes were 
6). In some experiments data points from individual neurons are plotted. In all experiments a paired parametric statistical test (Student's paired t-test) was used. P values that were <0.05 after they had been Bonferroni corrected for multiple comparisons (P value was multiplied by the number of comparisons) were considered significant. P values >0.001 are reported to three decimal places.
Single-cell molecular profiling
Single-cell reverse transcriptase–polymerase chain reaction (scRT-PCR) profiling was performed using protocols similar to those described previously (Tkatch et al. 2000). Individual acutely isolated neurons were aspirated into micropipettes containing 1 µl of diethylpyrocarbonate (DEPC)-treated water and 0.8 U/µl SUPERase-In (Ambion, Austin, TX). After aspirating the cell into the tip of the micropipette, the tip was broken off and the contents were expelled into an Eppendorf tube containing Superase-In (0.7 µl, 20 U/µl) (Ambion), DEPC-treated water (1.9 µl), BSA (0.7 µl, 143 ng/µl), dNTPs, (1.0 µl, 10 mM), and Oligo dT (0.7 µl, 0.5 µg/ml).
Single-stranded cDNA was generated by reverse transcription (RT) using the SuperScript III kit (Invitrogen). First, the neuron-containing mixture was heated to 65°C for 5 min to denature the nucleic acids, then cooled on ice for 1 min. To this mixture was added 10 x RT buffer (2 µl), MgCl2 (4 µl, 25 mM), DTT (2 µl, 0.1 M), RNAse Out (1 µl, 40 U/µl), SuperScript III (0.7 µl, 50 U/µl), and DEPC-treated water to bring the final volume to 20 µl. RT reactions were run at 50°C for 50 min. The temperature was then increased to 85°C for 5 min to terminate the reactions. Finally, to eliminate any residual RNA, RNAse H (0.5 µl, 2 U/µl) was added and the reaction mixtures were held at 37°C for 20 min.
The PCR primers for dopamine receptors (D2, D3, and D4) were developed from GenBank sequences using OLIGO software (National Biosciences). The primers for D2 cDNA (GenBank Accession Number M36831) were GCT CAG GAG CTG GAA ATG GAG AT (position 866) and CTT TCT GCG GCT CAT CGT CTTA (position 1108). The predicted product length was 264 bp. The primers for D3 cDNA (GenBank Accession Number X53944) were TCA ATA AGG GCC AGG TTT CTG TC (position 841) and GGG CTC AAG GAG TTC CGA GTC (position 1042). The predicted product length was 242 bp. The primers for D4 cDNA (GenBank Accession Number M84009) were GGC CTT CCT GAT GTG TTG GAC (position 1087) and CCC AGC GTT GAT AAA TGG TTAG (position 1381). The predicted product length was 316 bp. The primers for GAPDH (GenBank Accession Number X02231) were GGC ACA GTC AAG GCT GAG AATG (position 237) and TTC CAC GAT GCC AAA GTT GTC AT (position 559). The predicted product length was 345 bp.
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RESULTS |
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D2-like dopamine receptor modulation reduces the precision of autonomous activity and increases the firing of STN neurons in response to depolarizing input
As reported previously in brain slices, STN neurons exhibited autonomous, rhythmic, repetitive firing in the presence of AMPA, NMDA, GABAA, and GABAB receptor antagonists (Fig. 1 Ai; Baufreton et al. 2003; Beurrier et al. 1999; Bevan and Wilson 1999; Bevan et al. 2002; Do and Bean 2003; Hallworth et al. 2003; Overton and Greenfield 1995; Wigmore and Lacey 2000; Zhu et al. 2002a). Bath application of the broad spectrum D2-like dopamine receptor agonist quinpirole (10 µM) (Tsuruta et al. 1981) decreased the precision of firing (Fig. 1Aii) as assessed from the coefficient of variation (CV) of 100 interspike intervals (ISIs) recorded under control conditions and in the presence of quinpirole, in either the perforated (n = 4) or whole cell (n = 2) configurations. Because the effects of quinpirole were similar for each recording mode, the data were pooled (Student's paired t-test; control CV = 0.058 ± 0.009; quinpirole CV = 0.435 ± 0.354; n = 6; P = 0.037). Because the whole cell configuration can disrupt ion channel function and intracellular Ca2+ dynamics (Neher and Augustine 1992; Velumian and Carlen 1999; Velumian et al. 1997; Zhang et al. 1994, 1995), neurons whose firing in control conditions was disrupted by whole cell dialysis were not analyzed. Application of quinpirole also led to a significant depolarization in the average membrane potential (Fig. 1, A and B) (Student's paired t-test; control membrane potential = –62.8 ± 1.9 mV; membrane potential in quinpirole = –55.4 ± 7.3 mV; n = 6; P = 0.038), an increase in the average frequency of autonomous activity (Fig. 1, A and C) (Student's paired t-test; control = 7.8 ± 2.1 Hz; quinpirole = 16.1 ± 3.2 Hz; n = 6; P < 0.001) and depolarization of action potential threshold (Fig. 1, D–F) (Student's paired t-test; control = –54.0 ± 1.4 mV; quinpirole = –50.0 ± 1.3 mV; n = 6; P = 0.026), presumably due to increased inactivation of Nav channels (Baufreton et al. 2005).
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; Hallworth et al. 2003), and was composed of a low-sensitivity primary range (up to 
40 Hz), a higher-sensitivity secondary range, and a tertiary range at which the frequency of firing saturates (Fig. 2, A–C). Application of quinpirole shifted the frequency–intensity relationship to the left and eliminated the different sensitivities of the primary and secondary ranges (Fig. 2, A–C). Thus quinpirole increased the frequency of activity that was evoked by current pulses that gave rise to primary and secondary range firing in control media (Student's paired t-test; control response to 40 pA = 22.5 ± 6.0 Hz; response to 40 pA in quinpirole = 53.2 ± 13.4 Hz; n = 6; P < 0.001; Student's paired t-test; control response to 200 pA = 136.4 ± 19.6 Hz; response to 200 pA in quinpirole = 175.6 ± 37.7 Hz; n = 6; P = 0.022).
