J Neurophysiol 110: 2203–2216, 2013. First published August 14, 2013; doi:10.1152/jn.00161.2013.

Supersensitive presynaptic dopamine D2 receptor inhibition of the striatopallidal projection in nigrostriatal dopamine-deficient mice Wei Wei,1,2 Li Li,1 Guoliang Yu,1 Shengyuan Ding,1 Chengyao Li,2* and Fu-Ming Zhou1* 1

Department of Pharmacology, College of Medicine, University of Tennessee Health Science Center, Memphis, Tennessee; and 2School of Biotechnology, Southern Medical University, Guangzhou, China Submitted 5 March 2013; accepted in final form 14 August 2013

Wei W, Li L, Yu G, Ding S, Li C, Zhou FM. Supersensitive presynaptic dopamine D2 receptor inhibition of the striatopallidal projection in nigrostriatal dopamine-deficient mice. J Neurophysiol 110: 2203–2216, 2013. First published August 14, 2013; doi:10.1152/jn.00161.2013.—The dopamine (DA) D2 receptor (D2R)-expressing medium spiny neurons (D2MSNs) in the striatum project to and inhibit the GABAergic neurons in the globus pallidus (GP), forming an important link in the indirect pathway of the basal ganglia movement control circuit. These striatopallidal axon terminals express presynaptic D2Rs that inhibit GABA release and thus regulate basal ganglion function. Here we show that in transcription factor Pitx3 gene mutant mice with a severe DA loss in the dorsal striatum mimicking the DA denervation in Parkinson’s disease (PD), the striatopallidal GABAergic synaptic transmission displayed a heightened sensitivity to presynaptic D2R-mediated inhibition with the dose-response curve shifted to the left, although the maximal inhibition was not changed. Functionally, low concentrations of DA were able to more efficaciously reduce the striatopallidal inhibition-induced pauses of GP neuron activity in DA-deficient Pitx3 mutant mice than in wild-type mice. These results demonstrate that presynaptic D2R inhibition of the striatopallidal synapse becomes supersensitized after DA loss. These supersensitive D2Rs may compensate for the lost DA in PD and also induce a strong disinhibition of GP neuron activity that may contribute to the motor-stimulating effects of dopaminergic treatments in PD. basal ganglia; dopamine receptor; globus pallidus; presynaptic inhibition; Parkinson’s disease

is being increasingly appreciated as an important component of the basal ganglia motor control circuit in health and disease (Kita and Kita 2011a; Kravitz et al. 2010; Nambu et al. 2011). Studies have established that the GABAergic, phasically active medium spiny neurons (MSNs) expressing a high level of dopamine (DA) D2 receptors (D2Rs) project to and inhibit the globus pallidus (GP) in rodents or the external segment of the GP (GPe) in primates (Gerfen and Bolam 2010; Sano et al. 2013). Most GP neurons are GABAergic projection neurons and fire spikes spontaneously (Benhamou et al. 2012; Kita 2010; Soares et al. 2004; Starr et al. 2005). Classic and recent studies indicate that the activity of pallidal neurons is critical to the function and dysfunction of the basal ganglia (Bateup et al. 2010; DeLong 1990; Kravitz et al. 2010; Raz et al. 2000). In Parkinson’s disease (PD), GPe neuron activity may be decreased with increased burstiness (Kita and Kita 2011a, 2011b; Soares et al. 2004; Wichmann and Dostrovsky 2011). Anatomical studies

THE STRIATOPALLIDAL PROJECTION

* C. Li and F.-M. Zhou contributed equally to this study. Address for reprint requests and other correspondence: F.-M. Zhou, Dept. of Pharmacology, Univ. of Tennessee College of Medicine, Memphis, TN 38163 (e-mail: [email protected]). www.jn.org

have established that D2R-expressing MSNs (D2-MSNs) innervate GP neurons (Bolam et al. 2000; Fujiyama et al. 2011; Gerfen and Bolam 2010; Kita 2010; Lévesque and Parent 2005). Ultrastructural studies have demonstrated that striatopallidal axon terminals express D2Rs (Levey et al. 1993; Smith and Villalba 2008; Yung et al. 1995). Functionally, these presynaptic D2Rs inhibit GABA release from striatopallidal axon terminals (Chuhma et al. 2011; Cooper and Stanford 2001; Watanabe et al. 2009). Matching the strong D2R expression, the striatum receives an intense DA innervation originating in the DA neurons in the substantia nigra (Björklund and Lindvall 1984; Matsuda et al. 2009). The GPe (in primates) or GP (in rodents) also receives a DA innervation from axon collaterals of the nigrostriatal DA projection and dedicated nigropallidal DA neurons, although the pallidal DA innervation is much smaller than that in the striatum (Hedreen 1999; Jan et al. 2000; Smith and Kieval 2000). In postmortem late-stage PD brains, DA loss was up to 80% in GPe (Hornykiewicz 2001; Rajput et al. 2008). In comparison, the DA loss was 98% in the putamen. Severe DA loss can induce upregulated or supersensitive molecular and behavioral responses upon reintroduction of DA agonists (Creese et al. 1977; Schwarting and Huston 1996; Staunton et al. 1982; Ungerstedt 1971). A prominent receptor that is upregulated by DA loss in PD is the D2R (Hornykiewicz 2001). Studies indicate that the upregulation may result from an increased D2R expression and also an increased functionality such as higher D2R binding affinity and more efficient receptor-G protein coupling and downstream signaling (Betarbet and Greenamyre 2004; Cai et al. 2002; Geurts et al. 1999; Kostrzewa 1995; Qin et al. 1994; Seeman et al. 2005a). Supersensitive DA receptors may compensate for the lost DA (Bezard et al. 2010) and may be even the primary mediators of the therapeutic effects of dopaminergic treatments for PD (Seeman 2007; Seeman et al. 2005b). Despite the clear importance, the potential D2R supersensitivity at the striatopallidal axon terminals has not been studied and the functional consequences also remain unknown. We hypothesize that presynaptic D2Rs at the striatopallidal axon terminals are supersensitive after DA depletion, and during dopaminergic treatment these supersensitive presynaptic D2Rs may disinhibit GP neurons more potently in DA-deficient mice than in normal mice. We have tested these ideas in transcription factor Pitx3-mutant mice that have a severe and consistent nigral DA neuron loss (Li et al. 2013; Li and Zhou 2013; Nunes et al. 2003; van den Munckhof et al. 2003), a feature that is helpful to our experimental questions.

0022-3077/13 Copyright © 2013 the American Physiological Society

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MATERIALS AND METHODS

Animals. Two breeding pairs of heterozygous Pitx3⫹/⫺ mice were purchased from the Jackson Laboratory (Bar Harbor, ME), resulting in a small colony of homozygous Pitx3⫺/⫺ (Pitx3Null), heterozygous Pitx3⫹/⫺, and wild-type Pitx3⫹/⫹ (Pitx3WT) mice (Li et al. 2013; Li and Zhou 2013). Pitx3Null mice are aphakic and thus clearly identified (Fig. 1). The genotypes were further determined by PCRbased genotyping to identify WT, homozygotes, and heterozygotes (Li et al. 2013). The genotyping results are identical to our published results (Li et al. 2013). Since the Pitx3 gene is recessive, the nigrostriatal DA system is normal in heterozygous Pitx3⫹/⫺ mice (Hwang et al. 2003; Nunes et al. 2003; van den Munckhof et al. 2003). To avoid any potential ambiguity in data interpretation and also to avoid genotyping, heterozygous Pitx3⫹/⫺ mice were not used; only Pitx3WT mice (produced by mating Pitx3WT mice) and Pitx3Null mice (produced by mating Pitx3Null mice) were used in our experiments. Mice had free access to food and water. All procedures were approved by the Institutional Animal Care and Use Committee of the University of Tennessee Health Science Center in Memphis, TN. Reserpine treatment. Pitx3WT mice at postnatal 18 days of age received injections of reserpine according to published protocols (Day et al. 2006; Ding et al. 2006; Kreitzer and Malenka 2007; Taverna et al. 2008; Trugman and James 1992). Reserpine (5 mg) was first dissolved in glacial acetic acid (0.2 ml) and then mixed with a 1.8-ml mix of 2 parts (in volume) polyethylene glycol (PEG)-400 and 1 part Tween 20 (i.e., 2 ml vehicle). The reserpine dose was 2.5 mg/kg injected in 0.01 ml once per day in the morning about 10 AM. The mice started to display severe akinesia after the third reserpine injection. Two hours after the fourth reserpine injection, the mice were killed for in vitro electrophysiological experiments. Brain slice preparation. Parasagittal slices were prepared according to methods described in the literature (Beurrier et al. 2006; Ding et al. 2013). Mice of postnatal days 20 –24 were used in our experiments because brain slices from these mice at this age were highly viable and no age-dependent changes in cellular properties during these days were detected. These mice were killed by decapitation, and

