HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Supplemental Figures All Versions of this Article: 99/2/442    most recent 00998.2007v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (1) Citing Articles via Google Scholar Google Scholar Articles by Ramanathan, S. Articles by Bevan, M. D. Search for Related Content PubMed PubMed Citation Articles by Ramanathan, S. Articles by Bevan, M. D.

D₂-Like Dopamine Receptors Modulate SK_Ca Channel Function in Subthalamic Nucleus Neurons Through Inhibition of Ca_v2.2 Channels

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

	ABSTRACT

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
The activity patterns of subthalamic nucleus (STN) neurons are intimately related to motor function/dysfunction and modulated directly by dopaminergic neurons that degenerate in Parkinson's disease (PD). To understand how dopamine and dopamine depletion influence the activity of the STN, the functions/signaling pathways/substrates of D₂-like dopamine receptors were studied using patch-clamp recording. In rat brain slices, D₂-like dopamine receptor activation depolarized STN neurons, increased the frequency/irregularity of their autonomous activity, and linearized/enhanced their firing in response to current injection. Activation of D₂-like receptors in acutely isolated neurons reduced transient outward currents evoked by suprathreshold voltage steps. Modulation was inhibited by a D₂-like receptor antagonist and occluded by voltage-dependent Ca²⁺ (Ca_v) channel or small-conductance Ca²⁺-dependent K⁺ (SK_Ca) channel blockers or Ca²⁺-free media. Because Ca_v channels are targets of G_i/o-linked receptors, actions on step- and action potential waveform-evoked Ca_v channel currents were studied. D₂-like receptor activation reduced the conductance of Ca_v2.2 but not Ca_v1 channels. Modulation was mediated, in part, by direct binding of Gβ 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 SK_Ca 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 Ca_v2.2 and SK_Ca channels in STN neurons.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
The subthalamic nucleus (STN) is a key component of the basal ganglia, a group of subcortical brain nuclei that are critical for normal voluntary movement and associative/motor learning, and are the principal site of pathology in Parkinson's disease (PD) (Albin et al. 1989; 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⁺ (Ca_v1) 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.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
Experimental procedures were carried out in accordance with the policies of Northwestern University's Institutional Animal Care and Use Committee and the APS's Guiding Principles in the Care and Use of Animals.

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 Mg_1.5ATP, 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 (GABA_A and GABA_B, 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⁺ (Na_v) 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 Ca_v 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 Mg_1.5ATP, 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 Ca_v 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.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
A total of 124 STN neurons from 68 rats were selected for analysis. Data were obtained from 21 neurons that were recorded in brain slices and 103 acutely isolated neurons.

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, GABA_A, and GABA_B 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 Na_v channels (Baufreton et al. 2005).

View larger version (21K): [in this window] [in a new window]   FIG. 1. Activation of D2-like dopamine receptors depolarizes subthalamic nucleus (STN) neurons and increases the irregularity and frequency of their autonomous activity. A–F: data from brain slices. A–C: activation of D2-like dopamine receptors led to depolarization of the mean membrane potential (Vm), a reduction in the precision of autonomous activity [compare Ai with Aii; plots of interspike intervals (ISIs) vs. the number of interspike intervals n(ISI) further reveal the increased variability of ISIs in quinpirole] and an increase in the frequency of autonomous activity in a representative STN neuron (A) and the sample population (B, C) (n = 6). D2-like dopamine receptor activation additionally led to an increase in the threshold of action potentials (APth) in a representative neuron (D, E) and the sample population (F). D: zoom of an action potential in control conditions and in the presence of quinpirole. E: the phase plot (voltage vs. dV/dt) of the same APs in D also clearly illustrates the elevation in AP threshold in the presence of quinpirole. *P < 0.05.

View larger version (31K): [in this window] [in a new window]   FIG. 2. Activation of D2-like dopamine receptors enhances the activity of STN neurons in response to somatic current injection. A–C: data from brain slices. A–C: D2-like dopamine receptor activation enhanced the response of an individual STN neuron (A and B) and the sample population (C) (n = 6) to current injection. B: D2-like receptor activation linearized and shifted to the left the characteristic sigmoidal frequency-intensity relationship that was observed under control conditions. *P < 0.05.

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 Na_v 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.

View larger version (24K): [in this window] [in a new window]   FIG. 3. Activation of D2-like dopamine receptors reduces the magnitude of a transient outward current that is evoked by suprathreshold voltage steps. A–F: data from acutely isolated neurons. A: currents evoked by 5 mV depolarizing voltage steps from a holding voltage of –70 mV. Currents were evoked in the presence of tetrodotoxin (TTX) to eliminate Nav channel–mediated inward currents in the sub- and suprathreshold voltage ranges (Beurrier et al. 2000; Bevan and Wilson 1999). Note the transient outward current at the beginning of suprathreshold voltage steps (asterisk). B: application of the D2-like dopamine receptor agonist quinpirole (10 µM) reduced the peak amplitude of transient outward currents (asterisk) but had no effect on sustained outward currents. C: subtraction of the currents in B from A reveals the quinpirole-sensitive currents. D: quinpirole-sensitive transient and sustained outward currents normalized to cell capacitance plotted against voltage (n = 7 neurons). Significant reductions in peak transient but not sustained outward current were observed. E: the reduction in transient outward current that was mediated by quinpirole was reversed by application of the D2-like dopamine receptor antagonist sulpiride (10 µM). F: box plot (n = 3 neurons) of current density under control conditions, in the presence of quinpirole, in the presence of quinpirole and sulpiride, and on the removal of sulpiride. *P < 0.05 in D and F.

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 Ca_v channels conduct Ca²⁺ that activates Ca²⁺-dependent K⁺ (K_Ca) 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 Ca_v1 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.

View larger version (22K): [in this window] [in a new window]   FIG. 4. Elimination of Ca2+-dependent transient outward current occludes the action of D2-like dopamine receptor activation. A–F: data from acutely isolated neurons. A–C: application of CdCl2, a nonselective Cav1–2 channel blocker (n = 3) or reduction of the electrochemical gradient for Ca2+ (n = 3) reduced transient outward currents that were evoked at suprathreshold voltages in a representative cell (A, B) and the sample population (C). Subsequent application of quinpirole had no effect on the remaining transient outward current in a representative neuron (D, E) or in the sample population (F; n = 6). *P < 0.05.

Blockade of SK_Ca but not BK_Ca channels occludes the action of D₂-like dopamine receptor activation

To determine whether K_Ca 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) K_Ca 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 SK_Ca 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).

View larger version (21K): [in this window] [in a new window]   FIG. 5. Blockade of SKCa but not BKCa channels occludes the reduction of transient outward current by D2-like receptor activation. A–L: data from acutely isolated neurons. A–C: blockade of SKCa channels with the selective antagonist apamin (10 nM) reduced transient outward current that was evoked by suprathreshold voltage steps in a representative example (A, B) and in the sample population (C, n = 7). Blockade of SKCa channels also occluded the action of the D2-like receptor agonist quinpirole on transient outward current in a representative example (D, E) and the sample population (F; n = 6). G–I: blockade of BKCa channels with the selective antagonist iberiotoxin (Ibtx; 100 nM) also reduced transient outward current that was evoked by suprathreshold voltage steps in a representative example (G, H) and in the sample population (I, n = 3). However, blockade of BKCa channels failed to occlude the action of the D2-like receptor agonist quinpirole on transient outward current in a representative example (J, K) and the sample population (L; n = 4). *P < 0.05.