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Whole cell voltage-clamp experiments were carried out in acutely isolated STN neurons to further characterize the ion channels and signaling pathways associated with D2-like dopamine receptor activation. Whole cell currents were studied in the presence of TTX to eliminate inward currents at sub- and suprathreshold voltages that are generated by Nav channels in STN neurons (Beurrier et al. 2000; Bevan and Wilson 1999). In the presence of TTX, net outward currents were evoked by a series of 5 mV steps from a holding voltage of –70 to –20 mV. Figure 3 A illustrates the currents that were elicited by this protocol. Outward current was composed of two components: a transient and a sustained component. The transient outward current was observed at voltages of –40 mV or more and its amplitude increased with depolarization. The transient current peaked at 5.6 ± 0.9 ms (n = 7) following depolarization to –20 mV. Application of 10 µM quinpirole (Fig. 3, B–D) reversibly decreased transient outward current (Student's paired t-test; control current density at –20 mV = 65.1 ± 21.8 pA/pF; current density at –20 mV in quinpirole = 47.8 ± 21.3 pA/pF; n = 7; P < 0.001). In contrast, there was no effect on sustained outward current measured at the end of each voltage step (Student's paired t-test; current density in control at –20 mV = 36.2 ± 22.4 pA/pF; current density at –20 mV in quinpirole = 32.3 ± 22.7 pA/pF; n = 7; P = 0.47). Subtraction of currents evoked in quinpirole from currents evoked in control medium confirmed that the quinpirole-sensitive current was transient. The quinpirole-sensitive current had a time to peak (at –20 mV) of 5.6 ± 1.4 ms and its decay was fit by a monoexponential function (
decay at –20 mV = 67.6 ± 5.2 ms, R2 = 0.95 ± 0.02, n = 7). Quinpirole-sensitive currents also progressively increased in amplitude at voltages of –40 mV or more (Fig. 3, C and D). To confirm that the action of quinpirole was mediated via D2-like dopamine receptors, control solution, 10 µM quinpirole, and a mixture of 10 µM quinpirole and 10 µM sulpiride (a D2-like dopamine receptor antagonist; Jenner and Marsden 1984) were applied sequentially in three neurons. Figure 3E shows an example of outward currents elicited by stepping from –70 to –20 mV under the three conditions. The quinpirole-mediated reduction in transient outward current was reversed by coapplication of 10 µM sulpiride (Fig. 3, E and F; Student's paired t-test; control current density at –20 mV = 72.3 ± 3.1 pA/pF; current density at –20 mV in quinpirole = 61.5 ± 8.3 pA/pF; current density at –20 mV in quinpirole and sulpiride = 74.7 ± 1.0 pA/pF; n = 3; P = 0.007). In contrast, coapplication of sulpiride had no effect on sustained outward current compared with control values or following application of 10 µM quinpirole (data not shown). In each cell tested the effect of sulpiride on transient current was also reversible (Fig. 3F). Together these data demonstrate that D2-like dopamine receptors reduce a transient outward current that is evoked at suprathreshold voltages. No evidence for a D2-like dopamine receptor-mediated reduction of sustained outward current, as described previously by Zhu and colleagues (2002a), was obtained. Because the acute isolation procedure may disrupt the channels underlying this current and/or disrupt the signaling pathways underlying the modulation of these channels, experiments were also carried out in more intact neurons in brain slices. These experiments were carried out in the presence of TTX to improve voltage control and APV, DNQX, GABAzine, and CGP55845 to block synaptic transmission. Neurons were held at –60 mV and subjected to 1 s voltage steps ranging from –120 to –40 mV in control conditions, in the presence of 10 µM quinpirole and after the removal of quinpirole. Due to the difficulty in obtaining accurate measurements of whole cell capacitance in intact neurons, currents rather than current densities were compared. Quinpirole-sensitive current was observed during steps to –50 and –40 mV. The outward current elicited at these voltages had both a transient and a sustained component as observed in acutely isolated neurons. Quinpirole reversibly decreased transient outward current (Supplemental Fig. S1, A–C,1 Student's paired t-test; control current at –40 mV = 266.5 ± 125.6 pA; current at –40 mV in quinpirole = 239.2 ± 145.2 pA; n = 6; P = 0.038) but had no effect on sustained outward current measured at the end of the voltage step (Student's paired t-test; current in control at –40 mV = 205.1 ± 117.1 pA; current at –40 mV in quinpirole = 183.6 ± 107.7 pA; n = 6; P = 0.079). In three neurons, the membrane potential was stepped from –60 mV to voltages ranging from –120 to –20 mV (Supplemental Fig. S1, D–G). At suprathreshold voltages application of 10 µM quinpirole reduced transient outward current (Supplemental Fig. S1, D–G) (Student's paired t-test; control current at –20 mV = 941.9 ± 37.4 pA; current at –20 mV in quinpirole = 758.8 ± 13.6 pA; n = 3; P = 0.007) but had no significant effect on sustained outward current (Supplemental Fig. S1, D–G; Student's paired t-test; control current at –20 mV = 688.7 ± 125.7 pA; current at –20 mV in quinpirole = 636.8 ± 148.0 pA; n = 3; P = 0.066). Subtraction of currents evoked in quinpirole from currents evoked in control medium confirmed that quinpirole-sensitive currents were transient (Supplemental Fig. S1F). The quinpirole-sensitive current had a time to peak (at –20 mV) of 3.5 ± 0.1 ms and its decay was fit by a monoexponential function (decay at –20 mV = 53.4 ± 1.1 ms, R2 = 0.92 ± 0.06, n = 3). Quinpirole-sensitive currents also progressively increased in amplitude at voltages of –40 mV or more (Supplemental Fig. S1, F and G). Together these data suggest that (under the recording conditions used here) D2-like dopamine receptor activation reduces transient outward current at suprathreshold voltages in both acutely isolated neurons and neurons recorded in brain slices.
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Transient outward currents mediated by voltage-dependent K+ channels with rapid kinetics of activation and inactivation have not been reported in STN neurons. However, at suprathreshold voltages Cav channels conduct Ca2+ that activates Ca2+-dependent K+ (KCa) channels (Hallworth et al. 2003). To test for the Ca2+ dependence of transient outward current, the effect of reducing Ca2+ flux with 300 µM CdCl2 (a nonselective blocker of Cav1 and 2 channels; Brown and Griffith 1983) (n = 3) or external solution in which Ca2+ was replaced by Mg2+ (n = 3) was examined in acutely isolated neurons. Both treatments eliminated the transient component of outward currents evoked at suprathreshold voltages (Fig. 4, A–C) (Student's paired t-test; control current density at –20 mV = 79.7 ± 18.7 pA/pF; current density at –20 mV in the absence of Ca2+ flux = 37.0 ± 13.8 pA/pF; n = 6; P < 0.001) and sustained outward current was again not significantly altered (Student's paired t-test control current density at –20 mV = 33.6 ± 7.2 pA/pF; current density at –20 mV in the absence of Ca2+ flux = 30.0 ± 15.3 pA/pF; n = 6; P = 0.75). Together, these data suggest that transient outward current is mediated by Ca2+-dependent ion channels.
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Blockade of SKCa but not BKCa channels occludes the action of D2-like dopamine receptor activation
To determine whether KCa channels underlie the transient outward current that is reduced by D2-like dopamine receptor activation, selective blockers of these channels were applied to acutely isolated neurons. Application of 10 nM apamin, a selective blocker of small conductance (S) KCa channels (Blatz and Magleby 1986), significantly reduced the amplitude of transient outward current (Fig. 5, A–C) (Student's paired t-test; control current density at –20 mV = 110.4 ± 24.6 pA/pF; current density at –20 mV in apamin = 80.6 ± 27.1 pA/pF; n = 7; P < 0.001), but had no effect on the sustained component (data not illustrated) (Student's paired t-test; control current density at –20 mV = 41.7 ± 18.7 pA/pF; current density at –20 mV in apamin = 36.9 ± 21.4 pA/pF; n = 7; P = 0.10). Because the kinetics and current–voltage relationship of the apamin-sensitive transient current (Fig. 5, A–C) were similar to quinpirole-sensitive current (compare Fig. 3, C and D with Fig. 5, A–C) (kinetics of apamin-sensitive current: time to peak at –20 mV = 5.9 ± 1.2 ms; monoexponential decay at –20 mV = 62.1 ± 4.3 ms, R2 = 0.97 ± 0.07, n = 7), D2-like dopamine receptor activation may lead to a reduction in the current carried by SKCa channels. In support of this hypothesis, preapplication of apamin occluded the action of quinpirole (Fig. 5, D–F) (Student's paired t-test; current density at –20 mV in apamin = 56.4 ± 17.3 pA/pF; current density at –20 mV in apamin + quinpirole = 55.0 ± 16.2 pA/pF; n = 6; P = 0.53).