brains were dissected out quickly and immediately immersed in an oxygenated ice-cold cutting solution (in mM: 220 glycerol, 2.5 KCl, 1.25 NaH2PO4, 25 NaHCO3, 0.5 CaCl2, 7 MgCl2, 20 D-glucose) for 2 min. Three hundred-micrometer-thick, 15° angular parasagittal slices were cut with a Leica Zero Z VT1200S vibratome (Leica Microsystems, Wetzlar, Germany). The parasagittal slices were transferred to a standard extracellular bathing solution (in mM: 125 NaCl, 2.5 KCl, 25 NaHCO3, 1.25 NaH2PO4, 2.5 CaCl2, 1.3 MgCl2, and 10 D-glucose) that was continuously bubbled with 95% O2-5% CO2 for 30 min at 34°C in a holding chamber. After that, the holding chamber with the brain slices was kept at room temperature (25°C) for at least 30 min. Brain slices were used 1– 6 h after being prepared. Ascorbic acid (vitamin C, 0.4 mM) was added to all solutions including the cutting solution to protect the tissue and DA and to be consistent (Rice 1999). Fresh DA was added to the perfusing solution at desired concentrations immediately before use, and this DA-containing solution was used only for one DA application that lasted for 10 min. Synaptic stimulation. Conventional electrical stimulation was used to evoke inhibitory postsynaptic currents (IPSCs) or inhibitory postsynaptic potentials (IPSPs). A bipolar tungsten stimulating electrode (World Precision Instruments) was placed in the dorsal striatum. Stimulating pulses were controlled by a pulse generator (Master-8; AMPI) and delivered via a stimulus isolator (A365; World Precision Instruments). A single stimulating pulse or a pair of pulses was delivered every 20 s to evoke IPSCs or IPSPs. The stimulation intensity ranged from 20 to 100 ␮A, with a constant duration of 0.2 ms, and was adjusted to evoke roughly 70% of the maximal response. Electrophysiology. Slices were placed in a recording chamber and continuously perfused at 2 ml/min with the normal extracellular bathing solution saturated with 95% O2-5% CO2. Recordings were made under visual guidance of a video microscope (Olympus BX51WI and Zeiss Axiocam MRm digital camera) equipped with Nomarski optics and a ⫻60 water immersion lens. A Multiclamp 700B amplifier, pCLAMP 9.2 software, and Digidata 1322A interface (Molecular Devices, Sunnyvale, CA) were used to acquire data. Patch pipettes were pulled from borosilicate glass capillary tubing (KG-33,

Fig. 1. Visual identification of Pitx3⫹/⫹ (Pitx3WT) mice and Pitx3⫺/⫺ mutant (Pitx3Null) mice. Pitx3WT (A) and Pitx3Null (B) mice look identical except that Pitx3Null mice have malformed eyes that clearly identify Pitx3Null mice on or after postnatal day 14 when Pitx3WT and Pitx3⫺/⫹ mice open their eyes. These 2 example mice were 35 days old. A’ and B’ show that compared with the Pitx3WT mouse the eye of the Pitx3Null mouse (both mice were 30 days old) is smaller, with no clearly identifiable lens or cornea. Scale bar in B’ applies to A’. However, Pitx3Null mice eat well, are fertile, and do not need any special care.

J Neurophysiol • doi:10.1152/jn.00161.2013 • www.jn.org

SUPERSENSITIVE D2 RECEPTORS ON STRIATOPALLIDAL TERMINALS

1.1-mm ID, 1.65-mm OD; King Precision Glass, Claremont, CA) with a PC-10 puller (Narishige, Tokyo, Japan) and had resistances of ⬃2 M⍀ when filled with one of the following intracellular solutions. A low-Cl⫺ intracellular solution (in mM: 135 KSO3CH3, 5 KCl, 0.5 EGTA, 10 HEPES, 2 Mg-ATP, 0.2 Na-GTP, and 4 Na2-phosphocreatine, pH 7.25, 280 –290 mosM) was used for recording hyperpolarizing GABAA IPSPs and spiking activity. A high-Cl⫺ intracellular solution (in mM: 135 KCl, 0.5 EGTA, 10 HEPES, 2 Mg-ATP, 0.2 Na-GTP, and 4 Na2-phosphocreatine, pH 7.25, 280 –290 mosM) was used for recording inward GABAA IPSCs at ⫺70 mV. Series resistance was monitored by 10-mV, 50-ms pulses. The series resistance was 4 – 8 M⍀ among different cells, and the cells were discarded when series resistance increase was ⬎15%. The 8-mV liquid junction potential detected for the KSO3CH3-based intracellular solution used in the current-clamp experiments was subtracted from the membrane potential values presented in this report, whereas the 4-mV liquid junction potential for the KCl-based intracellular solution used in the voltage-clamp experiments was not corrected because its impact on the IPSCs at ⫺70 mV was minimal. Signals were filtered at 10 kHz with the built-in four-pole low-pass Bessel filter in the patch-clamp amplifier and digitized at 20 kHz. All recordings were made at 30 –32°C. At least five mice were used to obtain an averaged electrophysiological data point, with each mouse yielding one or two useful cells, depending on how difficult the experiment was. Immunohistochemistry. Conventional immunofluorescence methods were used to detect DA neurons and their axons according to our published procedures (Li et al. 2013; Li and Zhou 2013; Zhou et al. 2009). The brains were fixed in 4% paraformaldehyde dissolved in a phosphate buffer at 4°C overnight and then sectioned on a vibratome. The free-floating sections (50 ␮m in thickness) were incubated with 2% fat-free milk, 1% bovine serum albumin, and 0.4% Triton X-100 in a phosphate-buffered saline (PBS) for 1 h at room temperature to block nonspecific binding and permeabilize the cell membrane, respectively. After thorough rinsing, the free-floating sections were incubated for 48 h at 4°C with the primary antibody, a polyclonal tyrosine hydroxylase (TH) antibody raised in rabbit (diluted at 1:1,000; Novus Biologicals, Littleton, CO), and then rinsed in the PBS, followed by incubation with a donkey anti-rabbit secondary antibody conjugated with Alexa Fluor 488 (diluted at 1:200; Invitrogen) for 3 h at room temperature. Fluorescence images were acquired on a Zeiss 710 confocal laser scanning microscope. High-pressure liquid chromatography quantification of tissue DA contents. Brain slices (0.5 mm in thickness) containing the striatum and GP were cut in the same manner as the brain slices for electrophysiology. Immediately after cutting, an individual brain slice was placed on a block of agar gel partially submerged in the ice-cold cutting solution. With a glass pipette (outer diameter: 1.0 mm, inner diameter: 0.8 mm), tissue punches were obtained in the dorsal, middle, and ventral striatum and the dorsal GP. Tissue punches were collected into 1:50 diluted 70% perchloric acid (final concentration: 0.33 M) and fully homogenized with a Tissue Tearor. After 3-min centrifugation at 12,000 g and 4°C, the supernatant was aspirated and stored at ⫺80°C. For high-pressure liquid chromatography (HPLC) analysis, chromatographic separation of DA was achieved by using a 150 ⫻ 2-mm ODS C18 column (ESA, Chelmsford, MA) connected to an ESA 582 HPLC pump. The mobile phase was perfused at 0.2 ml/min and contained 80 mM NaH2PO4·H2O, 2.0 mM 1-octanesulfonic acid sodium salt, 100 ␮l/l triethylamine, 5 nM EDTA, and 10% acetonitrile (pH 3.0). The samples (20 ␮l) were automatically injected onto the column by an ESA 542 autosampler and were analyzed by an ESA Coulochem II 5200A electrochemical detector with an ESA 5041 high-sensitivity analytical cell. Electrochemical detection was performed at a potential of 220 mV with the current gain at 1.0 nA. Serially diluted DA standards containing known quantities were included in each assay. Under these conditions, the limit of detection for DA was 0.5 pg/injection.

