Characterization of acid-sensing ion channels in medium spiny neurons of mouse striatum

Neuroscience 162 (2009) 55– 66

CHARACTERIZATION OF ACID-SENSING ION CHANNELS IN MEDIUM SPINY NEURONS OF MOUSE STRIATUM Q. JIANG,a M.-H. LI,b C. J. PAPASIAN,a D. BRANIGAN,b Z.-G. XIONG,b J. Q. WANGa AND X.-P. CHUa*

domains are found on N- and C-termini (Waldmann et al., 1997b). Functional ASICs are a trimer complex assembled from mixtures of different subunits (Jasti et al., 2007) and most of these subunits can form into homomeric and/or heteromeric channels (Lingueglia et al., 1997; Waldmann and Lazdunski, 1998; Benson et al., 2002). ASICs are enriched in brain neurons (Alvarez de la Rosa et al., 2003; Wemmie et al., 2003), where three (ASIC1a, ASIC2a and ASIC2b) of the seven subunits can be found (Krishtal, 2003; Wemmie et al., 2006; Xiong et al., 2006, 2008). ASIC1a is the dominant subunit in brain and homomeric ASIC1a channels are permeable to both Na⫹ and Ca2⫹ ions (Waldmann et al., 1997a; Chu et al., 2002; Yermolaieva et al., 2004). ASIC1a channels localize at synapses and contribute to synaptic plasticity related to learning/memory and fear conditioning in brain (Wemmie et al., 2002, 2003). Recent studies indicate that activation of ASIC1a is involved in neuronal injury during brain ischemia (Xiong et al., 2004; Pignataro et al., 2007) and seizure (Ziemann et al., 2008). Moreover, ASIC1a channels also play critical roles in neurodegenerative diseases such as multiple sclerosis, Parkinson’s disease and Huntington’s disease (Friese et al., 2007; Arias et al., 2008; Wong et al., 2008). In contrast to homomeric ASIC1a channels, which have a pH for half-maximal activation (pH50) between 6.2 and 6.8 (Babini et al., 2002; Benson et al., 2002; Chu et al., 2002), homomeric ASIC2a channels are relatively insensitive to H⫹, with a pH50 of 4.4 (Price et al., 1996; Waldmann et al., 1996; Lingueglia et al., 1997). However, ASIC2a subunits can associate with ASIC1a to form heteromeric channels in brain (Askwith et al., 2004; Chu et al., 2004, 2006). Different from homomeric ASIC2a subunits, homomeric ASIC2b subunits do not form functional protongated channels by themselves, but may associate with other ASIC subunits to form heteromultimeric channels (Lingueglia et al., 1997; Hesselager et al., 2004). Functional studies suggest that transient global ischemia upregulates ASIC2a expression in surviving neurons (Johnson et al., 2001). Recent studies also suggest that activation of ASIC2 contributes to maintenance of retinal integrity in brain (Ettaiche et al., 2004). The electrophysiological properties and pharmacological profiles of ASICs have been extensively explored in many regions such as the cortex (Varming, 1998; Chu et al., 2004), hippocampus (Baron et al., 2002; Askwith et al., 2004), cerebellum (Allen and Attwell, 2002), retinal ganglion (Lilley et al., 2004), spinal cord (Wu et al., 2004; Baron et al., 2008), and dorsal root ganglion (Benson et al., 2002). Compared to these regions, ASICs in the striatum,

a Department of Basic Medical Science, University of Missouri–Kansas City School of Medicine, 2411 Holmes Street, Kansas City, MO 64108, USA b Robert S. Dow Neurobiology Laboratories, Legacy Research, 1225 Northeast 2nd Avenue, Portland, OR 97232, USA

Abstract—Acid-sensing ion channels (ASICs) regulate synaptic activities and play important roles in neurodegenerative diseases. They are highly expressed in the striatum, where medium spiny neurons (MSNs) are a major population. Given that the properties of ASICs in MSNs are unknown, in this study, we characterized ASICs in MSNs of the mouse striatum. A rapid drop in extracellular pH induced transient inward currents in all MSNs. The pH value for half-maximal activation was 6.25, close to that obtained in homomeric ASIC1a channels. Based on psalmotoxin 1 and zinc sensitivity, ASIC1a (70.5% of neurons) and heteromeric ASIC1a-2 channels (29.5% of neurons) appeared responsible for the acid-induced currents in MSNs. ASIC currents were diminished in MSNs from ASIC1, but not ASIC2, null mice. Furthermore, a drop in pH induced calcium influx by activating homomeric ASIC1a channels. Activation of ASICs increased the membrane excitability of MSNs and lowering extracellular Ca2ⴙ potentiated ASIC currents. Our data suggest that the homomeric ASIC1a channel represents a majority of the ASIC isoform in MSNs. The potential function of ASICs in the striatum requires further investigation. Published by Elsevier Ltd on behalf of IBRO. Key words: striatum, acid-sensing ion channels, medium spiny neurons, membrane excitability, patch-clamp, calcium imaging.