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), was applied to STN neurons. Transient outward current evoked at –20 mV under control conditions was also significantly reduced by application of iberiotoxin (Fig. 5, G–I) (Student's paired t-test; control current density at –20 mV = 135.0 ± 14.0 pA/pF; current density at –20 mV in iberiotoxin = 78.5 ± 22.6 pA/pF; n = 3; P = 0.008), whereas there was no effect on sustained outward current (data not illustrated) (Student's paired t-test; control current density at –20 mV = 58.4 ± 3.2 pA/pF; current density at –20 mV in iberiotoxin = 52.6 ± 13.7 pA/pF; n = 3; P = 0.44). In contrast to the effect of apamin, preapplication of iberiotoxin did not occlude the effects of subsequently applied quinpirole. Thus a significant quinpirole-mediated reduction in the amplitude of the transient current was observed in the presence of iberiotoxin in each of four cells tested (Fig. 5, J–L) (Student's paired t-test; current density in iberiotoxin = 57.9 ± 34.9 pA/pF; current density in iberiotoxin + quinpirole: 44.4 ± 31.4 pA/pF; P = 0.008; n = 4). These results suggest that the transient outward current evoked by suprathreshold voltage steps is due to the activation of SKCa and BKCa channels, but D2-like dopamine receptor activation specifically leads to a reduction in the current carried by SKCa channels. D2-like dopamine receptor activation reduces Cav channel current
Cav channels that are functionally coupled to SKCa channels (e.g., Bowden et al. 2001; Davies et al. 1996; Hallworth et al. 2003; Pineda et al. 1998; Wolfart and Roeper 2002) are targets of D2-like dopamine receptor modulation (e.g., Momiyama and Koga 2001; Svensson et al. 2003; Yan et al. 1997). To test for the modulation of Cav channels, currents flowing through Cav channels in acutely isolated neurons were isolated by eliminating currents flowing through Nav and Kv channels, and increased in magnitude by using Ba2+ rather than Ca2+ as a charge carrier. Under these conditions step depolarization from a holding potential of –70 mV evoked net inward currents (Fig. 6 A). Application of 300 µM CdCl2 abolished these inward currents (data not shown), confirming that the currents were due to Ba2+ flux through Cav channels. Cav channel currents evoked by depolarizing voltage steps from –70 to –20 mV were significantly reduced following application of 10 µM of quinpirole (Fig. 6, A–D) (Student's paired t-test; control Cav channel current density at –20 mV = –58.6 ± 22.4 pA/pF; Cav channel current density at –20 mV in quinpirole = –32.3 ± 12.3 pA/pF; n = 6; P = 0.002). Cav channel current density in quinpirole was 54.9 ± 5.4% of the control current density. Following the return to control conditions, Cav channel current density at –20 mV was 86.8 ± 3.5% of control current density (Fig. 6, C and D, n = 6). The failure of Cav channel currents to return to their initial values on return to control media may be attributed to the rundown of Cav channel currents and/or incomplete reversal of D2-like dopamine receptor-mediated modulation.
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) (Fig. 6, E–H). The action potential waveform also elicited Cav channel currents that were significantly and reversibly reduced in amplitude following the application of quinpirole (Fig. 6, E–H) (Student's paired t-test; control Cav channel current density = –83.7 ± 30.7 pA/pF; Cav channel current density in quinpirole: –44.0 ± 17.9 pA/pF; Cav channel current density on return to control media –77.0 ± 30.1 pA/pF; n = 6; P = 0.001). The Cav channel current density in the presence of quinpirole was 52.3 ± 5.3% of control current density. The reduction in waveform-evoked current was therefore similar to the reduction in step-evoked Cav channel current. Spike-triggered averaging revealed that Cav channels were activated by action potentials. Currents flowing through Cav channels increased on action potential repolarization and but then rapidly declined, presumably due to deactivation, such that they contributed minimal inward current during the majority of the ISI (Fig. 6H). The effect of quinpirole was consistent over the injected waveform of 37 action potentials. Indeed, the quinpirole-sensitive Cav channel current densities associated with the 1st, 15th, and 37th action potentials were not significantly different (Student's paired t-test; 1st action potential = –44.1 ± 18.2 pA/pF; 15th action potential: –42.0 ± 16.8 pA/pF; 37th action potential = –44.6 ± 17.0 pA/pF; n = 6; P = 0.962).
To determine whether modulation is consistent with activation of Gi/o proteins, NEM was applied, which is a broad-spectrum sulfydryl-alkylating reagent that alkylates cysteine residues on Gi/o proteins and prevents their coupling to receptors (Shapiro et al. 1994; Supplemental Fig. S2). As described earlier, depolarizing voltage steps evoked Cav channel currents that were reduced by 10 µM quinpirole (Supplemental Fig. S2, A and B) (Student's paired t-test; control Cav channel current density at –20 mV = –47.5 ± 10.3 pA/pF; Cav channel current density at –20 mV in quinpirole = –27.2 ± 6.3 pA/pF; n = 3; P = 0.001). Cav channel current density in quinpirole was 57.2 ± 1.3% of control. Cav channel currents returned to control values when returned to control solution containing 50 µM NEM (Supplemental Fig. S2, C, F, and G) (Student's paired t-test; control Cav channel current density at –20 mV = –47.5 ± 10.3 pA/pF; Cav channel current density at –20 mV in NEM = –47.3 ± 5.4 pA/pF; n = 3; P = 0.9425). The Cav channel current density in NEM was 98.9 ± 5.4% of control. After incubation in 50 µM NEM for 2 min, quinpirole no longer reduced Cav channel current (Supplemental Fig. S2, D–G) (Student's paired t-test; Cav channel current density at –20 mV in NEM = –44.4 ± 7.1 pA/pF; Cav channel current density at –20 mV in NEM + quinpirole = –45.4 ± 6.9 pA/pF; n = 3; P = 0.472). The Cav channel current density in NEM + quinpirole was 102.7 ± 1.2% of the current density in NEM. The effect of NEM is consistent with the activation of a Gi/Go-linked receptor.
D2-like dopamine receptor activation reduces current flow through Cav2.2 but not Cav1 channels
Although Cav2 channels and Cav1 channels are targets of D2-like receptors (Hernandez-Lopez et al. 2000; Momiyama and Koga 2001; Svensson et al. 2003; Yan et al. 1997), SKCa channels are more strongly activated by Ca2+ entry via Cav2.2 channels than Cav1 channels in STN neurons (Hallworth et al. 2003). We therefore hypothesized that Cav2.2 channels, which underlie the majority of high-voltage–activated Ca2+ current in STN neurons (Song et al. 2000) are the primary targets of D2-like dopamine receptor modulation. This hypothesis was tested by recording the effects of application of selective blockers of Cav1 and Cav2.2 channels on D2-like dopamine receptor-mediated modulation of Cav channel current in acutely isolated neurons.