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Chemicals and drugs. Routine chemicals were purchased from Sigma-Aldrich (St. Louis, MO). 6,7-Dinitroquinoxaline-2,3-dione (DNQX), 2-amino-5-phosphonopentanoic acid (AP5), picrotoxin, DA, reserpine, quinpirole, SKF81297 hydrobromide, and sulpiride were purchased from Sigma-Aldrich or Tocris. SKF81297 hydrobromide was also supplied by the Drug Supply Program of the National Institute of Mental Health, National Institutes of Health. Statistics. Data are expressed as means ⫾ SE. The paired Student’s t-test was used to compare changes before and after drug administration within the same group. The unpaired t-test, one-way ANOVA with post hoc Tukey test, and two-way ANOVA with post hoc least significant difference (LSD) test were used to compare the measurements among different groups. P ⬍ 0.05 was considered statistically significant. Calculations were performed with the IBM SPSS Statistics 21 program. RESULTS

Characterization of intrinsic membrane properties of GP neurons in DA-deficient Pitx3Null mice. Anatomical studies have established that D2-MSNs in the dorsal striatum project to the dorsal GP (Fujiyama et al. 2011; Hedreen and DeLong 1991; Lévesque and Parent 2005; Nambu 2011; Selemon and Goldman-Rakic 1990). Since our main goal was to determine whether the presynaptic D2Rs at the striatopallidal synapse are supersensitive, we chose to use Pitx3Null mice based on the fact that these mice have a consistent and severe loss in their substantia nigra pars compacta (SNc) DA neurons that normally innervate the GP and the dorsal striatum (Fig. 2). Since the DA loss is the severest in the dorsal striatum, with ⬃98% DA lost (Fig. 2C), as reported in published studies (Nunes et al. 2003; van den Munckhof et al. 2003), and DA receptor supersensitization is positively correlated to the severity of DA loss (Schwarting and Huston 1996), we focused on the striatopallidal projection from the dorsal striatum to the dorsal GP. The anatomical location of the dorsal striatum and dorsal GP were reliably identified in our angular sagittal brain slices (Fig. 3A). We first characterized the intrinsic membrane and action potential properties of dorsal GP neurons (Fig. 3, A and B). As illustrated in Fig. 3, C–G, and Table 1, injection of current pulses revealed that GP neurons in both Pitx3WT and Pitx3Null mice had widely distributed intrinsic and action potential properties, consistent with literature reports on the variation of GP neuron membrane properties (e.g., Deister et al. 2013; Günay et al. 2008). However, scatterplots of spike duration and input resistance placed our recorded GP neurons into two clusters or groups (Fig. 3C, Table 1). Type A GP neurons had a higher input resistance, a longer membrane time constant, and a longer spike duration than type B GP neurons (Fig. 3C, Table 1), consistent with the original characterization of Cooper and Stanford (2000). Equally important, for both cell types these parameters of intrinsic membrane and spike properties were similar in Pitx3WT mice and Pitx3Null mice (Table 1). Additionally, we also examined the Ih-mediated, hyperpolarization-induced depolarization sag in GP neurons. As illustrated in Fig. 3, D–G, the sag was detected in both type A and type B GP neurons in Pitx3WT mice and Pitx3Null mice. At a trough membrane potential about ⫺100 mV, the sag amplitude was larger in type A cells than in type B cells (9 mV vs. 5 mV, P ⬍ 0.01, unpaired t-test; Table 1). Similar values for the sag were obtained in type A cells and type B cells in Pitx3Null mice (Fig. 3, D–G, Table 1). These results indicate that loss of

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Fig. 2. The dopamine (DA) loss pattern in Pitx3Null mice. A and B: tyrosine hydroxylase (TH) immunofluorescent stain reveals intense DA innervation in the dorsal striatum (A1), modest DA innervation in the dorsal globus pallidus (GP) (A2), and densely packed DA neurons in the substantia nigra pars compacta (SNc) and in the ventral tegmental area (VTA) (A3) in WT mice. A1–A3 show the DA innervation in the dorsal striatum and the dorsal GP and DA neurons in the SNc and VTA DA neurons. Their counterparts in Pitx3Null mice are shown in B1–B3. These pictures are raw, unprocessed confocal images obtained at identical acquisition parameters. Note that the dorsal striatum is largely devoid of DA fibers (B1). Scale bar in A1 applies to A2, B1, and B2; scale bar in A3 applies to B3. SNr, substantia nigra pars reticulata. C: HPLC quantification of tissue DA content in dorsal (1.01 ⫾ 0.08 ␮mol/g wet tissue in Pitx3WT vs. 0.02 ⫾ 0.00 ␮mol/g in Pitx3Null), middle (0.93 ⫾ 0.10 ␮mol/g in Pitx3WT vs. 0.09 ⫾ 0.02 ␮mol/g in Pitx3Null), and ventral (0.84 ⫾ 0.11 ␮mol/g in Pitx3WT vs. 0.34 ⫾ 0.07 ␮mol/g in Pitx3Null) striatum and dorsal GP (0.07 ⫾ 0.01 ␮mol/g in Pitx3WT vs. 0.03 ⫾ 0.01 ␮mol/g in Pitx3Null) [all P ⬍ 0.01, 2-way ANOVA and least significant difference (LSD) post hoc test; 5 mice for each genotype]. Note that in Pitx3WT mice the DA content in the dorsal GP is only 6.5% of that in the dorsal striatum and the DA content in the dorsal GP in Pitx3Null mice is reduced to 45% of that in Pitx3WT mice. The DA loss is smaller in GP compared with the dorsal striatum at least partially because of the residual DA fibers of passage en route to the middle striatum. Inset C1 illustrates the locations for the dorsal striatum punch (D), the middle striatum punch (M), and the ventral striatum punch (V). Asterisk indicates anterior commissure. CC, corpus callosum.

the nigral DA system does not alter the intrinsic membrane properties of neurons in the dorsal GP. These results also pave the way for us to study the potential abnormalities in striatopallidal synaptic transmission. Characterization of striatopallidal IPSCs in DA-deficient Pitx3Null mice. Because our main goal was to determine whether the presynaptic D2Rs are supersensitive and because DA loss is severest in the dorsal striatum in Pitx3Null mice, we next focused on the dorsal striatum-evoked striatopallidal IPSCs in dorsal GP neurons. As indicated in Fig. 3, A and B, we placed the stimulating electrode in the dorsal striatum and recorded striatopallidal IPSCs in dorsal GP neurons in the presence of 10 ␮M DNQX and 20 ␮M D-AP5 to block ionotropic glutamate receptors. These IPSCs in Pitx3WT and

Pitx3Null mice were also blocked by 100 ␮M picrotoxin, confirming that they were GABAA receptor-mediated IPSCs. Additionally, under our experimental conditions the dorsal striatum-evoked IPSCs were facilitating (described below) in the majority of dorsal GP neurons and were thus predominantly striatopallidal IPSCs with minimal contamination from antidromically activated intra-GP recurrent IPSCs (Mallet et al. 2012; Miguelez et al. 2012; Voorn 2010). In ⬃20% of GP neurons, the striatum-evoked IPSCs were depressing or flat when two 20-Hz stimuli were used, indicating a significant contamination by antidromically activated intra-GP recurrent IPSCs (Miguelez et al. 2012). These depressing IPSCs were excluded from this report. Under our experimental conditions, the striatopallidal IPSCs were similar in type A and type B GP

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Fig. 3. Similar intrinsic membrane properties of GP neurons in Pitx3WT and DA-deficient Pitx3Null mice. A: a live sagittal brain slice showing the stimulating site in the dorsal striatum and the recording site in the dorsal GP, photographed with a ⫻4 objective. IC, internal capsule. Asterisk indicates anterior commissure. B: patch clamping of a typical GP neuron, photographed with a ⫻60 objective. C: scatterplot of input resistance and action potential base duration of type A and type B GP neurons in Pitx3WT and Pitx3Null mice. Inset shows the shorter spike duration in type B neurons. D–G: example recordings show intrinsic membrane and action potential properties of representative type A and type B GP neurons from a postnatal day (PN)20 Pitx3WT mouse (D and E) and representative type A and type B GP neurons from a PN20 Pitx3Null mouse (F and G). Ih-mediated sag is larger in these 2 type A neurons than in the 2 type B GP neurons, but no apparent difference between the genotypes is seen. See Table 1 for statistical comparisons.