Acid-sensing ion channels (ASICs), activated by a decrease in the extracellular pH, belong to the amiloridesensitive degenerin/epithelial Na⫹ channel (DEG/ENaC) superfamily (Kellenberger and Schild, 2002). Four genes (ASIC1–ASIC4), encoding seven different ASIC subunits, have been identified to date (Price et al., 1996; Waldmann et al., 1996, 1997a; Garcia-Anoveros et al., 1997; Lingueglia et al., 1997; Chen et al., 1998; de Weille et al., 1998; Babinski et al., 1999; Akopian et al., 2000; Grunder et al., 2000). Each subunit has two transmembrane domains with a large, cysteine rich extracellular loop; short intracellular *Corresponding author. Tel: ⫹1-816-235-2248; fax: ⫹1-816-2356517. E-mail address: [email protected] (X.-P. Chu). Abbreviations: ASIC, acid-sensing ion channel; [Ca2⫹]i, intracellular Ca2⫹ concentrations; CNQX, 6-cyano-7-nitroquinoxalinee-2,3-dione; ECF, extracellular solution; I–V, current–voltage; MSNs, medium spiny neurons; NMDA, N-methyl-D-aspartate; PcTX1, psalmotoxin 1; pH50, pH value for half-maximal activation. 0306-4522/09 $ - see front matter. Published by Elsevier Ltd on behalf of IBRO. doi:10.1016/j.neuroscience.2009.04.029

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a subcortical region that controls various limbic functions, have not been well characterized. Given the dense expression of ASICs in the striatum (Alvarez de la Rosa et al., 2003; Wemmie et al., 2003), the present study was initiated to characterize the electrophysiological and pharmacological properties of proton-triggered ASICs in mouse striatal neurons. Our results identified homomeric ASIC1a channels as a prime isoform of ASICs in striatal medium spiny neurons (MSNs).

EXPERIMENTAL PROCEDURES The care and use of animals in this study followed the guideline and protocol approved by the Institutional Animal Care and Use Committee of University of Missouri–Kansas City and Legacy Research. All procedures conformed to NIH guidelines on the ethical use of animals. Every effort was made to minimize the number of animals used and their suffering.

Primary striatal neuronal cultures Primary cultures of mouse striatal neurons were prepared according to previously described techniques (Chu et al., 2004, 2006; Xiong et al., 2004). Briefly, time-pregnant (embryonic day 16) Swiss mice were anesthetized with halothane followed by cervical dislocation. Fetuses were rapidly removed and placed in Ca2⫹and Mg2⫹-free cold Hanks’ solution. The striatum was dissected and incubated with 0.05% trypsin–EDTA for 10 min at 37 °C, followed by trituration with fire-polished glass pipettes, and plating on poly-L-ornithine-coated 35⫻35 mm culture dishes at a density of 1⫻106 cells per dish. Neurons were cultured with Neurobasal medium supplemented with B27 and maintained at 37 °C in a humidified 5% CO2 atmosphere incubator. Cultures were fed twice weekly and used for electrophysiological recording 11–14 days after plating.

Whole-cell currents or membrane potentials were recorded using Axopatch 200 B amplifiers (Axon CNS, Molecular Devices, CA, USA). Data were filtered at 2 kHz and digitized at 5 Hz using Digidata 1320 or 1440 DAC units (Axon CNS, Molecular Devices, CA, USA). The on-line acquisition was done using pCLAMP 10 software (Axon CNS, Molecular Devices). In general, to avoid current desensitization, ASIC channels were activated by reducing the pH from 7.4 to specific target levels every 2 min. During each experiment, a voltage step of ⫺10 mV from the holding potential was applied periodically to monitor the cell capacitance and the access resistance. Recordings in which either the access resistance or the capacitances changed by more than 10% during the experiment were excluded from data analysis.

Fura-2 fluorescent Ca2ⴙ-imaging Fura-2 fluorescent Ca2⫹ imaging was performed as described previously (Chu et al., 2002, 2004, 2006; Xiong et al., 2004). Striatal neurons grown on 25⫻25 mm glass coverslips were washed three times with ECF and incubated with 5 ␮M fura-2acetoxymethyl ester for ⬃40 min at room temperature, followed by a wash three times and incubation in normal ECF for 30 min. Coverslips with fura-2-loaded neurons were then transferred to a perfusion chamber on an inverted microscope (Nikon, Japan). Cells were illuminated using a xenon lamp and observed with a 40⫻ UV fluor oil-immersion objective lens. Video images were obtained using a cooled CCD camera (Cool SNAP HQ2, Photometrics, PA, USA). Digitized images were acquired, stored and analyzed in a PC controlled by Meta Imaging software (MIS, MetaFluo Version 7.5, Molecular Devices). The shutter and filter wheel (lambda 10 –2) were also controlled by MIS to allow timed illumination of cells at either 340 or 380 nm excitation wavelengths. Fura-2 fluorescence was detected at an emission wavelength of 510 nm. 340/380 Ratio images were analyzed by averaging pixel ratio values in circumscribed regions of cells in the field of view. The values were exported from MIS to SigmaPlot for further analysis and plotting.