Bath application of 5 µM isradipine, a selective Cav1 channel antagonist (Müller-Schweinitzer and Neumann 1983), reduced whole cell Ca2+ current evoked by a depolarizing step from –70 to –20 mV (Fig. 7, A and B) (Student's paired t-test; control Cav channel current density at –20 mV = –82.2 ± 2.6 pA/pF; Cav channel current density at –20 mV in isradipine = –70.6 ± 4.6 pA/pF; n = 3; P = 0.017). The Cav channel current density in isradipine was 85.8 ± 3.5% of control. Subsequent application of quinpirole reduced the Cav channel current observed in the presence of isradipine (Fig. 7, C and D) (Student's paired t-test; Cav channel current density at –20 mV in isradipine = –70.6 ± 4.6 pA/pF; Cav channel current density at –20 mV in isradipine + quinpirole = –38.5 ± 7.8 pA/pF; n = 3; P = 0.006). The Cav channel current density in isradipine and quinpirole was therefore 54.3 ± 7.8% of the Cav channel current density in isradipine. These data suggest that Cav1 channels are not a major target of D2-like dopamine receptor modulation in STN neurons.
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-conotoxin GVIA, a selective Cav2.2 channel blocker (Reynolds et al. 1986), significantly reduced whole cell Ca2+ current evoked by a depolarizing voltage step from –70 to –20 mV (Fig. 7, E and F) (Student's paired t-test; control Cav channel current density at –20 mV = –42.7 ± 14.0 pA/pF; Cav channel current density at –20 mV in -conotoxin GVIA = –24.2 ± 10.4 pA/pF; n = 3; P = 0.001). The Cav channel current density in -conotoxin GVIA was 56.5 ± 15.7% of the control Cav channel current density. Preapplication of 1 µM -conotoxin GVIA occluded the effect of quinpirole (Fig. 7, E–H) (Student's paired t-test; Cav channel current density at –20 mV in -conotoxin GVIA = –24.2 ± 10.4 pA/pF; Cav channel current density at –20 mV in -conotoxin GVIA + quinpirole = –23.3 ± 10.1 pA; n = 6; P = 0.1).
Similar results were obtained using a record of autonomous activity as the voltage-clamp waveform. Thus application of -conotoxin GVIA significantly reduced whole cell Ca2+ current (Fig. 7, I–L) (Student's paired t-test; control Cav channel current density = –85.8 ± 30.0 pA/pF; Cav channel current density in -conotoxin GVIA = –50.9 ± 19.2 pA/pF; n = 6; P = 0.001) and occluded the effect of subsequently applied quinpirole (Student's paired t-test; Cav channel current density in -conotoxin GVIA = –50.9 ± 19.2 pA/pF; Cav channel current density in -conotoxin GVIA + quinpirole = –49.0 ± 18.8 pA/pF; n = 6; P = 0.19). The Cav channel current density in -conotoxin GVIA was 58.5 ± 7.2% of the control Cav channel current density.
As for whole cell Cav channel current, Cav2.2 channel current increased during action potentials, peaked on repolarization, and then rapidly declined such that the current contributed by Cav2.2 channels during the majority of the ISI was minimal (Fig. 7L). Taken together, our data suggest that Cav2.2 but not Cav1 channels are potently modulated by D2-like dopamine receptors.
To confirm that dopamine reduces the conductance of Cav2.2 channels via the activation of D2-like dopamine receptors, sequential application of dopamine (10 µM), the D1-like receptor antagonist SCH23390 (1 µM; Barnett et al. 1986), and the D2-like receptor antagonist sulpiride (1 µM) was carried out in six neurons. Because dopamine increases the conductance of Cav1 channels through the activation of D5 receptors (Baufreton et al. 2003), isradipine (5 µM) was included in all solutions to block Cav1 channels. Application of voltage steps from –70 to –20 mV evoked Cav channel currents that were significantly reduced following application of 10 µM dopamine (Supplemental Fig. S3, A, B, and E) (Student's paired t-test; control Cav channel current density at –20 mV = –53.9 ± 11.5 pA/pF; Cav channel current density at –20 mV in dopamine = –36.5 ± 8.3 pA/pF; n = 6; P = 0.002). Cav channel current in dopamine was 62.6 ± 9.8% of control current density. Subsequent coapplication of 1 µM SCH23390 did not significantly alter the amplitude the Cav channel current (Supplemental Fig. S3, B, C, and E) (Student's paired t-test; Cav channel current density at –20 mV in dopamine = –36.5 ± 8.3 pA/pF; Cav channel current density at –20 mV in dopamine + SCH23390 = –35.0 ± 7.3 pA/pF; n = 6; P = 0.079). In each of four neurons tested the action of dopamine was reversed by the subsequent coapplication of sulpiride. The Cav channel currents recorded in dopamine, SCH23390, and sulpiride had values that were greater than those in dopamine and SCH23390 (Supplemental Fig. S3, C–E) (Student's paired t-test; Cav channel current density at –20 mV in dopamine + SCH23390 = –35.0 ± 7.3 pA/pF; Cav channel current density at –20 mV in dopamine + SCH23390 + sulpiride = –46.1 ± 11.2 pA/pF, n = 4, P = 0.003). The Cav channel current in dopamine, SCH23390, and sulpiride was similar to the current measured under control conditions (91.5 ± 8.2% of the control current density). Together these data suggest that dopamine potently reduces the conductance of Cav2.2 channels through the activation of D2-like dopamine not D1-like dopamine receptors.
Although the potent modulation of Cav2.2 channels seems likely to underlie the D2-like dopamine receptor-mediated reduction in SKCa channel-mediated outward current, direct modulation of SKCa channels could additionally contribute to modulation. To address this possibility, acutely isolated neurons were recorded with K-methylsulfate–filled electrodes in the presence of physiological external medium containing TTX. Application of 1 µM of -conotoxin GVIA significantly reduced the amplitude of transient outward current (Supplemental Fig. S4, A and B) (Student's paired t-test; control current density at –20 mV = 97.7 ± 29.4 pA/pF; current density at –20 mV in conotoxin = 52.3 ± 16.3 pA/pF; n = 3; P = 0.042), but had no effect on the sustained component (Student's paired t-test; control current density at –20 mV = 42.0 ± 11.2 pA/pF; current density at –20 mV in -conotoxin GVIA = 39.2 ± 11.8 pA/pF; n = 3; P = 0.20). The kinetics of the -conotoxin GVIA–sensitive transient current was similar to that of quinpirole-sensitive current (time to peak at –20 mV = 4.9 ± 0.9 ms; decay at –20 mV = 65.7 ± 9.6 ms; n = 3). Preapplication of -conotoxin GVIA occluded the action of subsequently applied quinpirole (10 µM) on remaining transient outward current (Supplemental Fig. S4, B–D) (Student's paired t-test; current density at –20 mV in -conotoxin GVIA = 52.3 ± 16.3 pA/pF; current density at –20 mV in conotoxin + quinpirole = 50.2 ± 15.5 pA/pF; n = 3; P = 0.14). Together, these data suggest that the D2-like dopamine receptor-mediated reduction in SKCa channel conductance is mediated through a reduction in the conductance of Cav2.2 channels.