neurons and thus data were pooled. Under these conditions, we found that the striatopallidal IPSCs had a similar 10 –90% rise time (1.2 ⫾ 0.1 ms vs. 1.2 ⫾ 0.1 ms) and decay time (␶ ⫽ 5.5 ⫾ 0.2 ms vs. ␶ ⫽ 5.4 ⫾ 0.2 ms, P ⬎ 0.05, unpaired t-test) in 20 GP neurons in Pitx3WT mice and in 20 GP neurons in Pitx3Null mice, consistent with the kinetic parameters of the striatopallidal IPSCs in the literature (Sims et al. 2008). We next examined the release properties of striatopallidal axon terminals by comparing the paired-pulse ratios (PPRs) of the striatopallidal IPSCs in dorsal GP neurons in Pitx3WT and Pitx3Null mice (Fig. 4). Since MSNs in freely moving WT and Pitx3Null mice may either be silent or fire at low frequencies (⬃1 Hz) or at bursty high frequencies (5– 40 Hz) (Hernandez et al. 2013; Miller et al. 2008; Sagot B and Zhou FM, unpublished data), we used paired pulses of 1, 2, 5, 10, 20, and 40 Hz. We found that the PPR was ⬃1 for paired pulses

separated by 1 or 0.5 s, indicating that low-frequency firing of MSNs at 1–2 Hz induces nonfacilitating, independent IPSCs in GP neurons in Pitx3WT and Pitx3Null mice. When the interval between the two pulses shortened to 200 ms, the paired IPSCs became facilitating, with the PPR increasing to ⬃1.4 in Pitx3WT and Pitx3Null mice (Fig. 4C). The maximal PPR was ⬃1.7, achieved with paired pulses separated by 50 ms, in Pitx3WT and Pitx3Null mice (Fig. 4). The PPR was ⬃1.5 for paired pulses separated by 25 ms in Pitx3WT and Pitx3Null mice (Fig. 4). These results indicate that MSN firing at 5– 40 Hz, the possible phasic firing frequencies for MSNs, may cause intensifying inhibition of GP neuron activity. There was no difference in the PPRs between Pitx3WT and Pitx3Null mice (Fig. 4C). These results show that the baseline properties of these striatopallidal axon terminals are similar in Pitx3WT and Pitx3Null mice, setting the stage for our main question:

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Table 1. Intrinsic membrane properties of GP neurons in Pitx3WT and Pitx3Null mice

Mouse Type Pitx3WT Pitx3WT Pitx3Null Pitx3Null

Input Resistance, M⍀

Membrane Time Constant, ms

AP Base Duration, ms

AP Amplitude, mV

AP Threshold, mV

fAHP Amplitude, mV

Ih Sag, mV (trough potential, mV)

Type A 373.8 ⫾ 28.7 (166.3–705.8) n ⫽ 37 Type B 167.2 ⫾ 10.5 (81.9–253.6) n ⫽ 26 Type A 377.6 ⫾ 24.7 (170.4–720.9) n ⫽ 35 Type B 171.7 ⫾ 10.4 (96.8–286.5) n ⫽ 30

37.4 ⫾ 2.3 (14.2–66.9) n ⫽ 37 20.2 ⫾ 1.8 (14.6–33.8) n ⫽ 26 37.2 ⫾ 2.5 (15.2–73.8) n ⫽ 35 21.1 ⫾ 1.3 (12.0–33.3) n ⫽ 30

1.34 ⫾ 0.06 (0.92–1.86) n ⫽ 37 0.73 ⫾ 0.01 (0.61–0.89) n ⫽ 26 1.38 ⫾ 0.04 (0.95–1.83) n ⫽ 35 0.72 ⫾ 0.02 (0.51–0.88) n ⫽ 30

70.5 ⫾ 1.8 (54.0–91.0) n ⫽ 37 71.4 ⫾ 1.8 (53.3–89.5) n ⫽ 26 70.8 ⫾ 1.4 (58.6–90.9) n ⫽ 35 71.2 ⫾ 1.2 (53.3–90.7) n ⫽ 30

⫺41.1 ⫾ 0.7 (⫺34.2 to ⫺50.0) n ⫽ 37 ⫺43.3 ⫾ 1.1 (⫺31.6 to ⫺58.4) n ⫽ 26 ⫺40.2 ⫾ 0.7 (⫺35.2 to ⫺51.4) n ⫽ 35 ⫺43.4 ⫾ 0.8 (⫺38.1 to ⫺56.1) n ⫽ 30

⫺17.4 ⫾ 0.7 (⫺11.0 to ⫺24.6) n ⫽ 37 ⫺21.5 ⫾ 0.8 (⫺16.4 to ⫺30.5) n ⫽ 26 ⫺17.6 ⫾ 0.6 (⫺10.8 to ⫺24.5) n ⫽ 35 ⫺21.1 ⫾ 0.7 (⫺16.5 to ⫺34.8) n ⫽ 30

8.9 ⫾ 0.7 (n ⫽ 25) (at ⫺100.1 ⫹ 0.9) (6.1–14.0) 4.8 ⫾ 0.6 (n ⫽ 11) (at ⫺97.7 ⫹ 0.9) (2.7–7.7) 8.8 ⫾ 0.6 (n ⫽ 31) (at ⫺100.1 ⫹ 0.9) (6.4–12.5) 4.9 ⫾ 0.5 (n ⫽ 14) (at ⫺98.8 ⫹ 1.2) (3.0–6.9)

P ⬎ 0.05 P ⬎ 0.05

P ⬎ 0.05 P ⬎ 0.05

P ⬎ 0.05 P ⬎ 0.05

P ⬎ 0.05 P ⬎ 0.05

P ⬎ 0.05 P ⬎ 0.05

P ⬎ 0.05 P ⬎ 0.05

P ⬎ 0.05 P ⬎ 0.05

P ⬍ 0.001 P ⬍ 0.001

P ⬍ 0.05 P ⬍ 0.05

P ⬍ 0.001 P ⬍ 0.001

P ⬎ 0.05 P ⬎ 0.05

P ⬎ 0.05 P ⬎ 0.05

P ⬍ 0.05 P ⬍ 0.05

P ⬍ 0.01 P ⬍ 0.01

Cell Type

Pitx3WT vs. Pitx3Null Type A Type B Type A vs. type B WT Homo

Values are means ⫾ SE for n cells. Values in parentheses indicate ranges of individual values. GP, globus pallidus; AP, action potential; fAHP, fast afterhyperpolarization; WT, wild type; Homo, Pitx3Null. P values are from unpaired t-tests.

is the presynaptic DA inhibition of striatopallidal IPSCs supersensitive? Low doses of DA induce stronger presynaptic inhibition of striatopallidal IPSCs in DA-deficient Pitx3Null mice than in Pitx3WT mice. First, we tested the effects of low doses of DA to determine whether DA is more efficacious in inhibiting striatopallidal IPSCs in DA-deficient mice than in Pitx3WT mice. At 0.1 and 0.5 ␮M, bath-applied DA had no significant effect on striatopallidal IPSCs in seven GP cells in Pitx3WT mice but significantly reduced the amplitude of striatopallidal IPSCs in Pitx3Null mice by 13.4 ⫾ 2.9% (n ⫽ 7 cells) and 40.9 ⫾ 4.8% (n ⫽ 7 cells), respectively. At 1 ␮M, DA reduced the peak amplitude of the striatopallidal IPSCs by 15.2 ⫾ 1.7% (n ⫽ 7 cells) in Pitx3WT mice and by 63.8 ⫾ 6.8% (n ⫽ 7 cells) in Pitx3Null mice (Fig. 5, A and D). At 3 ␮M, DA reduced the striatopallidal IPSC by 33.2 ⫾ 3.4% (n ⫽ 7 cells) in Pitx3WT mice and 68.3 ⫾ 7.2% (n ⫽ 7 cells) in Pitx3Null mice. At 5 ␮M, DA reduced the striatopallidal IPSC by 48.4 ⫾ 2.3% (n ⫽ 8 cells) in Pitx3WT mice and 72.2 ⫾ 1.5% (n ⫽ 7 cells) in Pitx3Null mice (Fig. 5, B and E). However, at 10, 20, and 50 ␮M, DA had a similar effect, reducing the peak amplitude of the striatopallidal IPSCs by ⬃72% in both Pitx3WT and Pitx3Null mice and indicating that DA had reached a saturating dose at 10 ␮M (Fig. 5, C, F, and G). The DA-induced inhibition was recovered upon washout. Two-way