Acute isolation of mouse striatal neurons

Solutions and compounds

Mouse striatal neurons were acutely isolated according to our previously described technique (Chu et al., 2004; Liu et al., 2006). Briefly, ASIC1 and -2 knockout mice (C57BL/6 genomic background), greater than 4 weeks of age, were anesthetized with halothane and sacrificed by decapitation using a guillotine. The whole brain was removed and placed in cold extracellular solution (ECF), and subsequently sectioned at 300⬃400 ␮m with a microtome (Leica VT 1000S, Nussloch, Germany). The slices were then incubated in ECF containing 3–5 mg/ml papain (from papaya latex, Sigma Chemical Co.) at room temperature for 20 –30 min. All solutions were bubbled with 100% O2. Following the enzymatic digestion, slices were washed three times and incubated in enzyme-free ECF for at least 30 min before dissociation. To isolate striatal neurons, individual slices were transferred into a 35 mm culture dishes containing 2 ml of ECF and each dish was placed on the stage of an inverted phase-contrast microscope. The striatal region of the slice was excised and single cells were mechanically dissociated using two fire-polished glass pipettes or fine forceps. Electrophysiological recording of isolated neurons began approximately 30 min after mechanical dissociation.

Standard ECF contained (mM): 140 NaCl, 5.4 KCl, 2.0 CaCl2, 1.0 MgCl2, 20 Hepes, 10 glucose (pH 7.4; 320 –330 mosM). For solutions with pH of 6.0 or lower, MES was used instead of Hepes for more reliable pH buffering (Chu et al., 2004, 2006). For voltage-clamp recordings, the pipette solution contained (mM): 140 CsF, 10 Hepes, 11 EGTA, 2 TEA, 1 CaCl2, 2 MgCl2 and 4 K2ATP (pH 7.2 ⬃7.3, 290⬃300 mosM). For current-clamp recording, CsF in the pipette solution was replaced with K-gluconate. Psalmotoxin 1 (PcTX1) was purchased from Peptides International Inc. (Louisville, KY, USA); fura-2-acetoxymethyl ester was purchased from Invitrogen (Eugene, OR, USA). Other chemicals were purchased from Sigma (Sigma Chemical Co., St. Louis, MO, USA). A multibarrel perfusion system (SF-77, Warner Instrument Co., CT, USA) was employed to achieve a rapid exchange of ECFs.

Whole-cell recordings Whole-cell patch-clamp recordings were performed as described previously (Chu et al., 2002, 2003, 2004, 2006; Liu et al., 2006). Patch electrodes, whose resistances ranged from 4 and 6 M⍀ when filled with intracellular solution, were constructed from thinwalled borosilicate glass (1.5 mm diameter, WPI, Sarasota, FL, USA) on a two-stage puller (PC-10, Narishige, Tokyo, Japan).

Statistical analysis All data were expressed as mean⫾SEM. The Student t-test was employed to examine the statistically significant differences between groups. Significant differences were set at either * (P⬍0.05), or ** (P⬍0.01). For pH activation curves, the ECF flowing out of one barrel of the perfusion system was pH 7.4 while the ECF flowing out of the second barrel was switched to pH 7.0, 6.5, 6.0, 5.0 and 4.0 sequentially using the SF-77B fast perfusion system (Warner Instrument Co., CT, USA). Acid-triggered currents at each pH were normalized to the peak current activated at pH 5.0. Normalized values were fitted to the Hill equation using SigmaPlot 10 software to obtain pH50 values and Hill coefficients.

Q. Jiang et al. / Neuroscience 162 (2009) 55– 66 For IC50 curves of amiloride, pH 6.0 –activated current without amiloride treatment was used as a control. pH 6.0 –triggered currents for each treatment level with amiloride (1, 3, 10, 30, 100, and 300 ␮M) were normalized to control values at the peak pH 6.0 current of the MSNs without amiloride treatment. Normalized values were fitted to the Hill equation to obtain IC50 values and Hill coefficients. To determine the time constant of the fit of the desensitizing portion of the ASIC currents triggered by a drop in pH 6.0, pH 6.0 –activated currents with or without PcTX1 treatment were fitted by a single, standard exponential equation using Clampfit 10.2.