D2-like dopamine receptor modulation of Cav2.2 channels is alleviated by brief depolarization
G-protein–coupled receptor (GPCR)–mediated inhibition of Cav2 channels is often mediated by a direct interaction between channels and Gβ subunits from Gi/Go proteins (Herlitze et al. 1996; Holz et al. 1986; Shapiro et al. 1994). This form of modulation has a characteristic biophysical signature: it can rapidly be reversed by strong depolarizing voltage steps (Bean 1989), in contrast to protein kinase/phosphatase-dependent modulation, which is relatively insensitive to depolarization. The effect of strong depolarizing voltage steps on D2-like dopamine receptor-mediated modulation of Cav channels was therefore tested in acutely isolated neurons.
Depolarizing voltage steps from –70 to –20 mV evoked Cav channel currents that were reduced by quinpirole (Fig. 8, A and B) (Student's paired t-test; control Cav channel current density at –20 mV = –51.5 ± 15.5 pA/pF; Cav channel current density at –20 mV in quinpirole = –25.5 ± 6.4 pA/pF; n = 7; P < 0.001). The Cav channel current density in quinpirole was 50.9 ± 8.8% of the control Cav channel current density.
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Another classical biophysical feature of Gβ-mediated modulation of Cav channels is a reduction in the speed of activation due to the stabilization of Cav channel closed states (Bean 1989; Carabelli et al. 1996; Kasai and Aosaki 1989; Lipscombe et al. 1989). Monoexponential curves better fit step-evoked whole cell Cav channel currents than biexponential fits both under control conditions and after application of quinpirole (monoexponential fit control, n = 7, R2 = 0.99 ± 0.01; biexponential fit control, n = 7, R2 = 0.98 ± 0.02; monoexponential fit quinpirole, n = 7, R2 = 0.97 ± 0.01; biexponential fit quinpirole, n = 7, R2 = 0.96 ± 0.01). However, the kinetics of activation of Cav channels under control conditions and in the presence of quinpirole was not slowed (Student's paired t-test; control activation at –20 mV = 7.2 ± 0.3 ms; activation at –20 mV in the presence of quinpirole = 10.3 ± 3.1 ms, n = 7, P = 0.159). An explanation for the absence of kinetic slowing is that modulated channels make no contribution to whole cell Cav channel current. Thus we compared the activation kinetics in control conditions and following the depolarizing prepulse, which leads to a partial alleviation of modulation. In support of our hypothesis, we observed that activation kinetics of whole cell Cav channel current was significantly slowed following the partial relief of modulation (Fig. 8E; Student's paired t-test, control activation at –20 mV = 7.2 ± 0.3 ms, R2 = 0.99 ± 0.01; activation at –20 mV after the depolarizing prepulse and in the presence of quinpirole = 16.8 ± 1.9 ms, R2 = 0.98 ± 0.03; n = 7, P = 0.008).
The reduction in D2-like dopamine receptor-mediated modulation of Cav channel current and the kinetic slowing that followed brief strong depolarization in each of seven neurons suggests that modulation was indeed mediated by the direct interaction of Gβ subunits with Cav2.2 channels.
D2 and/or D3 but not D4 dopamine receptors modulate Cav2.2 channels
To determine which type(s) of D2-like dopamine receptor underlie the modulation of Cav2.2 channels, a selective D4 agonist PD168077, which has a greater affinity for D4 receptors (8.7 nM) than D2 (3,740 nM) or D3 (2,810 nM) receptors (Glase 1997), was applied to acutely isolated neurons. Addition of 1 µM PD168077, however, did not alter the amplitude of Cav channel current evoked by step depolarization from –70 to –20 mV (Fig. 9, A, B, and D) (Student's paired t-test; control Cav channel current density at –20 mV = –29.3 ± 3.3 pA/pF; Cav channel current density at –20 mV in PD168077 = –28.7 ± 5.4 pA/pF; n = 3; P = 0.65). In contrast, subsequent application of 10 µM quinpirole reduced Cav channel current (Fig. 9, C and D) (Student's paired t-test; Cav channel current density at –20 mV in PD168077 = –28.7 ± 5.4 pA/pF; Cav channel current density at –20 mV in PD168077 + quinpirole = –16.4 ± 6.5 pA/pF; n = 3; P = 0.009). The Cav channel current density in quinpirole was 55.9 ± 16.4% of the control Cav channel current density.
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D2-like dopamine receptor modulation reduces the medium-duration component of action potential afterhyperpolarization
SKCa channels have been shown to underlie the "medium-duration" component of action potential afterhyperpolarization in STN neurons (Bevan and Wilson 1999; Hallworth et al. 2003). To investigate the D2-like dopamine receptor-dependent modulation of the functional contribution of these channels, in vitro slice experiments were conducted using the so-called hybrid clamp technique, in which a single unclamped action potential was induced by depolarization from –60 to 20 mV for 5 ms before returning to the holding voltage. The outward current evoked by this protocol was reduced in each of six neurons following application of 10 µM quinpirole (Fig. 10, A–C). The quinpirole-sensitive current peaked 16.6 ± 2.9 ms after termination of the voltage step and had a mean amplitude of 39.4 ± 11.7 pA. Using the time to peak value of the quinpirole-sensitive current as a guide, the amplitude of the outward currents under control conditions and in the presence of quinpirole were measured 15 ms after the voltage step. D2-like dopamine receptor activation reduced the amplitude of this outward current (Fig. 10C) (Student's paired t-test; control current = 72.4 ± 17.6 pA; current in quinpirole = 34.6 ± 6.7 pA; n = 6; P = 0.001). The decay of the quinpirole-sensitive outward current was fit by a monoexponential function with a decay of 58.4 ± 0.4 ms (R2 = 0.97 ± 0.02, n = 6). The decay kinetics of quinpirole-sensitive current evoked by the hybrid clamp protocol were therefore similar to quinpirole, apamin, and -conotoxin–sensitive currents described earlier that were evoked by traditional voltage steps to –20 mV.
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decay of 51.9 ± 1.0 ms (R2 = 0.96 ± 0.07; n = 3), and an amplitude of 27.2 ± 4.2 pA (n = 3), Comparison of the amplitude of outward currents 15 ms after each voltage step revealed that apamin also occluded the action of D2-like dopamine receptor activation (Fig. 10, E and F) (Student's paired t-test; current in apamin = 14.6 ± 5.7 pA; current in quinpirole = 14.5 ± 0.8 pA; n = 3; P = 0.987).
To determine whether D2-like dopamine receptor-mediated modulation of Cav2.2–SKCa coupling could account for the depolarization of STN neurons, the elevation in action potential threshold and alteration in the frequency–intensity relationship that was described initially, the effects of saturating concentrations of apamin and -conotoxin GVIA were analyzed and illustrated as for Figs. 1 and 2. Data from these experiments were published previously (Hallworth et al. 2003) and have been reanalyzed for direct comparison with the effects of D2-like dopamine receptor activation reported here.