D₂-like dopamine receptor activation reduces Ca_v channel current

Ca_v channels that are functionally coupled to SK_Ca 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 Ca_v channels, currents flowing through Ca_v channels in acutely isolated neurons were isolated by eliminating currents flowing through Na_v and K_v 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 Ca_v channels. Ca_v 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 Ca_v channel current density at –20 mV = –58.6 ± 22.4 pA/pF; Ca_v channel current density at –20 mV in quinpirole = –32.3 ± 12.3 pA/pF; n = 6; P = 0.002). Ca_v channel current density in quinpirole was 54.9 ± 5.4% of the control current density. Following the return to control conditions, Ca_v 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 Ca_v channel currents to return to their initial values on return to control media may be attributed to the rundown of Ca_v channel currents and/or incomplete reversal of D₂-like dopamine receptor-mediated modulation.

View larger version (18K): [in this window] [in a new window]   FIG. 6. D2-like dopamine receptor activation reduces Cav channel current. A–H: data from acutely isolated neurons. A–D: step-evoked steady-state and tail Cav channel currents were reversibly reduced by the application of quinpirole in a representative neuron (A–C) and the sample population (n = 6; D). E–H: when a record of autonomous activity of an STN neuron was used as the voltage-clamp waveform, quinpirole had a similar effect on action potential-evoked Cav channel currents. H: spike-triggered averaging revealed that control (Hi), quinpirole-resistant (Hi), and quinpirole-sensitive (Hii) Cav channel currents followed action potential with brief delay and declined rapidly following action potential afterhyperpolarization. *P < 0.05.

The effect of quinpirole was consistent over the injected waveform of 37 action potentials. Indeed, the quinpirole-sensitive Ca_v 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 G_i/o proteins, NEM was applied, which is a broad-spectrum sulfydryl-alkylating reagent that alkylates cysteine residues on G_i/o proteins and prevents their coupling to receptors (Shapiro et al. 1994; Supplemental Fig. S2). As described earlier, depolarizing voltage steps evoked Ca_v channel currents that were reduced by 10 µM quinpirole (Supplemental Fig. S2, A and B) (Student's paired t-test; control Ca_v channel current density at –20 mV = –47.5 ± 10.3 pA/pF; Ca_v channel current density at –20 mV in quinpirole = –27.2 ± 6.3 pA/pF; n = 3; P = 0.001). Ca_v channel current density in quinpirole was 57.2 ± 1.3% of control. Ca_v 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 Ca_v channel current density at –20 mV = –47.5 ± 10.3 pA/pF; Ca_v channel current density at –20 mV in NEM = –47.3 ± 5.4 pA/pF; n = 3; P = 0.9425). The Ca_v 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 Ca_v channel current (Supplemental Fig. S2, D–G) (Student's paired t-test; Ca_v channel current density at –20 mV in NEM = –44.4 ± 7.1 pA/pF; Ca_v channel current density at –20 mV in NEM + quinpirole = –45.4 ± 6.9 pA/pF; n = 3; P = 0.472). The Ca_v 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 G_i/G_o-linked receptor.

D₂-like dopamine receptor activation reduces current flow through Ca_v2.2 but not Ca_v1 channels

Although Ca_v2 channels and Ca_v1 channels are targets of D₂-like receptors (Hernandez-Lopez et al. 2000; Momiyama and Koga 2001; Svensson et al. 2003; Yan et al. 1997), SK_Ca channels are more strongly activated by Ca²⁺ entry via Ca_v2.2 channels than Ca_v1 channels in STN neurons (Hallworth et al. 2003). We therefore hypothesized that Ca_v2.2 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 Ca_v1 and Ca_v2.2 channels on D₂-like dopamine receptor-mediated modulation of Ca_v channel current in acutely isolated neurons.

Bath application of 5 µM isradipine, a selective Ca_v1 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 Ca_v channel current density at –20 mV = –82.2 ± 2.6 pA/pF; Ca_v channel current density at –20 mV in isradipine = –70.6 ± 4.6 pA/pF; n = 3; P = 0.017). The Ca_v channel current density in isradipine was 85.8 ± 3.5% of control. Subsequent application of quinpirole reduced the Ca_v channel current observed in the presence of isradipine (Fig. 7, C and D) (Student's paired t-test; Ca_v channel current density at –20 mV in isradipine = –70.6 ± 4.6 pA/pF; Ca_v channel current density at –20 mV in isradipine + quinpirole = –38.5 ± 7.8 pA/pF; n = 3; P = 0.006). The Ca_v channel current density in isradipine and quinpirole was therefore 54.3 ± 7.8% of the Ca_v channel current density in isradipine. These data suggest that Ca_v1 channels are not a major target of D₂-like dopamine receptor modulation in STN neurons.

View larger version (25K): [in this window] [in a new window]   FIG. 7. D2-like dopamine receptor activation reduces current flow through Cav2.2 but not Cav1 channels. A–L: data from acutely isolated neurons. A–D: blockade of Cav1 channels with the selective antagonist isradipine (5 µM) reduced step-evoked whole cell Cav channel current (A, B, and D) but did not prevent the D2-like receptor-mediated reduction in step-evoked whole cell Cav channel current that followed the application of quinpirole (C and D; n = 3). E–H: blockade of Cav2.2 channels with the selective antagonist -conotoxin GVIA (Ctx; 1 µM) greatly reduced step-evoked whole cell Cav channel current (E, F, and H) and largely occluded the D2-like receptor-mediated reduction in step-evoked whole cell Cav channel current that follows the application of quinpirole (G and H; n = 6). I–K: application of 1 µM Ctx greatly reduced action potential waveform-evoked whole cell Cav channel current (I, J, and Li) and largely occluded the D2-like receptor-mediated reduction in waveform-evoked whole cell Cav channel current that followed the application of quinpirole (K and Lii; n = 6). Lii: spike-triggered averaging revealed that Ctx-sensitive current lagged action potentials with brief delay and declined rapidly following action potential afterhyperpolarization. *P < 0.05.

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 Ca_v channel current density = –85.8 ± 30.0 pA/pF; Ca_v 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; Ca_v channel current density in -conotoxin GVIA = –50.9 ± 19.2 pA/pF; Ca_v channel current density in -conotoxin GVIA + quinpirole = –49.0 ± 18.8 pA/pF; n = 6; P = 0.19). The Ca_v channel current density in -conotoxin GVIA was 58.5 ± 7.2% of the control Ca_v channel current density.

As for whole cell Ca_v channel current, Ca_v2.2 channel current increased during action potentials, peaked on repolarization, and then rapidly declined such that the current contributed by Ca_v2.2 channels during the majority of the ISI was minimal (Fig. 7L). Taken together, our data suggest that Ca_v2.2 but not Ca_v1 channels are potently modulated by D₂-like dopamine receptors.

To confirm that dopamine reduces the conductance of Ca_v2.2 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 Ca_v1 channels through the activation of D₅ receptors (Baufreton et al. 2003), isradipine (5 µM) was included in all solutions to block Ca_v1 channels. Application of voltage steps from –70 to –20 mV evoked Ca_v 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 Ca_v channel current density at –20 mV = –53.9 ± 11.5 pA/pF; Ca_v channel current density at –20 mV in dopamine = –36.5 ± 8.3 pA/pF; n = 6; P = 0.002). Ca_v 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 Ca_v channel current (Supplemental Fig. S3, B, C, and E) (Student's paired t-test; Ca_v channel current density at –20 mV in dopamine = –36.5 ± 8.3 pA/pF; Ca_v 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 Ca_v 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; Ca_v channel current density at –20 mV in dopamine + SCH23390 = –35.0 ± 7.3 pA/pF; Ca_v channel current density at –20 mV in dopamine + SCH23390 + sulpiride = –46.1 ± 11.2 pA/pF, n = 4, P = 0.003). The Ca_v 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 Ca_v2.2 channels through the activation of D₂-like dopamine not D₁-like dopamine receptors.