RESULTS Electrophysiological properties of ASIC currents in cultured MSNs of the mouse striatum MSNs represent a major population of neurons in the striatum (up to 95%). They can be identified by their body size (diameter of 10 –20 ␮M) and resting membrane potential (⫺75 to ⫺85 mV) (Wilson and Groves, 1980). We thus selected the MSNs based on their body size and membrane potential. The resting membrane potential of cultured mouse MSNs, as measured in a subset of eight neurons, was ⫺79.7⫾2.6 mV. Based on this observation,

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we held the membrane potential at ⫺80 mV for whole-cell patch-clamp recording. A rapid drop in extracellular pH induced transient inward currents in all 167 MSNs tested. The average peak amplitude and current density of ASIC currents in MSNs, triggered by dropping the pH from 7.4 to 6.0, were ⫺3626.1⫾197.8 and 104.4⫾5.8 pA/PF (n⫽167), respectively. The mean cell capacitance of the recorded neurons was 34.74⫾0.6 pF (n⫽167). The dose-response curve for activation of ASICs revealed a pH50 value of 6.25⫾0.11 (varying from 6.06 to 6.50) and a Hill coefficient of 0.94⫾0.07 (n⫽6) (Fig. 1A, B). This pH50 value of MSNs is comparable to that obtained in homomeric ASIC1a channels. We next determined the ion selectivity of ASICs by recording the current–voltage (I–V curve) relationship. The I–V curve was conducted by plotting the peak value of the currents induced by a rapid decrease in pH from 7.4 to 6.0 at different membrane potentials. MSNs were clamped initially at ⫺80 mV and then increased at a step of ⫹20 mV every 2 min. The current activated by this step-wise drop in pH was recorded. In order to achieve this I–V curve, we added 10 mM NaCl into intracellular pipette solutions. Theoretically, according to the Nernst equation, the Na⫹

Fig. 1. Electrophysiological properties of ASICs in cultured mouse MSNs. (A) Representative traces showing pH-dependent activation of ASIC currents in MSNs. Transient inward currents were recorded with drops in pH from 7.4 to different levels in whole-cell configuration at ⫺80 mV. (B) Dose-response curve for activation of the currents by decreasing pH. The pH50 value is 6.25⫾0.11 (varying from 6.06 to 6.50) and the Hill coefficient is 0.94⫾0.07. Each point represents the average response of six neurons. All responses were normalized to the peak value of ASIC currents triggered by a drop in pH from 7.4 to 5.0. Pooled normalized values were fitted to the Hill equation using SigmaPlot 10 software to obtain pH50 values and Hill coefficients. (C) Representative traces showing the I–V relationship of acid-activated currents with different voltage holding levels by decreasing the pH from 7.4 to 6.0 in MSNs. (D) The I–V relationship (I–V curve). The extrapolated reversal potential was at ⬃60 mV (n⫽7), which is close to the Na⫹ equilibrium potential of ⫹68 mV according to the Nernst equation, with extra- and intracellular solutions containing 140 and 10 mM Na⫹, respectively. The peak values of ASIC currents at different potentials were normalized to that recorded at ⫺80 mV.

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equilibrium potential should be ⫹68 mV with extra- and intracellular solutions containing 140 and 10 mM Na⫹, respectively. The ASIC currents in MSNs (Fig. 1C, D) had a linear I–V relationship with a reversal potential close to ⫹60 mV (n⫽7), indicating that ASICs in MSNs are more permeable to Na⫹ than Cs⫹ ions. Pharmacological profiles of ASIC currents in cultured MSNs Amiloride, a diuretic agent that inhibits Na⫹/H⫹ and Na⫹/ Ca2⫹ antiporters and ENaC, is a non-selective inhibitor for ASICs. We tested the effects of amiloride on the ASIC current by dropping the pH from 7.4 to 6.0 at a holding potential of ⫺80 mV. Similar to ASIC currents in cortical neurons, the ASIC current in MSNs was dose-dependently and reversibly inhibited by amiloride (Fig. 2A). The IC50 value was 13.55⫾1.93 ␮M (n⫽5–11) with a Hill coefficient of 0.98⫾0.05 (Fig. 2B).