Bath application of saturating concentrations of apamin (either 10 or 100 nM) reduced single-spike afterhyperpolarization and the precision of autonomous firing (Supplemental Fig. S5A) as assessed from the CV of 100 ISIs (Student's paired t-test; control CV = 0.065 ± 0.037; apamin CV = 0.278 ± 0.212; n = 9; P = 0.018). SKCa channel block also depolarized the mean membrane potential (Supplemental Fig. S5B) (Student's paired t-test; control = –64.7 ± 2.4 mV; apamin = –57.2 ± 2.3 mV; n = 9; P < 0.001). The average frequency of autonomous activity, however, was not increased (Supplemental Fig. S5, A and C) (Student's paired t-test; control = 12.9 ± 6.0 Hz; apamin = 14.1 ± 10.5 Hz; n = 9; P = 0.663), presumably due to periods of depolarization block following complete blockade of SKCa channels (Hallworth et al. 2003). Indeed, blockade of SKCa channels also led to an elevation of action potential threshold (Supplemental Fig. S5, D–F) (Student's paired t-test; control = –52.5 ± 2.4 mV; apamin = –47.1 ± 3.6 mV; n = 9; P < 0.001). Blockade of SKCa channels also increased the firing rate of STN neurons during periods of applied positive current both in the primary and secondary ranges compared with control media (Supplemental Fig. S5, G–I; Student's paired t-test; control response to 40 pA = 31.6 ± 9.9 Hz; response to 40 pA in apamin = 48.4 ± 16.9 Hz; n = 9; P = 0.007; Student's paired t-test; control response to 200 pA = 143.3 ± 43.7 Hz; response to 200 pA in apamin = 164.3 ± 39.8 Hz; n = 9; P = 0.020), which led to a leftward shift and linearization of the frequency–intensity relationship (Supplemental Fig. S5H).
Blockade of the Cav2.2 channels also reduced the magnitude of single-spike afterhyperpolarization and reduced the rhythmicity of autonomous activity (Supplemental Fig. S6A) (Student's paired t-test; control CV = 0.067 ± 0.029; -conotoxin GVIA CV = 0.130 ± 0.038; n = 9; P < 0.001). Application of -conotoxin GVIA also depolarized STN neurons (Supplemental Fig. S6, A and B) (Student's paired t-test; control = –58.0 ± 3.6 mV; -conotoxin GVIA = –52.0 ± 2.3 mV; n = 9; P < 0.001), increased the average firing frequency (Supplemental Fig. S6, A and C) (Student's paired t-test; control = 12.1 ± 5.2 Hz; -conotoxin GVIA = 19.8 ± 9.5 Hz; n = 9; P = 0.008), and elevated the action potential threshold (Supplemental Fig. S6, D–F) (Student's paired t-test; control = –47.2 ± 3.3 mV; -conotoxin GVIA = –43.83 ± 2.44 mV; n = 9; P = 0.007). Cav2.2 channel blockade also increased the frequency of firing in response to current injection except for the highest frequencies (Supplemental Fig. S6, G–I). Thus Cav2.2 channel blockade increased primary range firing (Supplemental Fig. S6, G–I) (Student's paired t-test; control response to 40 pA = 35.4 ± 13.8 Hz; response to 40 pA in -conotoxin GVIA = 50.7 ± 19.2 Hz; n = 9; P = 0.01) but led to a reduction in higher secondary range firing (Supplemental Fig. S6, G–I) (Student's paired t-test; control response to 200 pA = 149.1 ± 27.6 Hz; response to 200 pA in -conotoxin GVIA = 125.1 ± 32.5 Hz; n = 9; P = 0.032). These data suggest that Cav2.2 channels, in addition to activating SKCa channels, contribute a significant inward current that augments high-frequency activity (Hallworth et al. 2003; Wilson et al. 2004). This discrepancy between the effects Cav2.2 channel blockade and Cav2.2 channel modulation/SKCa channel blockade suggests that Cav2.2 channel modulation might be partially alleviated at high frequencies of activity. However, taken together, the similarity of the effects of D2-like dopamine receptor mediated modulation and Cav2.2 or SKCa channel blockade indicate that modulation of Cav2.2-SKCa channel coupling largely underlies the effects described here.
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DISCUSSION |
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The data presented here suggest that a major effect of D2-like dopamine receptor activation in STN neurons is the potent and specific reduction in the conductance of Cav2.2 channels. Thus modulation of Cav channels was occluded by blockade of Cav2.2 channels, but was unaffected by blockade of Cav1 channels. Since Cav2.2 channels underlie approximately 45–48% of whole cell Ca2+ current in STN neurons (Song et al. 2000; this study), the roughly 42–44% reduction in whole cell Ca2+ current that followed the application of quinpirole (10 µM) further suggests that Cav2.2 channel conductance may be largely eliminated by D2-like dopamine receptor activation.
The modulation described here exhibited classical features of Gi/o-linked receptor-mediated membrane-delimited modulation of Cav2 channels that is mediated by the direct interaction of Gβ dimers with Cav2 channels (Dolphin 2003; Herlitze et al. 1996; Hille 1995; Tedford and Zamponi 2006). Thus modulation was blocked by brief preapplication with NEM, which disrupts Gi/o proteins (Shapiro et al. 1994), and attenuated by a brief, strong depolarizing prepulse (Bean 1989), which is thought to reflect the unbinding of Gβ dimers from modulated channels. The apparent failure to completely reverse modulation may reflect the rapid reassociation of Gβ dimers with Cav2.2 channels following the depolarizing prepulse and prior to the test voltage step (Zamponi and Snutch 1998; Zhou et al. 1997) and/or other modes of voltage-insensitive modulation, such as phosphorylation/dephosphorylation of Cav2.2 channels (Dolphin 2003; Herlitze et al. 1996; Hille 1995; Tedford and Zamponi 2006). Slow kinetics of activation that result from the stabilization of Cav channel closed states by Gβ subunits (Bean 1989; Carabelli et al. 1996; Kasai and Aosaki 1989; Lipscombe et al. 1989), however, were not generally observed, presumably because the current contribution of the modulated channels was largely eliminated. Indeed, when the modulation of Cav channels was partly alleviated by depolarizing prepulses, the kinetics of Cav channel activation during a subsequent test voltage step were slower than the kinetics of activation under control conditions.
The weight of evidence suggests that D2 and/or D3 rather than D4 dopamine receptors underlie the modulation of Cav2.2 channels described in this study. Sulpiride, applied at a concentration that does not antagonize D4 receptors (Price and Pittman 2001; Shin et al. 2003; Wang et al. 2002), inhibited the action of quinpirole, whereas PD168077, a selective agonist of D4 receptors (Glase et al. 1997), had no effect on Cav channel conductance. The results of mRNA expression analysis described previously (Flores et al. 1999) and here also suggest that STN neurons express relatively detectable levels of mRNA encoding D2 and D3 receptors but not D4 receptors. The failure to observe D2-like receptor mRNA expression in all harvested STN neurons, in contrast to the uniform effects of D2-like dopamine receptor activation, is likely to be a technical limitation related to the low abundance of mRNA encoding dopamine receptors in STN neurons (cf. Surmeier et al. 1996). Future experiments using D2 and D3 dopamine receptor-selective antagonists (Bristow et al. 1998; Joyce and Milan 2005; Stemp et al. 2000;) in STN neurons derived from wild-type and D2 and D3 dopamine receptor knockout mice (Holmes et al. 2004) will be necessary for the definitive characterization of the D2-like dopamine receptors mediating Cav2.2 (this study) and K+ channel (Zhu et al. 2002a) modulation in STN neurons. This endeavor may also be of translational interest given the specific functional roles of D2 and D3 receptors (reviewed by Joyce and Milan 2005).