ANOVA indicates a significant difference in the inhibitory DA effect on the striatopallidal IPSC amplitude in Pitx3Null mice and Pitx3WT mice [F(1,91) ⫽78.3, P ⬍ 0.001]. Post hoc LSD tests confirm that the DA effect was significantly stronger in Pitx3Null mice than in Pitx3WT mice (P ⬍ 0.001 for 1 and 3 ␮M DA and P ⬍ 0.05 for 5 ␮M DA), but the effect was not significantly different for DA concentrations ⱖ10 ␮M in the two types of mice (P ⬎ 0.01). As illustrated in Fig. 5G, fitting the data points to the Hill equation showed that while the maximal effect was similar, the IC50 was 3.22 ␮M for Pitx3WT mice and 0.36 ␮M for Pitx3Null mice, indicating a substantial increase in the efficacy of DA or supersensitivity in mediating inhibition of striatopallidal IPSCs in Pitx3Null mice. The Pitx3Null mouse offers a convenient mouse model to study the consequences of DA loss. However, its early loss of DA is different from the DA denervation in PD. To support our idea that DA loss is sufficient to induce supersensitive DA inhibition of the striatopallidal IPSCs, we treated Pitx3WT mice with a reserpine regimen (2.5 mg/kg, 1 injection/day) that has been widely used to deplete DA and induce akinesia (Day et al. 2006; Ding et al. 2006; Kreitzer and Malenka 2007; Taverna et al. 2008; Trugman and James 1992). The mice started to display severe akinesia after 3 days of reserpine treatment. Two hours after the fourth reserpine injection, the

Fig. 4. Similar baseline properties of striatopallidal inhibitory postsynaptic currents (IPSCs) in GP neurons in Pitx3WT and DA-deficient Pitx3Null mice. A and B: example striatopallidal IPSCs evoked by a pair of stimuli (P1 and P2) spaced 50 ms apart in a GP neuron in a Pitx3WT mouse (A) and a Pitx3Null mouse (B). Each trace is an average of 10 sweeps. C: summary of paired-pulse experiments at 1, 5, 10, 20, and 40 Hz in GP neurons in Pitx3WT and Pitx3Null mice. Holding potential was ⫺70 mV. There was no difference in the paired-pulse ratio (PPR) at each of these frequencies between Pitx3WT and Pitx3Null mice (P ⬎ 0.05, unpaired t-test). J Neurophysiol • doi:10.1152/jn.00161.2013 • www.jn.org

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sufficient to induce enhanced or supersensitive DA-mediated inhibition of the striatopallidal IPSCs. DA increases paired-pulse ratios of striatopallidal IPSCs in Pitx3WT mice and Pitx3Null mice. Striatopallidal axon terminals express D2Rs (Levey et al. 1993; Smith and Villalba 2008; Yung et al. 1995), providing a firm anatomical foundation for presynaptic D2R-mediated inhibition. It was also reported, however, that GP neurons may express D2-like receptors such as D4Rs that may reduce IPSCs postsynaptically (Shin et al. 2003), although this postsynaptic D2-like effect was not observed in other studies (Cooper and Stanford 2001; Miguelez et al. 2012). To determine whether the DA inhibition of striatopallidal IPSCs described above was presynaptic or postsynaptic in origin, we performed paired-pulse experiments. It is established that presynaptic inhibition of neurotransmitter release may increase the PPR whereas postsynaptic inhibition or enhancement of GABAA receptor function generally does not alter the PPR (Fioravante and Regehr 2011; Thomson 2000, 2003; Zucker and Regehr 2002). As shown in Fig. 7, during saturating 50 ␮M DA application the PPR of the striatopallidal IPSCs, evoked by a pair of stimuli spaced at 50 ms or 20 Hz, increased from 1.65 ⫾ 0.25 to 3.30 ⫾ 0.39 in seven GP neurons in Pitx3WT mice and from 1.56 ⫾ 0.15 to 3.03 ⫾ 0.36 in six GP neurons in Pitx3Null mice (paired t-test, P ⬍ 0.05 for both genotypes). These results indicate a presynaptic D2R-mediated reduction in GABA release. To further support this conclusion, we calculated the coefficient of variation (CV) of the evoked striatopallidal IPSCs by dividing the standard deviation by the mean of the 10 or more individual IPSCs, as described in Michaeli and Yaka (2010). Under control conditions, the baseline CV was 0.47 ⫾ 0.07 in Pitx3WT mice (n ⫽ 12) and 0.43 ⫾ 0.05 in Pitx3Null mice (n ⫽ 12). During 50 ␮M DA, the CV was 0.89 ⫾ 0.11 in Pitx3WT mice and 0.90 ⫾ 0.08 in Pitx3Null mice. The increase in CV was significant for each genotype with P ⬍ 0.05 (paired t-test), indicating a decreased vesicular release probability during 50 ␮M DA. D2R Agonist Quinpirole Mimics DA’s Inhibitory Effects on Striatopallidal IPSCs in Pitx3WT and Pitx3Null Mice Fig. 5. Low concentrations of DA reduce striatopallidal IPSCs more efficaciously in DA-deficient Pitx3Null mice than in WT mice. A–C: examples of the inhibitory effect of 1, 5, and 50 ␮M DA on striatopallidal IPSCs in Pitx3WT mice. D–F: examples of the inhibitory effect of 1, 5, and 50 ␮M DA on striatopallidal IPSCs in Pitx3Null mice. G: pooled data showing different dose-response relationships of DA inhibition of striatopallidal IPSCs in Pitx3WT mice and Pitx3Null (PitxHomo) mice. n ⫽ 7– 8 cells for each data point. Holding potential was ⫺70 mV. The 2 continuous lines are the fits to the Hill equation: y ⫽ A ⫻ xnH[1/(KnH ⫹ xnH)], where A is the maximal inhibition, x is the DA concentration, K is the IC50, and nH is the Hill number.

mice were killed for in vitro electrophysiological experiments. As shown in Fig. 6, at 1 ␮M bath-applied DA reduced the peak amplitude of the striatopallidal IPSC by 62.2 ⫾ 5.1% (n ⫽ 7 cells, paired t-test, P ⬍ 0.01) in reserpine-treated Pitx3WT mice and by only 15.6 ⫾ 1.9% (n ⫽ 7 cells, paired t-test, P ⬍ 0.01) in vehicle-treated Pitx3WT mice. Unpaired t-test indicated that the DA effect was significantly stronger in reserpinetreated Pitx3WT mice than in vehicle-treated Pitx3WT mice (P ⬍ 0.001). These results indicate that DA depletion is

Since it is firmly established that striatopallidal MSNs express only or predominantly D2Rs (Gerfen and Surmeier 2011), we did the following three experiments to confirm that it was the D2Rs that were mediating the heightened presynaptic DA inhibition of striatopallidal IPSCs in the DA-deficient Pitx3Null mice. First, as shown in Fig. 8, A and B, bath application of D2-like receptor agonist quinpirole mimicked the effect of DA. At 1 ␮M, quinpirole reduced the peak amplitude of the striatopallidal IPSCs by 39.67 ⫾ 3.1% in Pitx3Null mice (n ⫽ 9 cells, paired t-test, P ⬍ 0.01) and by 11.6 ⫾ 1.6% in Pitx3WT mice (n ⫽ 7 cells, paired t-test, P ⬍ 0.05). At 10 ␮M, quinpirole reduced the peak amplitude of the striatopallidal IPSCs by 63.0 ⫾ 3.8% (n ⫽ 6 cells, paired t-test, P ⬍ 0.01) in Pitx3Null mice and 56.3 ⫾ 2.2% (n ⫽ 6 cells, paired t-test, P ⬍ 0.01) in Pitx3WT mice. The effects of quinpirole were recovered upon washing. Two-way ANOVA indicates a significant difference in the inhibitory quinpirole effect on the striatopallidal IPSC amplitude in Pitx3Null mice and Pitx3WT mice [F(1,24) ⫽36.118, P ⬍ 0.001]. Post hoc LSD