Although the potent modulation of Ca_v2.2 channels seems likely to underlie the D₂-like dopamine receptor-mediated reduction in SK_Ca channel-mediated outward current, direct modulation of SK_Ca 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 SK_Ca channel conductance is mediated through a reduction in the conductance of Ca_v2.2 channels.

D₂-like dopamine receptor modulation of Ca_v2.2 channels is alleviated by brief depolarization

G-protein–coupled receptor (GPCR)–mediated inhibition of Ca_v2 channels is often mediated by a direct interaction between channels and Gβ subunits from G_i/G_o 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 Ca_v channels was therefore tested in acutely isolated neurons.

Depolarizing voltage steps from –70 to –20 mV evoked Ca_v channel currents that were reduced by quinpirole (Fig. 8, A and B) (Student's paired t-test; control Ca_v channel current density at –20 mV = –51.5 ± 15.5 pA/pF; Ca_v channel current density at –20 mV in quinpirole = –25.5 ± 6.4 pA/pF; n = 7; P < 0.001). The Ca_v channel current density in quinpirole was 50.9 ± 8.8% of the control Ca_v channel current density.

View larger version (15K): [in this window] [in a new window]   FIG. 8. D2-like dopamine receptor modulation of Cav channels was alleviated by brief depolarization. A–E: data from acutely isolated neurons. A, B, and D: step-evoked whole cell Cav channel current was reduced by D2-like receptor activation (n = 7). C and D: a depolarizing prepulse from –70 to +120 mV for 50 ms partially reversed the reduction in Cav channel current that was evoked by the test voltage step from –70 to –20 mV. E: application of 10 µM quinpirole slowed the activation kinetics of whole cell Cav channel current only when the test voltage step was preceded by a depolarizing prepulse. *P < 0.05.

Another classical biophysical feature of Gβ-mediated modulation of Ca_v channels is a reduction in the speed of activation due to the stabilization of Ca_v 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 Ca_v 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 Ca_v 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 Ca_v 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 Ca_v 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 Ca_v 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 Ca_v2.2 channels.

D₂ and/or D₃ but not D₄ dopamine receptors modulate Ca_v2.2 channels

To determine which type(s) of D₂-like dopamine receptor underlie the modulation of Ca_v2.2 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 Ca_v channel current evoked by step depolarization from –70 to –20 mV (Fig. 9, A, B, and D) (Student's paired t-test; control Ca_v channel current density at –20 mV = –29.3 ± 3.3 pA/pF; Ca_v 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 Ca_v channel current (Fig. 9, C and D) (Student's paired t-test; Ca_v channel current density at –20 mV in PD168077 = –28.7 ± 5.4 pA/pF; Ca_v channel current density at –20 mV in PD168077 + quinpirole = –16.4 ± 6.5 pA/pF; n = 3; P = 0.009). The Ca_v channel current density in quinpirole was 55.9 ± 16.4% of the control Ca_v channel current density.

View larger version (19K): [in this window] [in a new window]   FIG. 9. D2/3 but not D4 dopamine receptors modulate Cav2.2 channels. A–F: data from acutely isolated neurons. A, B, and D: addition of the selective D4 dopamine receptor agonist PD168077 (1 µM) did not alter the amplitude of Cav channel current that was evoked by step depolarization from –70 to –20 mV in a representative neuron (A, B) or in each of 3 neurons tested (D). C and D: subsequent application of 10 µM quinpirole reduced Cav channel current (n = 3). E: scRT-PCR analysis of D2-like dopamine receptor mRNA in a single neuron. The neuron expressed mRNA encoding D2 and D3 but not D4 dopamine receptors. F: scRT-PCR analysis revealed that STN neurons consistently expressed detectable levels of D2 (37%) and D3 (20%) but not D4 (0%) dopamine receptors.

D₂-like dopamine receptor modulation reduces the medium-duration component of action potential afterhyperpolarization

SK_Ca 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.

View larger version (15K): [in this window] [in a new window]   FIG. 10. D2-like dopamine receptor modulation reduces the medium-duration component of action potential afterhyperpolarization. A–F: data from brain slices. To evoke an unclamped action potential, the membrane potential was stepped from –60 to +20 mV for 5 ms. On return to the holding voltage of –60 mV an outward current was observed that was reduced in amplitude by D2-like dopamine receptor activation in a representative example (A, B) and across the sample population (C; n = 6). Blockade of SKCa channels with the selective antagonist apamin (10 nM) occluded D2-like dopamine receptor modulation in a representative neuron (D, E) and the sample population (F, n = 3). The quinpirole-sensitive current observed under control conditions (B) and the apamin-sensitive current (E) were similar in amplitude and kinetics. *P < 0.05.

To determine whether D₂-like dopamine receptor-mediated modulation of Ca_v2.2–SK_Ca 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). SK_Ca 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 SK_Ca channels (Hallworth et al. 2003). Indeed, blockade of SK_Ca 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 SK_Ca 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 Ca_v2.2 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). Ca_v2.2 channel blockade also increased the frequency of firing in response to current injection except for the highest frequencies (Supplemental Fig. S6, G–I). Thus Ca_v2.2 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 Ca_v2.2 channels, in addition to activating SK_Ca channels, contribute a significant inward current that augments high-frequency activity (Hallworth et al. 2003; Wilson et al. 2004). This discrepancy between the effects Ca_v2.2 channel blockade and Ca_v2.2 channel modulation/SK_Ca channel blockade suggests that Ca_v2.2 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 Ca_v2.2 or SK_Ca channel blockade indicate that modulation of Ca_v2.2-SK_Ca channel coupling largely underlies the effects described here.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
Ca_v2.2 channels are potently modulated by D₂-like dopamine receptors

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 Ca_v2.2 channels. Thus modulation of Ca_v channels was occluded by blockade of Ca_v2.2 channels, but was unaffected by blockade of Ca_v1 channels. Since Ca_v2.2 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 Ca_v2.2 channel conductance may be largely eliminated by D₂-like dopamine receptor activation.

The modulation described here exhibited classical features of G_i/o-linked receptor-mediated membrane-delimited modulation of Ca_v2 channels that is mediated by the direct interaction of Gβ dimers with Ca_v2 channels (Dolphin 2003; Herlitze et al. 1996; Hille 1995; Tedford and Zamponi 2006). Thus modulation was blocked by brief preapplication with NEM, which disrupts G_i/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 Ca_v2.2 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 Ca_v2.2 channels (Dolphin 2003; Herlitze et al. 1996; Hille 1995; Tedford and Zamponi 2006). Slow kinetics of activation that result from the stabilization of Ca_v 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 Ca_v channels was partly alleviated by depolarizing prepulses, the kinetics of Ca_v 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 Ca_v2.2 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 Ca_v 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 Ca_v2.2 (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 Ca_v2.2 channels leads to a reduction in the conductance of SK_Ca channels