D2-like dopamine receptor modulation of Cav2.2 channels leads to a reduction in the conductance of SKCa channels
Cav2.2 channels conduct Ca2+ ions that lead to the selective activation of SKCa channels in STN neurons (Bevan and Wilson 1999; Hallworth et al. 2003). Thus D2-like dopamine receptor activation leads, through a reduction in the conductance of Cav2.2 channels, to a reduction in the conductance of SKCa channels, as evidenced by a reduction in transient outward current at suprathreshold voltages and occlusion of D2-like dopamine receptor modulation by the selective Cav2.2 channel blocker -conotoxin GVIA or the selective SKCa channel antagonist apamin. Although BKCa channels also underlie a portion of transient outward current at suprathreshold voltages, blockade of these channels did not occlude the effects of D2-like dopamine receptor activation. Thus BKCa channels are likely to be activated by Ca2+ arising from different Cav channels and/or intracellular Ca2+ stores. Selective coupling of KCa channels to specific and diverse classes of Cav channels has been widely reported and may reflect the precise positioning of Cav channels and KCa channels (e.g., Bloodgood and Sabatini 2007; Davies et al. 1996; Hallworth et al. 2003; Marrion and Tavalin 1998; Pineda et al. 1998; Wolfart and Roeper 2002).
Brain slice recordings of neurons with relatively intact dendritic processes supported findings from acutely isolated neurons. During suprathreshold voltage steps or following the generation of an unclamped action potential, apamin- and/or quinpirole-sensitive outward currents were observed, which possessed similar voltage-dependent and/or kinetic properties to those observed in isolated neurons (see also Hallworth et al. 2003). Furthermore, occlusion of D2-like dopamine receptor modulation by apamin confirmed that the ultimate action of D2-like dopamine receptor modulation was a reduction in the conductance of SKCa channels. The concordance of data derived from brain slice and acutely isolated neuron recordings further suggests that D2-like dopamine receptors, Cav2.2, and SKCa channels are coexpressed in the proximal parts of STN neurons. Furthermore, dissections of the STN do not appear to have been greatly contaminated by surrounding structures like the zona incerta, which relatively weakly express D2-like dopamine receptors (Flores et al. 1999).
Although suprathreshold voltage steps were associated with the prolonged activation Cav2.2 channels (as evidenced by recordings with Ba2+ as a charge carrier), SKCa channel current declined rapidly with a time course similar to that observed following single action potentials. A number of factors may relate to the apparent mismatch between Cav2.2 and SKCa channel current during prolonged voltage steps: 1) Cav channels may exhibit Ca2+-dependent inactivation when Ca2+ rather than Ba2+ is the dominant charge carrier (Budde et al. 2002; unpublished observations); 2) reduction in the concentration of Ca2+ in the vicinity of SKCa channels due to diffusion and/or binding to intrinsic buffers and/or removal by pumps may limit the activation of SKCa channels (Augustine et al. 2003; Helmchen et al. 1996; Marty and Neher 1985; Neher and Augustine 1992; Wilson and Callaway 2000); 3) the conductance of SKCa channels exhibits a steep (slope >4) dependence on intracellular [Ca2+] within the range of about 100–1,000 nM, which suggests that small changes in [Ca2+] in the microdomain of SKCa channels will have large and possibly nonlinear effects on SKCa channel conductance (Hirschberg et al. 1999; Maylie et al. 2004); and 4) prolonged increases in intracellular [Ca2+] may paradoxically reduce the sensitivity of SKCa channels to Ca2+ (Allen et al. 2007; Marty and Neher 1985).
D2-like dopamine receptor modulation reduces the negative feedback control of firing that is mediated by Cav2.2/SKCa channel coupling in STN neurons
Application of autonomous activity as a voltage-clamp waveform allowed us to determine the precise timing of Cav2.2 (-conotoxin GVIA–and quinpirole-sensitive) channel currents with respect to action potentials. Thus Cav2.2 channel currents increased during action potentials, reached their peak on repolarization, and then declined rapidly such that they were negligible during the majority of the intervals between action potentials. Whole cell Cav channel and non-Cav2.2 channel currents exhibited similar kinetics (Do and Bean 2003; this study). The electrophysiological data are therefore concordant with Ca2+ imaging, which demonstrated that intracellular [Ca2+] peaks shortly after autonomously generated action potentials (Hallworth et al. 2003). However, the relatively rapid decline in Cav channel current compared with intracellular [Ca2+] suggests that intracellular Ca2+ dynamics in STN neurons is also influenced by factors such as Ca2+ buffering and/or sequestration and extrusion of Ca2+ and/or Ca2+-induced Ca2+ release from intracellular stores (Augustine et al. 2003; Helmchen et al. 1996; Marty and Neher 1985; Neher and Augustine 1992; Wilson and Callway 2000; Wolfart and Roeper 2002; M Teagarden, JF Atherton, MD Bevan, and CJ Wilson, unpublished observations).
Cav2.2–SKCa channel coupling is critical for the precision of autonomous activity in STN neurons. Blockade of Cav2.2 or SKCa channels (Bevan et al. 1999; Hallworth et al. 2003) or activation of D2-like dopamine receptors (this study) leads to an increase in the CV of ISIs associated with autonomous activity. SKCa channels have been shown to be similarly critical for the precision of autonomous activity of neurons in other brain regions, including the striatum (Bennett et al. 2000), substantia nigra (Atherton and Bevan 2005; Wolfart and Roeper 2002), and cerebellum (Womack et al. 2004). Irregular firing in STN neurons in the presence of -conotoxin GVIA or apamin or quinpirole may arise from increased inactivation of the Nav channels that drive normal pacemaker activity, as evidenced by a consistent increase in action potential threshold. Reduction in the magnitude of action potential afterhyperpolarization presumably reduces the degree to which Nav channels deactivate and recover from inactivation in the intervals between action potentials (Baufreton et al. 2005; Do and Bean 2003).
Although strong depolarization can alleviate Cav2.2 channel modulation, depolarization due to autonomous activity was apparently not sufficient for alleviation. Thus action potential–associated Cav channel currents were modulated to a similar degree during the application of 5 s duration waveforms of autonomous activity. However, it remains to be determined whether weaker modulation that leads to the liberation of fewer Gβ subunits for reassociation (Zamponi and Snutch 1998; Zhou et al. 1997) can be alleviated by repetitive firing.