PcTX1 is a 40 amino acid peptide toxin that specifically blocks homomeric ASIC1a channels (Escoubas et al., 2000), so we next tested whether PcTX1 can block the ASIC currents in MSNs. Based on their sensitivity to PcTX1, we assigned the 78 MSNs tested to three distinct populations (type 1–3 neurons). Type 1 neurons (n⫽55) were highly PcTX1-sensitive; the peak amplitude of ASIC currents elicited by a drop in pH from 7.4 to 6.0 was inhibited by 53.2% (from ⫺3015.2⫾278.9 to ⫺1412.2⫾ 189.3 pA; P⬍0.01) in the presence of 10 nM PcTX1 (Fig. 3A, B). In addition, the time constant of ASICs in these neurons was significantly decreased from 2.24⫾0.13 to 1.21⫾0.08 s (P⬍0.01) (Fig. 3G). Since PcTX1 is a specific inhibitor of ASIC1a homomers, these data suggest that ASICs in type 1 neurons are composed primarily of ASIC1a homomers. In type 2 neurons (n⫽12), the peak amplitudes of ASIC currents were not changed by treatment with 10 nM PcTX1 (from ⫺3402⫾431.8 to ⫺3068.5⫾ 404.3 pA; P⬎0.05), but the time constant of ASICs was dramatically decreased from 1.11⫾0.12 to 0.77⫾0.09 s (Fig. 3C, D, G; P⬍0.05). The remaining type 3 neurons (n⫽11) were insensitive to PcTX1 treatment; PcTX1 affected neither the peak amplitude (control: ⫺3142.1⫾ 498.3 pA; PcTX1: ⫺3024.5⫾493.5 pA; P⬎0.05) nor the time constant (control: 0.73⫾0.03 s; PcTX1: 0.70⫾0.02 s; P⬎0.05) of ASIC currents (Fig. 3E–G). Since PcTX1 does not inhibit the ASIC currents recorded from type 2 or 3 neurons, it appears that ASICs of these neurons are made up of isoforms other than ASIC1a homomers. Nevertheless, type 1 neurons predominate within the striatum (70.5% of MSNs recovered), suggesting that ASIC1a homomers are the predominant ASIC population in striatal MSNs.

Modulation of ASIC currents by zinc in cultured MSNs

Fig. 2. Dose-dependent blockade of ASIC currents in cultured MSNs by amiloride, a non-specific ASIC blocker. (A) Representative traces showing the dose-dependent block of ASIC currents triggered by dropping the pH from 7.4 to 6.0. (B) Dose-inhibition curve of the acid-induced currents by amiloride. The IC50 of amiloride blockade is 13.55⫾1.93 ␮M and Hill coefficient is 0.98⫾0.05. pH 6.0 –triggered currents for each treatment level with amiloride (1, 3, 10, 30, 100, and 300 ␮M) were normalized to control values at the peak pH 6.0 current of the MSNs without amiloride treatment. Pooled normalized values were fitted to the Hill equation using SigmaPlot 10 software to obtain IC50 values and Hill coefficients. Each point represents the average response of five to 11 neurons.

Previous studies by Baron et al. (2001) indicate that zinc can potentiate ASIC currents mediated by ASIC2a homomers or ASIC2a-containing channels (e.g. ASIC1a/ ASIC2a). To investigate the potential contribution of ASIC2a subunits to ASIC currents in MSNs, we recorded ASIC currents induced by a pH drop from 7.4 to 6.0 in the presence and absence of 300 ␮M zinc chloride. For type 1 neurons (Fig. 4A, B; n⫽28), zinc failed to potentiate ASIC currents, and in fact it induced a minor inhibition (control: ⫺2253.9⫾255.3 pA; zinc: ⫺1977.3⫾ 253.1 pA; P⬎0.05), indicating that ASIC2a is poorly expressed in those neurons. These results are consistent with the findings of PcTX1 experiments presented above, further indicating that homomeric ASIC1a is the dominant isoform of ASICs in type 1 neurons. For type 2 neurons (Fig. 4C, D; n⫽6), zinc did not affect the ASIC currents (control: ⫺2678.4⫾534.5 pA; zinc: ⫺2499.7⫾ 585.0 pA; n⫽6, P⬎0.05), also indicating the lack of ASIC2a in these neurons. In type 3 neurons (Fig. 4E, F; n⫽10), however, zinc significantly potentiated the ASIC currents from ⫺2715.5⫾628.3 to ⫺3326.7⫾726.8 pA (n⫽10; P⬍0.05), indicating the presence of ASIC2a sub-

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Fig. 3. Effects of PcTX1, a specific inhibitor of ASIC1a homomers, on ASIC currents recorded from cultured MSNs. (A) Original current traces showing inhibition by 10 nM PcTX1 on a type 1 neuron. (B) Inhibition of the mean peak value of ASIC currents by PcTX1 on type 1 neurons (** P⬍0.01, compared with CTRL; n⫽55). (C) Original current traces showing the effect of 10 nM PcTX1 on a type 2 neuron. (D) PcTX1 had no effect on peak amplitude of type 2 neurons (P⬎0.05 compared with CTRL; n⫽12). (E) Original current traces showing the effect of 10 nM PcTX1 on a type 3 neuron. (F) PcTX1 had no effect on peak amplitudes of type 3 neurons (P⬎0.05 compared with CTRL, n⫽11). (G) Effects of PcTX1 on the time constant (␶) of ASICs on three types of neurons. PcTX1 decreased the ␶ of ASIC currents in both type 1 and type 2, but not type 3, neurons. Upright inset traces came from A, C and E in this figure; ␶type3⬍␶type2⬍␶type1. “⵨” Bar represents 2 s and 1.0 nA. All ASIC currents were recorded by dropping pH from 7.4 to 6.0. MSNs were clamped at ⫺80 mV. pH 6.0 –activated current with or without PcTX1 treatment was fitted by a single, standard exponential equation using Clampfit 10.2. CTRL represents control.