Ca_v2.2 channels conduct Ca²⁺ ions that lead to the selective activation of SK_Ca 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 Ca_v2.2 channels, to a reduction in the conductance of SK_Ca channels, as evidenced by a reduction in transient outward current at suprathreshold voltages and occlusion of D₂-like dopamine receptor modulation by the selective Ca_v2.2 channel blocker -conotoxin GVIA or the selective SK_Ca channel antagonist apamin. Although BK_Ca 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 BK_Ca channels are likely to be activated by Ca²⁺ arising from different Ca_v channels and/or intracellular Ca²⁺ stores. Selective coupling of K_Ca channels to specific and diverse classes of Ca_v channels has been widely reported and may reflect the precise positioning of Ca_v channels and K_Ca 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 SK_Ca channels. The concordance of data derived from brain slice and acutely isolated neuron recordings further suggests that D₂-like dopamine receptors, Ca_v2.2, and SK_Ca 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 Ca_v2.2 channels (as evidenced by recordings with Ba²⁺ as a charge carrier), SK_Ca 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 Ca_v2.2 and SK_Ca channel current during prolonged voltage steps: 1) Ca_v 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 SK_Ca channels due to diffusion and/or binding to intrinsic buffers and/or removal by pumps may limit the activation of SK_Ca 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 SK_Ca 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 SK_Ca channels will have large and possibly nonlinear effects on SK_Ca channel conductance (Hirschberg et al. 1999; Maylie et al. 2004); and 4) prolonged increases in intracellular [Ca²⁺] may paradoxically reduce the sensitivity of SK_Ca 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 Ca_v2.2/SK_Ca channel coupling in STN neurons

Application of autonomous activity as a voltage-clamp waveform allowed us to determine the precise timing of Ca_v2.2 (-conotoxin GVIA–and quinpirole-sensitive) channel currents with respect to action potentials. Thus Ca_v2.2 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 Ca_v channel and non-Ca_v2.2 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 Ca_v 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).

Ca_v2.2–SK_Ca channel coupling is critical for the precision of autonomous activity in STN neurons. Blockade of Ca_v2.2 or SK_Ca 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. SK_Ca 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 Na_v 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 Na_v 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 Ca_v2.2 channel modulation, depolarization due to autonomous activity was apparently not sufficient for alleviation. Thus action potential–associated Ca_v 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 Ca_v2.2 or SK_Ca 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 Ca_v2.2 channels are engaged by action potentials and conduct Ca²⁺ over a range of firing frequencies (Hallworth et al. 2003), these channels "report" activity to SK_Ca channels, which in turn limit firing. The fact that D₂-like dopamine receptor modulation produced a similar alteration in firing to blockade of Ca_v2.2 or SK_Ca channels provides further evidence that modulation acts through a reduction in the negative feedback control of firing that is mediated through Ca_v2.2/SK_Ca 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 Ca_v2.2 channels. However, at the highest firing rates selective blockade of Ca_v2.2 channels reduced activity in contrast to the effects of D₂-like receptor modulation or blockade of SK_Ca channels. At these frequencies Ca_v2.2 channels, in addition to activating SK_Ca 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 Ca_v2.2 channel blockade and Ca_v2.2 channel modulation/SK_Ca channel blockade suggests that Ca_v2.2 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 SK_Ca 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 Ca_v2.2-SK_Ca channel coupling (Bevan and Wilson 1999; Hallworth et al. 2003).

Functional implications

By reducing the conductance of Ca_v2.2 channels (this study) that are coupled to SK_Ca channels (Hallworth et al. 2003) and increasing the conductance of Ca_v1 channels (Baufreton et al. 2003) that are not functionally coupled to SK_Ca 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 Ca_v2.2 and Ca_v1 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 Ca_v1 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 Ca_v2.2–SK_Ca channel coupling enhances the frequency of action potentials generated in response to excitation (Bevan and Wilson 1999; Hallworth et al. 2003), dopaminergic modulation of Ca_v1 and Ca_v2.2 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 Ca_v2.2 channels is also likely to reduce the resting availability of Ca_v3 channels and the capability of both excitatory and inhibitory synaptic inputs to generate Ca_v3 channel–mediated burst firing (Beurrier et al. 1999, 2000; Bevan et al. 2002; Hallworth and Bevan 2005; Kass and Mintz 2006). Modulation of Ca_v2.2 and Ca_v1 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 Ca_v1 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 Ca_v3 channels and the capability of inhibitory and excitatory inputs to trigger Ca_v3 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 Ca_v2.2/SK_Ca 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).

	GRANTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
This study was supported by National Institute of Neurological Disorders and Stroke Grants NS-047085, NS-041280, and NS-20702 and the National Parkinson Foundation.

	ACKNOWLEDGMENTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
We thank D. J. Surmeier for helpful discussions throughout this study.

	FOOTNOTES

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

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)

	REFERENCES

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
Abe Y, Furukawa K, Itoyama Y, Akaike N. Glycine response in acutely dissociated ventromedial hypothalamic neurone of the rat: new approach with gramicidin perforated patch-clamp technique. J Neurophysiol 72: 1530–1537, 1994.[Abstract/Free Full Text]

Albin RL, Young AB, Penney JB. The functional anatomy of basal ganglia disorders. Trends Neurosci 12: 366–375, 1989.[CrossRef][Web of Science][Medline]

Allen D, Fakler B, Maylie J, Adelman JP. Organization and regulation of small conductance Ca²⁺-activated K⁺ channel multiprotein complexes. J Neurosci 27: 2369–2376, 2007.[Abstract/Free Full Text]

Alvarez L, Macias R, Lopez G, Alvarez E, Pavon N, Rodriguez-Oroz MC, Juncos JL, Maragoto C, Guridi J, Litvan I, Tolosa ES, Koller W, Vitek J, DeLong MR, Obeso JA. Bilateral subthalamotomy in Parkinson's disease: initial and long-term response. Brain 128: 570–583, 2005.[Abstract/Free Full Text]

Atherton JF, Bevan MD. Ionic mechanisms underlying autonomous action potential generation in the somata and dendrites of GABAergic substantia nigra pars reticulata neurones in vitro. J Neurosci 25: 8272–8281, 2005.[Abstract/Free Full Text]

Augustine GJ, Santamaria F, Tanaka K. Local calcium signaling in neurons. Neuron 40: 331–346, 2003.[CrossRef][Web of Science][Medline]

Barnett A, Ahn HS, Billard W, Gold EH, Kohli JD, Glock D, Goldberg LI. Relative activities of SCH 23390 and its analogs in three tests for D1/DA1 dopamine receptor antagonism. Eur J Pharmacol 128: 249–253, 1986.[CrossRef][Web of Science][Medline]

Baufreton J, Atherton JF, Surmeier DJ, Bevan MD. Enhancement of excitatory synaptic integration by GABAergic inhibition in the subthalamic nucleus. J Neurosci 25: 8505–8517, 2005.[Abstract/Free Full Text]

Baufreton J, Garret M, Rivera A, de la Calle A, Gonon F, Dufy B, Bioulac B, Taupignon A. D5 (not D1) dopamine receptors potentiate burst-firing in neurons of the subthalamic nucleus by modulating an L-type calcium conductance. J Neurosci 23: 816–825, 2003.[Abstract/Free Full Text]

Bean BP. Neurotransmitter inhibition of neuronal calcium currents by changes in channel voltage dependence. Nature 340: 153–156, 1989.[CrossRef][Medline]

Benabid AL. Deep brain stimulation for Parkinson's disease. Curr Opin Neurobiol 13: 696–706, 2003.[CrossRef][Web of Science][Medline]

Bennett BD, Callaway JC, Wilson CJ. Intrinsic membrane properties underlying spontaneous tonic firing in neostriatal cholinergic interneurons. J Neurosci 20: 8493–8503, 2000.[Abstract/Free Full Text]