Under control conditions in vitro STN neurons exhibit a sigmoidal relationship between applied current and evoked activity (Bevan and Wilson 1999; Hallworth et al. 2003; Wilson et al. 2004). Thus STN neurons exhibit a primary firing range where they are relatively less sensitive to current injection, a secondary range characterized by higher sensitivity, and a tertiary range associated with the saturation of firing and depolarization block. Blockade of Cav2.2 or SKCa channels increases the sensitivity of firing to current and abolishes the different sensitivities of primary and secondary range firing (Bevan and Wilson 1999; Hallworth et al. 2003). Because Cav2.2 channels are engaged by action potentials and conduct Ca2+ over a range of firing frequencies (Hallworth et al. 2003), these channels "report" activity to SKCa channels, which in turn limit firing. The fact that D2-like dopamine receptor modulation produced a similar alteration in firing to blockade of Cav2.2 or SKCa channels provides further evidence that modulation acts through a reduction in the negative feedback control of firing that is mediated through Cav2.2/SKCa channel coupling. Because firing sensitivity was increased across a range of firing frequencies, these data further suggest that modulation was not generally alleviated by action potential–mediated dissociation of Gβ subunits from Cav2.2 channels. However, at the highest firing rates selective blockade of Cav2.2 channels reduced activity in contrast to the effects of D2-like receptor modulation or blockade of SKCa channels. At these frequencies Cav2.2 channels, in addition to activating SKCa channels, may therefore contribute a significant inward current that augments high-frequency activity (Hallworth et al. 2003; Wilson et al. 2004). The discrepancy between the effects Cav2.2 channel blockade and Cav2.2 channel modulation/SKCa channel blockade suggests that Cav2.2 channel modulation may be partially alleviated by action potentials evoked during high-frequency secondary range activity.
Our data are inconsistent with the findings of Zhu and colleagues (2002a) who reported that D2-like dopamine receptor modulation of STN neurons was mediated by the closure of voltage-independent K+ channels. However, it should be noted that in that study, analysis was restricted to subthreshold voltages, which would not have revealed the modulatory pathway identified here. Our failure to observe a current similar to that observed by Zhu and colleagues (2002a) may also relate to the different intracellular anions used in the two studies. Thus gluconate may reduce the contribution of SKCa channels, whereas methylsulfate may block some types of K+ channel (Kaczorowski et al. 2007; Velumian et al. 1997; Zhang et al. 1994). The different strategies for intracellular Ca2+ chelation may have also have contributed to the relative expression of two signaling pathways engaged by the activation of D2-like dopamine receptors in STN neurons (Velumian and Carlen 1999; Zhang et al. 1995). That said, the specific manner in which D2-like dopamine receptors modulated the frequency–intensity curve of neurons that were recorded with the relatively noninvasive perforated-patch technique (Abe et al. 1994; Hallworth et al. 2003; Kyrozis and Reichling 1995) is consistent with the modulation of Cav2.2-SKCa channel coupling (Bevan and Wilson 1999; Hallworth et al. 2003).
Functional implications
By reducing the conductance of Cav2.2 channels (this study) that are coupled to SKCa channels (Hallworth et al. 2003) and increasing the conductance of Cav1 channels (Baufreton et al. 2003) that are not functionally coupled to SKCa channels (Hallworth et al. 2003), dopamine acting at D2-like and D5 receptors, respectively, can powerfully modulate the autonomous firing and integrative properties of STN neurons (Beurrier et al. 1999; Gillies and Wilshaw 2006; Hallworth et al. 2003; Otsuka et al. 2001; Wilson et al. 2004). D2-like dopamine receptor-mediated modulation may through an increase in the variability of autonomous firing contribute to the decorrelation of STN neuronal activity that is observed under normal conditions (Magill et al. 2000; Wichmann et al. 1994). Together D2-like and/or D5 dopamine receptor-mediated modulation of Cav2.2 and Cav1 channels should also produce a leftward shift in the frequency–intensity curve of STN neurons (Bevan and Wilson 1999; Hallworth et al. 2003) and thus enhance the frequency of firing in response to excitatory synaptic input (Fujimoto and Kita 1993; Kitai and Deniau 1981; Mouroux et al. 1995; Nakanishi et al. 1988). Because Cav1 channels underlie plateau potentials in STN neurons that can be triggered by excitatory synaptic inputs (Kass and Mintz 2006; Otsuka et al. 2001) and inhibition of Cav2.2–SKCa channel coupling enhances the frequency of action potentials generated in response to excitation (Bevan and Wilson 1999; Hallworth et al. 2003), dopaminergic modulation of Cav1 and Cav2.2 channels may also amplify not only the frequency but also the duration of firing in response to excitatory inputs. Linearization of the frequency–intensity curve further suggests that D2-like dopamine receptor-mediated modulation will cause the firing responses of STN neurons to grade more smoothly with the intensity of incoming excitatory inputs. The depolarization of STN neurons that accompanies D2-like dopamine receptor-mediated inhibition of Cav2.2 channels is also likely to reduce the resting availability of Cav3 channels and the capability of both excitatory and inhibitory synaptic inputs to generate Cav3 channel–mediated burst firing (Beurrier et al. 1999, 2000; Bevan et al. 2002; Hallworth and Bevan 2005; Kass and Mintz 2006). Modulation of Cav2.2 and Cav1 channels in the axon/terminal regions of STN neurons may also contribute to the dopaminergic regulation of transmission at subthalamopallidal and subthalamonigral synapses recently reported (Hernandez et al. 2006; Ibanez-Sandoval et al. 2006).
By augmenting STN activity, substantia nigra dopamine neurons could through positive feedback with STN neurons potentiate their resting tonic activity and transient high-frequency activity during associative learning (Chergui et al. 1994; Schultz 2002). Dopaminergic enhancement of electrogenesis in STN neurons could also contribute to the long-term potentiation of excitatory synaptic inputs that trigger high-frequency activity in STN neurons. Indeed, Ca2+ associated with Cav1 channel activity is necessary for some forms of long-term synaptic potentiation (Kapur et al. 1998; Remy and Spruston 2007; Rosanova and Ulrich 2005).
In PD, STN neurons exhibit abnormal, correlated, low-frequency, rhythmic, burst activity that is coherent with resting tremor and/or cortical β-band activity (Bergman et al. 1994; Hutchison et al. 2004; Levy et al. 2002). Hypoactivation of postsynaptic STN D2-like dopamine receptors may contribute to the pathological pattern of STN activity. STN neurons may be relatively hyperpolarized by the absence of dopamine (Tofighy et al. 2003; Wilson et al. 2006; Zhu et al. 2002a,b; this study), in which case the resting availability of Cav3 channels and the capability of inhibitory and excitatory inputs to trigger Cav3 channel-mediated burst firing may be enhanced (Bevan et al. 2002; Kass and Mintz 2006; Otsuka et al. 2001; Wichmann and Soares 2006). Pathological enhancement in the coupling of Cav2.2/SKCa channels may also enhance self-terminating/phasic/ bursting STN activity in response to synaptic input. The differential sensitivities of STN neurons to excitatory input under conditions of reduced dopaminergic modulation (Bevan and Wilson 1999; Hallworth et al. 2003; Wilson et al. 2004) could also oppose the smooth transition to coherent -band activity that occurs in the basal ganglia and cortex during the execution of normal voluntary movement (Hutchison et al. 2004; Williams et al. 2002).
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ACKNOWLEDGMENTS |
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1 The online version of this article contains supplemental data. 
Address for reprint requests and other correspondence: M. D. Bevan, Northwestern University, Department of Physiology, Feinberg School of Medicine, 303 E. Chicago Avenue, Chicago, IL 60611 (E-mail: m-bevan{at}northwestern.edu)
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