units in these neurons. Collectively, these data support the concept that homomeric ASIC1a is the dominant ASIC expressed by most striatal MSNs, and that ASIC2a is expressed by a relatively minor population of MSNs (i.e. type 3 neurons). However, the effect of zinc on ASIC2b containing channels has not been thoroughly

investigated; therefore we cannot exclude the potential contribution of this subunit to zinc potentiation in type 3 MSNs. The composition of ASICs expressed by striatal MSN, differs markedly from the isoforms found in hippocampal and spinal cord neurons (Baron et al., 2002, 2008; Askwith et al., 2004).

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Fig. 4. Effects of zinc on ASIC currents on three types of cultured MSNs. (A) Representative traces showing that 300 ␮M zinc chloride produced relatively little inhibition on ASIC currents in type 1 neurons. (B) Summary data showing that zinc had no effect on type 1 neurons (n⫽28; P⬎0.05 compared with control group). (C, D) Original current traces and summary data showing that 300 ␮M zinc had no effect on ASIC currents in type 2 neurons (n⫽6; P⬎0.05 compared with control group). (E, F) Representative traces and summary data showing that 300 ␮M zinc potentiated ASIC currents in type 3 neurons (n⫽10; * P⬍0.05 compared with control group). CTRL represents control.

Properties of ASIC currents in ASIC1ⴚ/ⴚ and ASIC2ⴚ/ⴚ To further characterize the ASIC composition in striatal MSNs, we measured ASIC currents in neurons from ASIC1 and ASIC2 knockout mice. Acutely dissociated neurons were prepared for whole-cell recordings from 1 to 2 month old ASIC1 and ASIC2 null mice. They are considered to be much more mature than cultured neurons. Therefore, acutely dissociated MSNs used here to verify the properties of ASIC currents observed in cultured MSNs. As shown in Fig. 5A, dropping the pH from 7.4 to 5.0 failed to produce a detectable ASIC current in 30 neurons from ASIC1⫺/⫺ mice, while 100 ␮M N-methyl-Daspartate (NMDA), which activates glutamate receptors, triggered large inward currents (Fig. 5B). These results are consistent with our earlier experiments, suggesting that homomeric ASIC1a channels are the major ASIC configuration in striatal MSNs. Further, in two out of 32 neurons from ASIC1⫺/⫺ mice, small inward currents were triggered by a drop in pH from 7.4 to 5.0, and the currents were potentiated by 100 ␮M zinc (Fig. 5C), indicating the presence of ASIC2a subunits in a minor population of MSNs. In contrast to ASIC1 knockout mice, ASIC currents were induced in all MSNs from ASIC2⫺/⫺ mice by dropping

the pH from 7.4 to 6.5 (Fig. 5D; n⫽27). Bath application of 3 nM PcTX1 markedly inhibited ASIC currents in all neurons tested (n⫽6), indicating that ASIC1a is the major subunit expressed in these neurons. Calcium influx by activation of ASIC1a in cultured MSNs Intracellular Ca2⫹ concentration ([Ca2⫹]i) is critical to neuronal function; overload of [Ca2⫹]i induces neuronal cell death in many neurological diseases. Previous studies indicate that activation of Ca2⫹-permeable ASIC1a plays an important role in acidosis-induced, glutamate-independent neuronal injury (Xiong et al., 2004; Yermolaieva et al., 2004). To further characterize the ASICs in MSNs, we examined whether a drop in pH can induce Ca2⫹ entry through ASICs. These experiments are complicated by the possibility that a drop in pH may induce membrane depolarization, with consequent activation of other (i.e. nonASICs) ion channels and receptors, thereby permitting Ca2⫹ influx. To preclude this complication, we added a cocktail to the ECF that blocks both voltage-gated Ca2⫹ channels (10 ␮M nimodipine and 3 ␮M ␻-conotoxin MVIIC) and glutamate receptors (10 ␮M MK-801 and 20 ␮M

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Fig. 5. Properties of ASIC currents recorded in acutely dissociated MSNs from ASIC1⫺/⫺ and ASIC2⫺/⫺ mice. (A) Original current trace showing that there was no inward current induced by a drop in pH from 7.4 to 5.0 in ASIC1⫺/⫺ mice. In 30 of 32 recorded neurons there was no response to drop in pH to 5.0. (B) Inward currents triggered by application of 100 ␮M NMDA in the same neurons as (A) from ASIC1⫺/⫺ mice. (C) Original current traces showing the small inward currents activated by a drop in pH from 7.4 to 5.0. The current was potentiated by 100 ␮M zinc recorded in a neuron from ASIC1⫺/⫺ mouse. We observed two neurons responding to pH 5.0. (D) Representative traces showing the inward currents recorded in neurons from ASIC2⫺/⫺ mice by a drop in pH from 7.4 to 6.5. We recorded 27 neurons in total and all responded to a drop to pH 6.5. The ASIC currents were inhibited by 3 nM PcTX1 (n⫽6). Acutely dissociated MSNs were prepared from one to two month old ASIC1 or ASIC2 null mice.