Bergman H, Wichmann T, DeLong MR. Reversal of experimental parkinsonism by lesions of the subthalamic nucleus. Science 249: 1436–1438, 1990.[Abstract/Free Full Text]

Bergman H, Wichmann T, Karmon B, DeLong MR. The primate subthalamic nucleus. II. Neuronal activity in the MPTP model of parkinsonism. J Neurophysiol 72: 507–520, 1994.[Abstract/Free Full Text]

Beurrier C, Bioulac B, Hammond C. Slowly inactivating sodium current (I_NaP) underlies single-spike activity in rat subthalamic neurons. J Neurophysiol 83: 1951–1957, 2000.[Abstract/Free Full Text]

Beurrier C, Congar P, Bioulac B, Hammond C. Subthalamic nucleus neurons switch from single-spike activity to burst-firing mode. J Neurosci 19: 599–609, 1999.[Abstract/Free Full Text]

Bevan MD, Atherton JF, Baufreton J. Cellular principles underlying normal and pathological activity in the subthalamic nucleus. Curr Opin Neurobiol 16: 621–628, 2006.[CrossRef][Web of Science][Medline]

Bevan MD, Magill PJ, Hallworth NE, Bolam JP, Wilson CJ. Regulation of the timing and pattern of action potential generation in rat subthalamic neurons in vitro by GABA_A IPSPs. J Neurophysiol 87: 1348–1362, 2002.[Abstract/Free Full Text]

Bevan MD, Wilson CJ. Mechanisms underlying spontaneous oscillation and rhythmic firing in rat subthalamic neurons. J Neurosci 19: 7617–7628, 1999.[Abstract/Free Full Text]

Blatz AL, Magleby KL. Single apamin-blocked Ca-activated K⁺ channels of small conductance in cultured rat skeletal muscle. Nature 323: 718–720, 1986.[CrossRef][Medline]

Bloodgood BL, Sabatini BL. Nonlinear regulation of unitary synaptic signals by CaV(2.3) voltage-sensitive calcium channels located in dendritic spines. Neuron 53: 249–260, 2007.[CrossRef][Medline]

Bowden SE, Fletcher S, Loane DJ, Marrion NV. Somatic colocalization of rat SK1 and D class (Ca(v)1.2) L-type calcium channels in rat CA1 hippocampal pyramidal neurons. J Neurosci 21: RC 175 (1–6), 2001.

Bristow LJ, Cook GP, Patel S, Curtis N, Mawer I, Kulagowski JJ. Discriminative stimulus properties of the putative dopamine D3 receptor agonist, (+)-PD128907: role of presynaptic dopamine D2 autoreceptors. Neuropharmacology 37: 793–802, 1998.[CrossRef][Web of Science][Medline]

Brown DA, Griffith WH. Persistent slow inward calcium current in voltage-clamped hippocampal neurones of the guinea-pig. J Physiol 337: 303–320, 1983.[Abstract/Free Full Text]

Brown P, Oliviero A, Mazzone P, Insola A, Tonali P, Di Lazzaro V. Dopamine dependency of oscillations between subthalamic nucleus and pallidum in Parkinson's disease. J Neurosci 21: 1033–1038, 2001.[Abstract/Free Full Text]

Budde T, Meuth S, Pape HC. Calcium-dependent inactivation of neuronal calcium channels. Nat Rev Neurosci 3: 873–883, 2002.[CrossRef][Web of Science][Medline]

Carabelli V, Lovallo M, Magnelli V, Zucker H, Carbone E. Voltage dependent modulation of single N-type Ca²⁺ channel kinetics by receptor agonists in IMR32 cells. Biophys J 70: 2144–2154, 1996.[Web of Science][Medline]

Chergui K, Akaoka H, Charlety PJ, Saunier CF, Buda M, Chouvet G. Subthalamic nucleus modulates burst firing of nigral dopamine neurons via NMDA receptors. Neuroreport 5: 1185–1188, 1994.[Web of Science][Medline]

Cossette M, Lévesque M, Parent A. Extrastriatal dopaminergic innervation of human basal ganglia. Neurosci Res 34: 51–54, 1999.[CrossRef][Web of Science][Medline]

Cragg SJ, Baufreton J, Xue Y, Bolam JP, Bevan MD. Synaptic release of dopamine in the subthalamic nucleus. Eur J Neurosci 20: 1788–1802, 2004.[CrossRef][Web of Science][Medline]

Davies PJ, Ireland DR, McLachlan EM. Sources of Ca²⁺ for different Ca(²⁺)-activated K⁺ conductances in neurones of the rat superior cervical ganglion. J Physiol 495: 353–366, 1996.[Abstract/Free Full Text]

DeLong MR. Primate models of movement disorders of basal ganglia origin. Trends Neurosci 13: 281–285, 1990.[CrossRef][Web of Science][Medline]

Do MT, Bean BP. Subthreshold sodium currents and pacemaking of subthalamic neurons: modulation by slow inactivation. Neuron 39: 109–120, 2003.[CrossRef][Web of Science][Medline]

Dolphin AC. G protein modulation of voltage-gated calcium channels. Pharmacol Rev 55: 607–627, 2003.[Abstract/Free Full Text]

Filion M. Effects of interruption of the nigrostriatal pathway and of dopaminergic agents on the spontaneous activity of globus pallidus neurons in the awake monkey. Brain Res 178: 425–441, 1979.[CrossRef][Web of Science][Medline]

Flores G, Liang JJ, Sierra A, Martinez-Fong D, Quirion R, Aceves J, Srivastava LK. Expression of dopamine receptors in the subthalamic nucleus of the rat: characterization using reverse transcriptase-polymerase chain reaction and autoradiography. Neuroscience 91: 549–556, 1999.[CrossRef][Web of Science][Medline]

Fogelson N, Pogosyan A, Kühn AA, Kupsch A, van Bruggen G, Speelman H, Tijssen M, Quartarone A, Insola A, Mazzone P, Di Lazzaro V, Limousin P, Brown P. Reciprocal interactions between oscillatory activities of different frequencies in the subthalamic region of patients with Parkinson's disease. Eur J Neurosci 22: 257–266, 2005.[CrossRef][Web of Science][Medline]

François C, Savy C, Jan C, Tande D, Hirsch EC, Yelnik J. Dopaminergic innervation of the subthalamic nucleus in the normal state, in MPTP-treated monkeys, and in Parkinson's disease patients. J Comp Neurol 425: 121–129, 2000.[CrossRef][Web of Science][Medline]

Fujimoto K, Kita H. Response characteristics of subthalamic neurons to the stimulation of the sensorimotor cortex in the rat. Brain Res 609: 185–192, 1993.[CrossRef][Web of Science][Medline]

Galvez A, Gimenez-Gallego G, Reuben JP, Roy-Contancin L, Feigenbaum P, Kaczorowski GJ, Garcia ML. Purification and characterization of a unique, potent, peptidyl probe for the high conductance calcium-activated potassium channel from venom of the scorpion Buthus tamulus. J Biol Chem 265: 11083–11090, 1990.[Abstract/Free Full Text]

Gillies A, Willshaw D. Membrane channel interactions underlying rat subthalamic projection neuron rhythmic and bursting activity. J Neurophysiol 95: 2352–2365, 2006.[Abstract/Free Full Text]

Glase SA, Akunne HC, Georgic LM, Heffner TG, MacKenzie RG, Manley PJ, Pugsley TA, Wise LD. Substituted [(4-phenylpiperazinyl)-methyl]benzamides: selective dopamine D4 agonists. J Med Chem 40: 1771–1772, 1997.[CrossRef][Web of Science][Medline]