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Fig. 6. DA reduces striatopallidal IPSCs more efficaciously in reserpine-treated WT mice. A: averaged traces of striatopallidal IPSCs before, during, and after bath application of 1 ␮M DA in vehicle-treated Pitx3WT mice. B: averaged traces of striatopallidal IPSCs before, during, and after bath application of 1 ␮M DA in reserpine-treated Pitx3WT mice. C: pooled data showing 1 ␮M DA effects on striatopallidal IPSCs in vehicle- and reserpine-treated Pitx3WT mice.

tests confirm that the effect of 1 ␮M quinpirole effect was stronger in Pitx3Null mice than in Pitx3WT mice (P ⬍ 0.05), but the effect of 10 ␮M quinpirole was not significantly different in the two types of mice. Second, bath application of 10 ␮M D1-like agonist SKF81297 had no detectable effect on striatopallidal IPSCs in Pitx3WT mice (6 neurons) or Pitx3Null mice (6 neurons) (Fig. 8D). Third, in the presence of 10 ␮M sulpiride, a D2-like antagonist, 10 ␮M DA did not inhibit the striatopallidal IPSCs in five cells in WT mice and five cells in Pitx3Null mice (Fig. 8D). (Sulpiride did not cause a detectable effect on the baseline striatopallidal IPSCs. This is likely due to the lack of sufficient ambient extracellular DA in the GPe in a 300-␮m-thick brain slice preparation, where the modest amount of DA may dissipate easily.) These results together with the paired-pulse data indicate that DA inhibited the peak amplitudes of the striatopallidal IPSCs in both Pitx3WT mice

Fig. 7. DA increases PPR of striatopallidal IPSCs in Pitx3WT and Pitx3Null mice. A: averaged traces of striatopallidal paired IPSCs (evoked by 2 stimuli 50 ms apart) before and during bath application of saturating 50 ␮M DA in Pitx3WT mice. These 2 traces were then scaled to the peak of their 1st IPSC to show the relative increase of the 2nd IPSC peak. B: summary showing a clear DA-induced increase in PPR in Pitx3WT mice. C: averaged traces of striatopallidal paired IPSCs (evoked by 2 stimuli 50 ms apart) before and during bath application of saturating 50 ␮M DA in Pitx3Null mice. These 2 traces were then scaled to the peak of their 1st IPSC to show the relative increase of the 2nd IPSC peak. D: summary showing a clear DA-induced increase in PPR in Pitx3Null mice. Holding potential was ⫺70 mV for all cells. *P ⬍ 0.05, paired t-test.

and Pitx3Null mice through the D2Rs at the striatopallidal axon terminals. DA disinhibits GP neuron firing more efficaciously in DAdeficient Pitx3Null mice than in Pitx3WT mice. Striatopallidal GABAergic inputs induce pauses in the spontaneous firing in GP neurons (Elias et al. 2007; Kita and Kita 2011a, 2011b; Sani et al. 2009). We reasoned that the supersensitive presynaptic D2R inhibition of the striatopallidal terminals in Pitx3Null mice may enable low concentrations of DA to more effectively reduce the pausing effect on GP neuron firing. To test this possibility, we evoked striatopallidal IPSPs that caused pauses in the spontaneous firing in GP neurons both in Pitx3WT mice and Pitx3Null mice (Fig. 9, A1 and B1). A KSO3CH3-based pipette solution was used such that the GABAA IPSPs were hyperpolarizing. To quantify the DA effect, We measured the duration of the firing pause induced by the striatopallidal IPSPs. Here the pause duration was defined as the time window between the stimulus artifact and the first spike in each sweep (Fig. 9, A1 and B1). We found that bath application of 1 ␮M DA reduced the pause duration by 10.7 ⫾ 0.8% (n ⫽ 6 GP neurons, P ⬍ 0.05, paired t-test) [from control duration of 250.8 ⫾ 41.7 ms (range: 394 –140 ms)] in Pitx3WT mice but by 32.7 ⫾ 3.3% (n ⫽ 6 GP neurons, P ⬍ 0.05, paired t-test) [from control duration of 244.5 ⫾ 34.3 ms (range: 371–132 ms)] in Pitx3Null mice (Fig. 9C). Increasing DA to 3 ␮M reduced the pause duration by 25.4 ⫾ 2.9% (n ⫽ 9 GP cells, paired t-test, P ⬍ 0.05) [from control duration of 239.9 ⫾ 24.5 ms (range: 387–158 ms)] in Pitx3WT mice and by 54.7 ⫾ 4.8% (n ⫽ 6 GP cells, paired t-test, P ⬍ 0.01) [from control duration of 273.4 ⫾ 38.0 ms (range: 372–150 ms)] in Pitx3Null mice (Fig. 9). When saturating 50 ␮M DA was bath applied, the pause duration was reduced by 57.1 ⫾ 5.1% (n ⫽ 10 GP cells, paired t-test, P ⬍ 0.01) [from control duration of 225.1 ⫾ 20.7 ms (range: 341–109 ms)] in Pitx3WT mice and 56.6 ⫾ 3.8% (n ⫽ 10 GP cells, paired t-test, P ⬍ 0.01) [from control duration of 201.1 ⫾ 19.9 ms (range: 315–108 ms)] in Pitx3Null mice (Fig. 9C). Two-way ANOVA indicates a significant difference in the DA effect on striatopallidal IPSP-induced pause duration in GP neuron firing in Pitx3WT mice and Pitx3Null mice [F(1,41) ⫽ 28.067, P ⬍ 0.001]. Post hoc LSD tests confirm that at 1 and 3 ␮M the DA effect was significantly stronger in Pitx3Null mice than in Pitx3WT mice (P ⬍ 0.001), whereas there was no significant difference at 50 ␮M (P ⬎ 0.05). These results indicate that low concentrations of DA were more efficacious in reducing the pausing effect of the striatopallidal IPSP in the DA-deficient Pitx3Null mice, although the maximal inhibition under saturating doses of DA was similar, consistent with our data in the preceding sections showing that the D2Rs on the striatopallidal axon terminals were supersensitive to DA with a leftward shift in the doseresponse curve in Pitx3Null mice (Fig. 5).

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Pitx3Null mice are a suitable model to study DA receptor supersensitivity. In this study, we used Pitx3Null mice that have a severe and consistent loss of the nigral DA neurons and hence the nigrostriatal DA projection and other nigra-originated DA innervation (Hwang et al. 2003; Li et al. 2013; Li and Zhou 2013). The DA neuron loss occurs early in Pitx3Null mice such that the majority of the nigral DA neurons are lost when the animal is born, even though a substantial number of DA neurons in the ventral tegmental area survive into adulthood (Nunes et al. 2003; Smidt et al. 2004; van den Munckhof et al. 2003). It is known that the expression levels of D1Rs and D2Rs in MSNs start to increase rapidly in the first two postnatal weeks (Jung and Bennett 1996; Rao et al. 1991; Schambra et al. 1994). Consequently, most of the D1Rs and D2Rs in the dorsal striatum in Pitx3Null mice have little or no exposure to DA. This raises a concern as to the suitability of Pitx3Null mice as a model to study DA receptor supersensitivity. We argue that a lack of prior exposure to DA does not affect the supersensitization of DA receptors for the following reasons. First, DA depletion by reserpine treatment in normal mice led to a similar supersensitive DA inhibition of the striatopallidal IPSCs. Second, literature evidence and our prior work have shown that Pitx3Null mice have the behavioral and molecular hallmarks of typical DA denervation supersensitivity (Ding et al. 2007; Hwang et al. 2005; Li et al. 2013; Li and Zhou 2013; van den Munckhof et al. 2006). Third, studies have indicated that prior DA innervation is not required for DA

Fig. 8. D2-like agonist quinpirole mimics the DA effects on striatopallidal IPSCs in Pitx3Null mice. A: averaged traces of striatopallidal IPSCs before, during, and after bath application of 1 (A1) and 10 (A2) ␮M quinpirole in Pitx3WT mice. B: averaged traces of striatopallidal IPSCs before, during, and after bath application of 1 (B1) and 10 (B2) ␮M quinpirole in Pitx3Null mice. C: pooled data showing that 1 ␮M quinpirole reduced the striatopallidal IPSCs more strongly in Pitx3Null mice than in Pitx3WT mice but the effects of 10 ␮M quinpirole were not significantly different in the 2 genotypes. *P ⬍ 0.01, post hoc LSD test following 2-way ANOVA. D: pooled data showing that 10 ␮M SKF81297 had no effect on the striatopallidal IPSCs in Pitx3WT or Pitx3Null mice. Holding potential was ⫺70 mV for all cells. DISCUSSION

The main finding of this study is that in mice with severe nigral DA neuron loss resulting from transcription factor Pitx3 gene null mutation, the D2Rs at the striatopallidal axon terminals are supersensitive, leading to reduced inhibitory output from indirect pathway MSNs and consequently reduced pausing in GP neuron activity upon introduction of D2R agonism. In the following sections, we first discuss the suitability of Pitx3 mutant mice as a model to study DA receptor supersensitivity and then discuss our main findings.