6-cyano-7-nitroquinoxalinee-2,3-dione [CNQX]). Reducing the pH from 7.4 to 6.0 induced a substantial increase in [Ca2⫹]i in MSNs, as indicated by an increase in the intensity of 340/380 nm ratio image of fura-2 fluorescence from 0.71⫾0.08 to 2.24⫾0.25 (n⫽8; P⬍0.01) (Fig. 6A, B). This pH-induced increase in [Ca2⫹]i was blocked significantly by 30 ␮M amiloride, as indicated by a reduction in the 340/380 ratio from 2.24⫾0.25 to 1.08⫾0.11 (n⫽8; P⬍ 0.01) (Fig. 6C, D). Similarly, 10 nM PcTX1 also significantly inhibited the pH induced Ca2⫹ influx as indicated by a reduction in the 340/380 ratio from 2.50⫾0.30 to 1.10⫾0.11 (n⫽7; P⬍0.01). Collectively, these data support the conclusion that a drop in pH induces Ca2⫹ influx into MSNs through homomeric ASIC1a channels.

pH drop triggers membrane depolarization and action potentials of cultured MSNs by activation of ASICs In addition to increasing [Ca2⫹]i, activation of ASICs has been shown to induce membrane depolarization in cortical, hippocampal, retinal ganglion and spinal cord neurons (Baron et al., 2002, 2008; Chu et al., 2004; Lilley et al., 2004; Wu et al., 2004). Membrane depolarization by activation of ASICs facilitates glutamate receptor-mediated

excitatory neurotransmission and neuronal injury through its effect on NR2B of NMDA receptors (Gao et al., 2005). Our next experiment was to determine whether a drop in pH triggers membrane depolarization by activating ASICs in MSNs. Current-clamp experiments were conducted to record membrane potential as described previously (Chu et al., 2004, 2006). A minor drop in extracellular pH from 7.4 to 6.8, induced significant membrane depolarization from a holding potential of ⫺80 to ⫺42.8⫾3.1 mV (Fig. 7A, C; n⫽8, P⬍0.01), which accompanied trains of action potentials. This pH-induced membrane depolarization was significantly attenuated by either 100 ␮M amiloride (from ⫺42.8⫾3.1 to ⫺62.1⫾1.7 mV; n⫽5; P⬍0.01), a non-specific ASIC blocker, or 10 nM PcTX1 (from ⫺43.5⫾3.0 to ⫺58.1⫾3.4 mV; n⫽5, P⬍0.01), a specific ASIC1a inhibitor (Fig. 7B, D). Tetrodotoxin (0.3 ␮M), a voltage-gated Na⫹ channel blocker, had little effect on the membrane depolarization but completely diminished the action potentials triggered by a drop in pH from 7.4 to 6.8. Since this depolarization was recorded in the presence of cocktails that block the voltage-gated Ca2⫹ channels (10 ␮M nimodipine and 3 ␮M ␻-conotoxin MVIIC) and glutamate receptor-gated channels (10 ␮M MK-801 and 20 ␮M CNQX), these results indicate that ASICs contribute to neuronal excitation of MSNs.

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Fig. 6. Ca2⫹ influx through ASICs is ASIC1a-dependent in cultured MSNs. (A, B) Representative 340/380 ratios and summary data showing increases in [Ca2⫹]i by a drop in pH from 7.4 to 6.0 and inhibition of pH 6.0 –induced increases in [Ca2⫹]i by 30 ␮M amiloride, a non-specific ASIC blocker (n⫽8; ** P⬍0.01 compared with pH 6.0 group). (C, D) Representative 340/380 ratios and summary data showing increases in [Ca2⫹]i by a drop in pH from 7.4 to 6.0 and inhibition of pH 6.0 –induced increases in [Ca2⫹]i by 3.0 nM PcTX1, a specific ASIC1a blocker (n⫽7; ** P⬍0.01 compared with pH 6.0 group). Neurons were bathed in normal ECF containing 2.0 mM CaCl2 with blockers for voltage-gated Ca2⫹ channels (10 ␮M nimodipine and 3 ␮M ␻-conotoxin MVIIC) and glutamate receptors (10 ␮M MK-801 and 20 ␮M CNQX).