Hallworth NE, Bevan MD. Globus pallidus neurons dynamically regulate the activity pattern of subthalamic nucleus neurons through the frequency-dependent activation of postsynaptic GABA_A and GABA_B receptors. J Neurosci 25: 6304–6315, 2005.[Abstract/Free Full Text]

Hallworth NE, Wilson CJ, Bevan MD. Apamin-sensitive small conductance calcium-activated potassium channels, through their selective coupling to voltage-gated calcium channels, are critical determinants of the precision, pace, and pattern of action potential generation in rat subthalamic nucleus neurons in vitro. J Neurosci 23: 7525–7542, 2003.[Abstract/Free Full Text]

Hamani C, Neimat J, Lozano AM. Deep brain stimulation for the treatment of Parkinson's disease. J Neural Transm Suppl 70: 393–399, 2006.[CrossRef][Medline]

Hassani OK, François C, Yelnik J, Féger J. Evidence for a dopaminergic innervation of the subthalamic nucleus in the rat. Brain Res 749: 88–94, 1997.[CrossRef][Web of Science][Medline]

Heimer G, Bar-Gad I, Goldberg JA, Bergman H. Dopamine replacement therapy reverses abnormal synchronization of pallidal neurons in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine primate model of parkinsonism. J Neurosci 22: 7850–7855, 2002.[Abstract/Free Full Text]

Helmchen F, Imoto K, Sakmann B. Ca²⁺ buffering and action potential-evoked Ca²⁺ signaling in dendrites of pyramidal neurons. Biophys J 70: 1069–1081, 1996.[Web of Science][Medline]

Herlitze S, Garcia DE, Mackie K, Hille B, Scheuer T, Catterall WA. Modulation of Ca²⁺ channels by G-protein subunits. Nature 380: 258–262, 1996.[CrossRef][Medline]

Hernandez A, Ibanez-Sandoval O, Sierra A, Valdiosera R, Tapia D, Anaya V, Galarraga E, Bargas J, Aceves J. Control of the subthalamic innervation of the rat globus pallidus by D2/3 and D4 dopamine receptors. J Neurophysiol 96: 2877–2888, 2006.[Abstract/Free Full Text]

Hernandez-Lopez S, Tkatch T, Perez-Garci E, Galarraga E, Bargas J, Hamm H, Surmeier DJ. D2 dopamine receptors in striatal medium spiny neurons reduce L-type Ca²⁺ currents and excitability via a novel PLC[beta]1-IP3-calcineurin-signaling cascade. J Neurosci 20: 8987–8995, 2000.[Abstract/Free Full Text]

Hille B, Beech DJ, Bernheim L, Mathie A, Shapiro MS, Wollmuth LP. Multiple G-protein-coupled pathways inhibit N-type Ca channels of neurons. Life Sci 56: 989–992, 1995.[CrossRef][Web of Science][Medline]

Hirschberg B, Maylie J, Adelman JP, Marrion NV. Gating properties of single SK channels in hippocampal CA1 pyramidal neurons. Biophys J 77: 1905–1913, 1999.[Web of Science][Medline]

Holmes A, Lachowicz JE, Sibley DR. Phenotypic analysis of dopamine receptor knockout mice; recent insights into the functional specificity of dopamine receptor subtypes. Neuropharmacology 47: 1117–1134, 2004.[Web of Science][Medline]

Holz GG 4th, Rane SG, Dunlap K. GTP-binding proteins mediate transmitter inhibition of voltage-dependent calcium channels. Nature 319: 670–672, 1986.[CrossRef][Medline]

Hornykiewicz O. The discovery of dopamine deficiency in the parkinsonian brain. J Neural Transm Suppl 70: 9–15, 2006.[CrossRef][Medline]

Hutchison WD, Dostrovsky JO, Walters JR, Courtemanche R, Boraud T, Goldberg J, Brown P. Neuronal oscillations in the basal ganglia and movement disorders: evidence from whole animal and human recordings. J Neurosci 24: 9240–9243, 2004.[Free Full Text]

Ibanez-Sandoval O, Hernandez A, Floran B, Galarraga E, Tapia D, Valdiosera R, Erlij D, Aceves J, Bargas J. Control of the subthalamic innervation of substantia nigra pars reticulata by D1 and D2 dopamine receptors. J Neurophysiol 95: 1800–1811, 2006.[Abstract/Free Full Text]

Jenner P, Marsden CD. Multiple dopamine receptors in brain and the pharmacological action of substituted benzamide drugs. Acta Psychiatr Scand Suppl 311: 109–123, 1984.[Medline]

Joyce JN, Millan MJ. Dopamine D₃ receptor antagonists as therapeutic agents. Drug Discov Today 10: 917–925, 2005.[CrossRef][Web of Science][Medline]

Kaczorowski CC, Disterhoft J, Spruston N. Stability and plasticity of intrinsic membrane properties in hippocampal CA1 pyramidal neurons: effects of internal anions. J Physiol 578: 799–818, 2007.[Abstract/Free Full Text]

Kapur A, Yeckel MF, Gray R, Johnston D. L-Type calcium channels are required for one form of hippocampal mossy fiber LTP. J Neurophysiol 79: 2181–2190, 1998.[Abstract/Free Full Text]

Kasai H, Aosaki T. Modulation of Ca-channel current by an adenosine analog mediated by a GTP-binding protein in chick sensory neurons. Eur J Physiol 414: 145–149, 1989.[CrossRef][Web of Science][Medline]

Kass JI, Mintz IM. Silent plateau potentials, rhythmic bursts, and pacemaker firing: three patterns of activity that coexist in quadristable subthalamic neurons. Proc Natl Acad Sci USA 103: 183–188, 2006.[Abstract/Free Full Text]

Kita H, Kitai ST. Efferent projections of the subthalamic nucleus in the rat: light and electron microscopic analysis with the PHA-L method. J Comp Neurol 260: 435–452, 1987.[CrossRef][Web of Science][Medline]

Kita H, Nambu A, Kaneda K, Tachibana Y, Takada M. Role of ionotropic glutamatergic and GABAergic inputs on the firing activity of neurons in the external pallidum in awake monkeys. J Neurophysiol 92: 3069–3084, 2004.[Abstract/Free Full Text]

Kitai ST, Deniau JM. Cortical inputs to the subthalamus: intracellular analysis. Brain Res 214: 411–415, 1981.[CrossRef][Web of Science][Medline]

Kitai ST, Kita H. Anatomy and physiology of the subthalamic nucleus: a driving force of the basal ganglia. In: The Basal Ganglia II. Structure and Function: Current Concepts, edited by Carpenter MB, Jayaraman A. New York: Plenum, 1987, p. 357–373.