Fig. 9. Low concentrations of DA reduce the pausing effect of striatopallidal inhibitory postsynaptic potentials (IPSPs) on GP neuron spike firing more efficaciously in DA-deficient Pitx3Null mice than in WT mice. A: in normal Pitx3WT mice, striatopallidal IPSPs induced a pause in the spontaneous firing in GP neurons (A1) that was reduced by bath application of 3 ␮M DA (A2). B: in DA-deficient Pitx3Null mice, striatopallidal IPSPs induced a pause in the spontaneous firing in GP neurons (B1) that was strongly reduced by bath application of 3 ␮M DA (B2). C: summary showing the effects of 1, 3, and 50 ␮M DA on the IPSP-induced pause duration in GP neuron firing in Pitx3WT mice and Pitx3Null mice. **P ⬍ 0.001, post hoc LSD test following 2-way ANOVA.

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receptor supersensitivity to develop. For example, fetal MSNs that had no or very little DA innervation developed DA receptor supersensitivity when these fetal MSNs were transplanted into the cortex or thalamus (Guerra et al. 1996). DA receptor supersensitivity also occurred in animals whose DA system was lesioned during the neonatal period before the expression of the majority of DA receptors (Kostrzewa 1995). Fourth, studies using mice with null TH gene in their DA neurons suggested that D1R and D2R supersensitivity at behavioral and molecular levels can be reversed or resensitized, depending on DA availability, regardless of prior history of DA innervation or exposure (Kim et al. 2000, 2002). These studies (Kim et al. 2000, 2002) even suggested that DA receptors expressed during the embryonic and first few postnatal days in normal animals were supersensitive, because of a lack of sufficient DA innervation, until the arrival of the massive nigrostriatal DA projection. Fifth, the induction of normal movements and dyskinesias by the first dose of L-DOPA in young children with TH deficiency (Pons et al. 2013) also indicates that the intensity of DA loss, not age or repeated use of L-DOPA, is critical to the supersensitive DA response. For these reasons, it is reasonable to conclude that the DA receptor supersensitivity caused by an early loss of DA in Pitx3Null mice is similar or identical to the DA supersensitivity induced by a DA loss at a later point of the animal’s life. Therefore, Pitx3Null mice are suitable for studying the cellular neurophysiological aspects of DA denervation supersensitivity. In fact, the premade and consistent DA loss in Pitx3Null mice that are viable without the need for any special care is a highly convenient feature to study cellular experimental questions that usually require a large number of animals (Ding et al. 2011; present study). Nigral DA neuron loss does not alter GP neuron intrinsic membrane properties in Pitx3Null mice. We detected the Ih-mediated depolarizing sag in both type A and type B GP neurons, although the amplitude of the sag was larger in type A neurons than in type B neurons. This is in partial agreement with Cooper and Stanford (2000), who reported that type A GP neurons had a strong Ih sag whereas type B neurons had no Ih, although Fig. 5A of that paper shows a clear Ih sag in a type B GP neuron. We also found that the severe loss of nigral DA neurons in Pitx3Null mice did not induce any detectable change in the Ih sag or other intrinsic properties such as input resistance in GP neurons, indicating that Ih amplitude was not affected in GP neurons in the DA-deficient Pitx3Null mice. This is in contrast with Chan et al. (2011), who reported that DA depletion with 6-OHDA or reserpine reduced Ih. Despite this discrepancy, our data are solid and also entirely possible for the following two reasons. First, even though the GP receives a dopaminergic input from SNc DA neurons (Hedreen 1999; Prensa and Parent 2001; Smith and Kieval 2000), this DA innervation is very small compared with the massive DA innervation in the striatum in both rodents and primates including humans (Hornykiewicz 2001; Rajput et al. 2008). Furthermore, a significant portion of the modest DA content in the GP clearly belongs to the DA fibers of passage en route to the striatum (Jan et al. 2000), leaving the DA fibers truly innervating GP even more sparse. Second, the expression of DA receptors in GP neurons is very low compared with the intense DA receptor expression in the striatum (Levey et al. 1993; Mansour et al. 1990; Yung et al. 1995). These two anatomical

facts suggest that it is entirely possible that DA loss in the GP may not induce any detectable effect in the intrinsic properties of GP neurons simply because the DA innervation and DA receptor expression are too low. In contrast, the drastic loss of the intense DA innervation in the striatum and strong D2R expression in striatopallidal neurons provide a rich substrate for DA loss to induce robust homeostatic responses. Indeed, increased D2R functionality (due to increased receptor expression and signaling efficiency) at the somatodendritic area, after severe DA loss, is an established observation (Betarbet and Greenamyre 2004; Cai et al. 2002; Geurts et al. 1999; Graham et al. 1990a, 1990b; Kostrzewa 1995; Mandel et al. 1993; Newman-Tancredi et al. 2001; Qin et al. 1994). These changes may spread to the axon terminals of D2-MSNs, leading to the increased functionality of D2Rs at the striatopallidal axon terminals. Presynaptic D2R inhibition of striatopallidal synapse is supersensitive in DA-deficient mice. DA is known to reduce the striatopallidal synaptic transmission through the inhibitory presynaptic D2Rs (Chuhma et al. 2011; Cooper and Stanford 2001; Kim and Kita 2013; Watanabe et al. 2009). As the D2R-expressing striatopallidal MSNs need to be inhibited for the basal ganglia motor circuit to function properly (Sano et al. 2013), these presynaptic D2Rs serve as the final layer of negative control on these indirect pathway MSNs. Despite the apparent importance, the effect of DA loss on these D2Rs at the striatopallidal axon terminals was not known. A recent study indicated that 6-OHDA-induced DA loss did not alter the baseline striatopallidal synaptic transmission but did not examine whether DA sensitivity was altered (Miguelez et al. 2012). In our present study, we found that in the DA-deficient Pitx3Null mice the baseline striatopallidal synaptic transmission was identical to that in Pitx3WT mice, consistent with Miguelez et al. (2012); however, the presynaptic DA inhibition of the striatopallidal synapse was much more efficacious in Pitx3Null mice (IC50 ⬃ 0.35 ␮M) than in Pitx3WT mice (IC50 ⬃ 3 ␮M). Thus, to our knowledge, these data provide the first evidence that the presynaptic D2Rs at the striatopallidal axon terminals become supersensitive after a severe loss of nigral DA neurons. This upregulation of D2R functionality at the striatopallidal axon terminal may result from the loss of DA innervation in both striatum and GP. First, a loss of the massive DA innervation in the striatum, coupled with the strong expression of D2Rs in D2-MSNs, may induce substantial homeostatic responses in D2-MSN somata that are likely to spread to axon terminals. Second, the loss of DA in GP may further increase the presynaptic D2R supersensitivity at the striatopallidal axon terminals. The normal DA innervation in GP is low (see the discussion in the preceding section). Therefore, even a moderate DA loss in the GP may reduce the synaptic DA level to a DA-deficient state, contributing to the supersensitization of D2Rs at the striatopallidal axon terminals. Presynaptic D2Rs inhibit GABA release from D2-MSNs in normal animals (Delgado et al. 2000; Geldwert et al. 2006; Guzmán et al. 2003; Kohnomi et al. 2012; Salgado et al. 2005; Tecuapetla et al. 2007, 2009). Ca2⫹ imaging studies in MSNs in the striatum indicate that D2Rs inhibited the action potential-induced Ca2⫹ signal in the axon terminals (Mizuno et al. 2007; Wu et al. 2006), Ca2⫹ currents in the somatodendritic area, and the Ca2⫹ signal in the dendritic spines (Hernandez-Lopez et al. 2000; Higley and Sabatini 2010; Olson et al. 2005; Rakhilin et al.