Kress GJ, Reynolds IJ. Dopaminergic neurotoxins require excitotoxic stimulation in organotypic cultures. Neurobiol Dis 20: 639–645, 2005.[CrossRef][Web of Science][Medline]

Kyrozis A, Reichling DB. Perforated-patch recording with gramicidin avoids artifactual changes in intracellular chloride concentration. J Neurosci Methods 57: 27–35, 1995.[CrossRef][Web of Science][Medline]

Levy R, Hutchison WD, Lozano AM, Dostrovsky JO. High-frequency synchronization of neuronal activity in the subthalamic nucleus of parkinsonian patients with limb tremor. J Neurosci 20: 7766–7775, 2000.[Abstract/Free Full Text]

Levy R, Hutchison WD, Lozano AM, Dostrovsky JO. Synchronized neuronal discharge in the basal ganglia of parkinsonian patients is limited to oscillatory activity. J Neurosci 22: 2855–2861, 2002.[Abstract/Free Full Text]

Levy R, Lang AE, Dostrovsky JO, Pahapill P, Romas J, Saint-Cyr J, Hutchison WD, Lozano AM. Lidocaine and muscimol microinjections in subthalamic nucleus reverse parkinsonian symptoms. Brain 124: 2105–2118, 2001.[Abstract/Free Full Text]

Lipscombe D, Kongsamut S, Tsien RW. Adrenergic inhibition of sympathetic neurotransmitter release mediated by modulation of N-type calcium channel gating. Nature 340: 639–642, 1989.[CrossRef][Medline]

Lozano AM, Lang AE, Levy R, Hutchison W, Dostrovsky J. Neuronal recordings in Parkinson's disease patients with dyskinesias induced by apomorphine. Ann Neurol 47: S141–S146, 2000.[CrossRef][Web of Science][Medline]

Magill PJ, Bolam JP, Bevan MD. Relationship of activity in the subthalamic nucleus-globus pallidus network to cortical electroencephalogram. J Neurosci 20: 820–833, 2000.[Abstract/Free Full Text]

Marrion NV, Tavalin SJ. Selective activation of Ca²⁺-activated K⁺ channels by co-localized Ca²⁺ channels in hippocampal neurons. Nature 395: 900–905, 1998.[CrossRef][Medline]

Marty A, Neher E. Potassium channels in cultured bovine adrenal chromaffin cells. J Physiol 367: 117–141, 1985.[Abstract/Free Full Text]

Maylie J, Bond CT, Herson PS, Lee WS, Adelman JP. Small conductance Ca²⁺-activated K⁺ channels and calmodulin. J Physiol 554: 255–261, 2004.[Abstract/Free Full Text]

Mintz I, Hammond C, Féger J. Excitatory effect of iontophoretically applied dopamine on identified neurons of the rat subthalamic nucleus. Brain Res 375: 172–175, 1986.[CrossRef][Web of Science][Medline]

Momiyama T, Koga E. Dopamine D(2)-like receptors selectively block N-type Ca(2+) channels to reduce GABA release onto rat striatal cholinergic interneurons. J Physiol 533: 479–492, 2001.[Abstract/Free Full Text]

Mouroux M, Hassani OK, Féger J. Electrophysiological study of the excitatory parafascicular projection to the subthalamic nucleus and evidence for ipsi- and contralateral controls. Neuroscience 67: 399–407, 1995.[CrossRef][Web of Science][Medline]

Müller-Schweinitzer E, Neumann P. In vitro effects of calcium antagonists PN 200–110, nifedipine, and nimodipine on human and canine cerebral arteries. J Cereb Blood Flow Metab 3: 344–361, 1983.

Myers VB, Haydon DA. Ion transfer across lipid membranes in the presence of gramicidin A. II. The ion selectivity. Biochim Biophys Acta 274: 313–322, 1972.[Medline]

Nakanishi H, Kita H, Kitai ST. An N-methyl-D-aspartate receptor mediated excitatory postsynaptic potential evoked in subthalamic neurons in an in vitro slice preparation of the rat. Neurosci Lett 95: 130–136, 1988.[CrossRef][Web of Science][Medline]

Neher E, Augustine GJ. Calcium gradients and buffers in bovine chromaffin cells. J Physiol 450: 273–301, 1992.[Abstract/Free Full Text]

Otsuka T, Murakami F, Song WJ. Excitatory postsynaptic potentials trigger a plateau potential in rat subthalamic neurons at hyperpolarized states. J Neurophysiol 86: 1816–1825, 2001.[Abstract/Free Full Text]

Overton PG, Greenfield SA. Determinants of neuronal firing pattern in the guinea-pig subthalamic nucleus: an in vivo and in vitro comparison. J Neural Transm Park Dis Dement Sect 10: 41–54, 1995.[CrossRef][Web of Science][Medline]

Pineda JC, Waters RS, Foehring RC. Specificity in the interaction of HVA Ca²⁺ channel types with Ca²⁺-dependent AHPs and firing behavior in neocortical pyramidal neurons. J Neurophysiol 79: 2522–2534, 1998.[Abstract/Free Full Text]

Price CJ, Pittman QJ. Dopamine D4 receptor activation inhibits presynaptically glutamatergic neurotransmission in the rat supraoptic nucleus. J Neurophysiol 86: 1149–1155, 2001.[Abstract/Free Full Text]

Remy S, Spruston N. Dendritic spikes induce single-burst long-term potentiation. Proc Natl Acad Sci USA 104: 17192–17197, 2007.[Abstract/Free Full Text]

Reynolds IJ, Wagner JA, Snyder SH, Thayer SA, Olivera BM, Miller RJ. Brain voltage-sensitive calcium channel subtypes differentiated by omega-conotoxin fraction GVIA. Proc Natl Acad Sci USA 83: 8804–8807, 1986.[Abstract/Free Full Text]

Rosanova M, Ulrich D. Pattern-specific associative long-term potentiation induced by a sleep spindle-related spike train. J Neurosci 25: 9398–9405, 2005.[Abstract/Free Full Text]

Sanchez-Vives MV, McCormick DA. Cellular and network mechanisms of rhythmic recurrent activity in neocortex. Nat Neurosci 3: 1027–1034, 2000.[CrossRef][Web of Science][Medline]

Schultz W. Getting formal with dopamine and reward. Neuron 36: 241–263, 2002.[CrossRef][Web of Science][Medline]

Shapiro MS, Wollmuth LP, Hille B. Modulation of Ca²⁺ channels by PTX-sensitive G-proteins is blocked by N-ethylmaleimide in rat sympathetic neurons. J Neurosci 14: 7109–7116, 1994.[Abstract]

Shen KZ, Johnson SW. Presynaptic dopamine D2 and muscarine M3 receptors inhibit excitatory and inhibitory transmission to rat subthalamic neurones in vitro. J Physiol 525: 331–341, 2000.[Abstract/Free Full Text]

Shin RM, Masuda M, Miura M, Sano H, Shirasawa T, Song WJ, Kobayashi K, Aosaki T. Dopamine D4 receptor-induced postsynaptic inhibition of GABAergic currents in mouse globus pallidus neurons. J Neurosci 23: 11662–11672, 2003.[Abstract/Free Full Text]

Song WJ, Baba Y, Otsuka T, Murakami F. Characterization of Ca(2+) channels in rat subthalamic nucleus neurons. J Neurophysiol 84: 2630–2637, 2000.[Abstract/Free Full Text]

Stemp G, Ashmeade T, Branch CL, Hadley MS, Hunter AJ, Johnson CN, Nash DJ, Thewlis KM, Vong AKK, Austin NE, Jeffrey P, Avenell KY, Boyfield I, Hagan JJ, Middlemiss DN, Reavill C, Riley GJ, Routledge C, Wood M. Design and synthesis of trans-N-[4-[2-(6-cyano-1,2,3,4-tetrahydroisoquinolin-2-yl)ethyl]cyclohexyl]-4-quinolinecarboxamide (SB-277011): a potent and selective dopamine D3 receptor antagonist with high oral bioavailability and CNS penetration in the rat. J Med Chem 43: 1878–1885, 2000.[CrossRef][Web of Science][Medline]

Surmeier DJ, Song WJ, Yan Z. Coordinated expression of dopamine receptors in neostriatal medium spiny neurons. J Neurosci 16: 6579–6591, 1996.[Abstract/Free Full Text]

Svensson E, Wikstrom MA, Hill RH, Grillner S. Endogenous and exogenous dopamine presynaptically inhibits glutamatergic reticulospinal transmission via an action of D2-receptors on N-type Ca²⁺ channels. Eur J Neurosci 17: 447–454, 2003.[CrossRef][Web of Science][Medline]

Tedford HW, Zamponi GW. Direct G protein modulation of Ca_v2 calcium channels. Pharmacol Rev 58: 837–836, 2006.[Abstract/Free Full Text]

Tkatch T, Baranauskas G, Surmeier DJ. K_v4.2 mRNA abundance and A-type K(+) current amplitude are linearly related in basal ganglia and basal forebrain neurons. J Neurosci 20: 579–588, 2000.[Abstract/Free Full Text]

Tofighy A, Abbott A, Centonze D, Cooper AJ, Noor E, Pearce SM, Puntis M, Stanford IM, Wigmore MA, Lacey MG. Excitation by dopamine of rat subthalamic nucleus neurones in vitro—a direct action with unconventional pharmacology. Neuroscience 116: 157–166, 2003.[CrossRef][Web of Science][Medline]

Tsuruta K, Frey EA, Grewe CW, Cote TE, Eskay RL, Kebabian JW. Evidence that LY-141865 specifically stimulates the D-2 dopamine receptor. Nature 292: 463–465, 1981.[CrossRef][Medline]

Velumian AA, Carlen PL. Differential control of three after-hyperpolarizations in rat hippocampal neurons by intracellular calcium buffering. J Physiol 517: 201–216, 1999.[Abstract/Free Full Text]

Velumian AA, Zhang L, Pennefather P, Carlen PL. Reversible inhibition of IK, IAHP, Ih and ICa currents by internally applied gluconate in rat hippocampal pyramidal neurons. Pfluegers Arch 433: 343–350, 1997.[CrossRef][Web of Science][Medline]

Wang X, Zhong P, Yan Z. Dopamine D4 receptors modulate GABAergic signaling in pyramidal neurons of prefrontal cortex. J Neurosci 22: 9185–9193, 2002.[Abstract/Free Full Text]

Wichmann T, Bergman H, DeLong MR. The primate subthalamic nucleus. I. Functional properties in intact animals. J Neurophysiol 72: 494–506, 1994.[Abstract/Free Full Text]

Wichmann T, Soares J. Neuronal firing before and after burst discharges in the monkey basal ganglia is predictably patterned in the normal state and altered in parkinsonism. J Neurophysiol 95: 2120–2133, 2006.[Abstract/Free Full Text]

Wigmore MA, Lacey MG. A K_v3-like persistent, outwardly rectifying, Cs⁺-permeable, K⁺ current in rat subthalamic nucleus neurons. J Physiol 527: 493–506, 2000.[Abstract/Free Full Text]

Williams D, Tijssen M, Van Bruggen G, Bosch A, Insola A, Di Lazzaro V, Mazzone P, Oliviero A, Quartarone A, Speelman H, Brown P. Dopamine-dependent changes in the functional connectivity between basal ganglia and cerebral cortex in humans. Brain 125: 1558–1569, 2002.[Abstract/Free Full Text]

Wilson CJ, Callaway JC. Coupled oscillator model of the dopaminergic neuron of the substantia nigra. J Neurophysiol 83: 3084–3100, 2000.[Abstract/Free Full Text]

Wilson CJ, Weyrick A, Terman D, Hallworth NE, Bevan MD. A model of reverse spike frequency adaptation and repetitive firing of subthalamic nucleus neurons. J Neurophysiol 91: 1963–1980, 2004.[Abstract/Free Full Text]

Wilson CL, Cash D, Galley K, Chapman H, Lacey MG, Stanford IM. Subthalamic nucleus neurones in slices from 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned mice show irregular, dopamine-reversible firing pattern changes, but without synchronous activity. Neuroscience 143: 565–572, 2006.[CrossRef][Web of Science][Medline]

Wolfart J, Roeper J. Selective coupling of T-type calcium channels to SK potassium channels prevents intrinsic bursting in dopaminergic midbrain neurons. J Neurosci 22: 3404–3413, 2002.[Abstract/Free Full Text]

Womack MD, Chevez C, Khodakhah K. Calcium-activated potassium channels are selectively coupled to P/Q-type calcium channels in cerebellar Purkinje neurons. J Neurosci 24: 8818–8822, 2004.[Abstract/Free Full Text]

Yan Z, Song WJ, Surmeier J. D2 dopamine receptors reduce N-type Ca²⁺ currents in rat neostriatal cholinergic interneurons through a membrane-delimited, protein-kinase-C-insensitive pathway. J Neurophysiol 77: 1003–1015, 1997.[Abstract/Free Full Text]

Zamponi GW, Snutch TP. Decay of prepulse facilitation of N type calcium channels during G protein inhibition is consistent with binding of a single Gbeta subunit. Proc Natl Acad Sci USA 95: 4035–4039, 1998.[Abstract/Free Full Text]

Zhang L, Pennefather P, Velumian A, Tymianski M, Charlton M, Carlen PL. Potentiation of a slow Ca²⁺-dependent K⁺ current by intracellular Ca²⁺ chelators in hippocampal CA1 neurons of rat brain slices. J Neurophysiol 74: 2225–2241, 1995.[Abstract/Free Full Text]

Zhang L, Weiner JL, Valiante TA, Velumian AA, Watson PL, Jahromi SS, Schertzer S, Pennefather P, Carlen PL. Whole-cell recording of the Ca²⁺-dependent slow afterhyperpolarization in hippocampal neurons: effects of internally applied anions. Pfluegers Arch 426: 247–253, 1994.[CrossRef][Web of Science][Medline]

Zhou J, Shapiro MS, Hille B. Speed of Ca²⁺ channel modulation by neurotransmitters in rat sympathetic neurons. J Neurophysiol 77: 2040–2048, 1997.[Abstract/Free Full Text]

Zhu Z, Bartol M, Shen K, Johnson SW. Excitatory effects of dopamine on subthalamic nucleus neurons: in vitro study of rats pretreated with 6- hydroxydopamine and levodopa. Brain Res 945: 31–40, 2002b.[CrossRef][Web of Science][Medline]

Zhu ZT, Shen KZ, Johnson SW. Pharmacological identification of inward current evoked by dopamine in rat subthalamic neurons in vitro. Neuropharmacology 42: 772–781, 2002a.[CrossRef][Web of Science][Medline]

This article has been cited by other articles:

				C. A. Deister, C. S. Chan, D. J. Surmeier, and C. J. Wilson Calcium-Activated SK Channels Influence Voltage-Gated Ion Channels to Determine the Precision of Firing in Globus Pallidus Neurons J. Neurosci., July 1, 2009; 29(26): 8452 - 8461. [Abstract] [Full Text] [PDF]

				J. Baufreton and M. D. Bevan D2-like dopamine receptor-mediated modulation of activity-dependent plasticity at GABAergic synapses in the subthalamic nucleus J. Physiol., April 15, 2008; 586(8): 2121 - 2142. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Supplemental Figures All Versions of this Article: 99/2/442    most recent 00998.2007v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (1) Citing Articles via Google Scholar Google Scholar Articles by Ramanathan, S. Articles by Bevan, M. D. Search for Related Content PubMed PubMed Citation Articles by Ramanathan, S. Articles by Bevan, M. D.