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2004; Salgado et al. 2005). Furthermore, it has been documented that toxin-induced DA denervation causes a supersensitive D2R inhibition of voltage-activated Ca2⫹ currents in the somatodendritic area of MSNs (Prieto et al. 2009). DA denervation also led to supersensitive DA presynaptic inhibition of GABA and glutamate release in the subthalamic nucleus (Shen et al. 2003). Therefore, the supersensitive D2R is fully capable of inhibiting GABA release from striatopallidal axon terminals in a heightened manner. Although not the subject of our present study, several mechanisms have been proposed to underlie D2R supersensitivity at D2-MSN somata, including an increased D2R expression at the cell surface and an increased functionality such as higher D2R binding affinity and more efficient receptor-G protein coupling and downstream signaling (Betarbet and Greenamyre 2004; Cai et al. 2002; Geurts et al. 1999; Kostrzewa 1995; Qin et al. 1994; Seeman et al. 2005a). How DA loss in Pitx3Null mice leads to supersensitive D2Rs on the striatopallidal axon terminals remains to be determined. However, the fact that the IC50 of DA inhibition of the striatopallidal IPSCs was decreased while the maximal inhibition remained unchanged indicates that DA receptor affinity probably was increased after DA loss, as a homeostatic response to the decreased DA availability (Seeman et al. 2005a). Supersensitive DA inhibition of inhibitory striatopallidal synapse in DA-deficient Pitx3Null mice: functional implications. Preclinical and clinical studies indicate an importance of GP neuronal activity and its temporary inhibition (pausing) in the physiology and pathophysiology of the basal ganglia and hence movement control (Kita and Kita 2011a, 2011b; Nambu et al. 2011; Obeso et al. 2008; Sano et al. 2013; Soares et al. 2004; Wichmann and Dostrovsky 2011). DA, via presynaptic D2Rs, inhibits this inhibitory effect of striatopallidal IPSPs, leading to a disinhibition of, or a smaller pausing in, GP neuron firing in normal mice. This result indicates that in normal animals the presynaptic D2Rs on striatopallidal axon terminals work, in concert with the somatic inhibitory D2Rs (West and Grace 2002), to reduce the output from D2-MSNs. Such a coordinated D2R-mediated inhibition of D2-MSNs likely facilitates movements, since inhibition or ablation of D2-MSNs is known to increase motor activity (Bateup et al. 2010; Durieux et al. 2009; Sano et al. 2003). In DA-deficient animals, owing to the supersensitive presynaptic D2Rs, low concentrations of DA may be able to disinhibit GP neurons from the striatopallidal inhibition more efficaciously than in normal animals. This supersensitive presynaptic D2R-mediated disinhibition of GP neurons has important functional implications for PD pathophysiology and dopaminergic treatments. First, it may compensate for the lower DA level in the GP in PD, thus minimizing a possible decrease in GP neuron activity under a DA-deficient or PD condition and minimizing motor deficits. Second, during treatment with L-DOPA [converted to DA by residual DA axon terminals and the rich 5-HT axon terminals in the GP (Parent et al. 2011)] or a D2-like agonist, the striatopallidal synapse may be inhibited more strongly because of the supersensitive presynaptic D2Rs. Consequently, GP neurons may also be more strongly disinhibited, potentially contributing to the motor-stimulating effects of these dopaminergic treatments.

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ACKNOWLEDGMENTS The authors thank Drs. Robert Foehring and Doug Guan for help with measuring liquid junction potentials. GRANTS This work was supported by National Institute of Neurological Disorders and Stroke Grants R01 NS-058850 and R03 NS-085380 to F.-M. Zhou. DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s). AUTHOR CONTRIBUTIONS Author contributions: W.W. and F.-M.Z. conception and design of research; W.W., L.L., G.Y., S.D., and F.-M.Z. performed experiments; W.W. and F.-M.Z. analyzed data; W.W. and F.-M.Z. interpreted results of experiments; W.W. and F.-M.Z. prepared figures; W.W. and F.-M.Z. drafted manuscript; W.W. and F.-M.Z. edited and revised manuscript; W.W., L.L., G.Y., S.D., C.L., and F.-M.Z. approved final version of manuscript. REFERENCES Bateup HS, Santini E, Shen W, Birnbaum S, Valjent E, Surmeier DJ, Fisone G, Nestler EJ, Greengard P. Distinct subclasses of medium spiny neurons differentially regulate striatal motor behaviors. Proc Natl Acad Sci USA 107: 14845–14850, 2010. Benhamou L, Bronfeld M, Bar-Gad I, Cohen D. Globus pallidus external segment neuron classification in freely moving rats: a comparison to primates. PLoS One 7: e45421, 2012. Betarbet R, Greenamyre JT. Regulation of dopamine receptor and neuropeptide expression in the basal ganglia of monkeys treated with MPTP. Exp Neurol 189: 393– 403, 2004. Beurrier C, Ben-Ari Y, Hammond C. Preservation of the direct and indirect pathways in an in vitro preparation of the mouse basal ganglia. Neuroscience 140: 77– 86, 2006. Bezard E, Porras G, Blesa J, Obeso J. Compensatory mechanisms in experimental and human parkinsonism: potential for new therapies. In: Handbook of Basal Ganglia Structure and Function, edited by Steiner H, Tseng KY. London: Academic, 2010. Björklund A, Lindvall O. Dopamine-containing systems in the CNS. In: ¨ okfelt T. Classical Transmitters in the CNS, Part I, edited by Björklund A, H Amsterdam: Elsevier, 1984. Bolam JP, Hanley JJ, Booth PA, Bevan MD. Synaptic organisation of the basal ganglia. J Anat 196: 527–542, 2000. Cai G, Wang HY, Friedman E. Increased dopamine receptor signaling and dopamine receptor-G protein coupling in denervated striatum. J Pharmacol Exp Ther 302: 1105–1112, 2002. Chan CS, Glajch KE, Gertler TS, Guzman JN, Mercer JN, Lewis AS, Goldberg AB, Tkatch T, Shigemoto R, Fleming SM, Chetkovich DM, Osten P, Kita H, Surmeier DJ. HCN channelopathy in external globus pallidus neurons in models of Parkinson’s disease. Nat Neurosci 14: 85–92, 2011. Chuhma N, Tanaka KF, Hen R, Rayport S. Functional connectome of the striatal medium spiny neuron. J Neurosci 31: 1183–1192, 2011. Cooper AJ, Stanford IM. Electrophysiological and morphological characteristics of three subtypes of rat globus pallidus neurone in vitro. J Physiol 527: 291–304, 2000. Cooper AJ, Stanford IM. Dopamine D2 receptor mediated presynaptic inhibition of striatopallidal GABAA IPSCs in vitro. Neuropharmacology 41: 62–71, 2001. Creese I, Burt DR, Snyder SH. Dopamine receptor binding enhancement accompanies lesion-induced behavioral supersensitivity. Science 197: 596 – 598, 1977. Day M, Wang Z, Ding J, An X, Ingham CA, Shering AF, Wokosin D, Ilijic E, Sun Z, Sampson AR, Mugnaini E, Deutch AY, Sesack SR, Arbuthnott GW, Surmeier DJ. Selective elimination of glutamatergic synapses on striatopallidal neurons in Parkinson disease models. Nat Neurosci 9: 251– 259, 2006. Deister CA, Dodla R, Barraza D, Kita H, Wilson CJ. Firing rate and pattern heterogeneity in the globus pallidus arise from a single neuronal population. J Neurophysiol 109: 497–506, 2013.

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Supersensitive presynaptic dopamine D2 receptor inhibition of the striatopallidal projection in nigrostriatal dopamine-deficient mice.

The dopamine (DA) D2 receptor (D2R)-expressing medium spiny neurons (D2-MSNs) in the striatum project to and inhibit the GABAergic neurons in the glob...
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