Potentiation of ASIC currents by lowering extracellular Ca2ⴙ in cultured MSNs Extracellular Ca2⫹ concentrations in the CNS decrease substantially in both physiological and pathological conditions. Changes in extracellular Ca2⫹ have been reported to affect ASIC currents in neurons from many regions (de Weille et al., 2001; Babini et al., 2002; Chu et al., 2003; Immke and McCleskey, 2003; Paukert et al., 2004), but its effect on ASICs in the striatum has not been investigated. Thus, our next experiment was conducted to determine the effect of decreased extracellular Ca2⫹ levels on ASIC currents in striatal MSNs. ASIC currents in MSNs induced by a pH drop from 7.4 to 6.5 were recorded in the presence of 2.0, 0.5 and 0.1 mM Ca2⫹. As shown in Fig. 8, decreasing the extracellular Ca2⫹ concentration from 2.0 to either 0.5 or 0.1 mM significantly increased pH-induced ASIC currents in MSNs.

DISCUSSION The striatum plays critical roles in extrapyramidal motor activity, recognition and emotion. In addition to other ion channels and receptors, ASICs are densely expressed in this region (Alvarez de la Rosa et al., 2003; Wemmie et al., 2003). Given the contribution of ASICs to neurodegeneration in Parkinson’s disease and Huntington’s disease (Arias et al., 2008; Wong et al., 2008), detailed characterization of the electrophysiological and pharmacological properties of ASICs in MSNs has become critically important. The data presented here reveal that homomeric ASIC1a is the major isoform of ASICs in striatal MSNs.

Homomeric ASIC1a is the major isoform in MSNs In the present study, we demonstrate that MSNs respond to a drop in pH with a rapid and transient inward current. These ASIC currents were blocked by amiloride, a nonspecific ASIC blocker, and the reversal potential was close to the Na⫹ equilibrium potential, indicating that functional ASICs are present in MSNs. We characterized three types of neurons in MSNs (types 1–3) according to their electrophysiological and pharmacological properties of ASICs. Type 1 neurons, which represent the majority of MSNs (55 out of 78 neurons), show high sensitivity to PcTX1. The following evidence supports the conclusion that ASICs in type 1 neurons are composed mainly of homomeric ASIC1a channels: (1) the pH50 value for ASICs is 6.25, which corresponds to that obtained with homomeric ASIC1a channels; (2) acid-induced ASIC currents, Ca2⫹ influx, and membrane depolarization were blocked by PcTX1, a specific ASIC1a inhibitor; (3) ASIC currents were insensitive to zinc, a metal ion that potentiates ASIC2a, but not ASIC1a currents; (4) a drop in pH from 7.4 to 5.0, which induced significant ASIC currents in neurons from wildtype mice, failed to activate any current in the majority of MSNs from ASIC1⫺/⫺ mice. In type 2 neurons, the peak amplitude of ASIC currents was not altered by PcTX1 or zinc, indicating that ASICs in these neurons are not homomers of ASIC1a and that they do not contain the ASIC2a. However, the time constant of ASICs in type 2 neurons was markedly decreased by PcTX1. We suspect that ASICs in type 2 neurons may represent ASIC1a/2b heteromers. Further studies are

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Fig. 7. pH drop triggered membrane depolarization and action potentials by activation of ASICs in cultured MSNs. (A, C) Representative traces and summary data showing the membrane depolarization induced by a drop in pH from 7.4 to 6.8 and the inhibition of membrane depolarization by amiloride (AMI, 100 ␮M) in MSNs (n⫽8; ** P⬍0.01 compared with control group). pH 6.8 –induced membrane depolarization subsequently triggered trains of action potentials. (B, D) Representative traces and summary data showing the membrane depolarization induced by a drop in pH from 7.4 to 6.8 and inhibition of membrane depolarization by PcTX1 (10 nM) in MSNs (n⫽5, ** P⬍0.01 compared with control group). pH 6.8 –triggered membrane depolarization subsequently accompanied by trains of action potentials. All responses were normalized to the peak value of membrane depolarization triggered by a drop in pH from 7.4 to 6.8. CTRL represents control. MSNs were bathed in normal ECF containing blockers for voltage-gated Ca2⫹ channels (10 ␮M nimodipine and 3 ␮M ␻-conotoxin MVIIC) and glutamate receptors (10 ␮M MK-801 and 20 ␮M CNQX).

needed to confirm whether heteromeric ASIC1a/2b channels expressed in heterologous systems have a similar response to PcTX1 and zinc. Type 3 neurons represent a small population of striatal MSNs that display PcTX1-insensitivity but whose currents are potentiated by zinc. The following evidence supports the presence of functional heteromeric ASIC1a/ASIC2a channels in these neurons: (1) ASIC currents were induced by dropping the pH to 6.0 in all type 3 neurons, indicating the presence of ASIC1a subunits in these neurons (homomeric ASIC2a channels do not become activated until the pH drops from 7.4 to below 5.5); (2) ASIC currents were potentiated by 300 ␮M zinc; (3) ASIC currents were not affected by PcTX1; (4) ASIC currents were triggered in a few neurons recorded from ASIC1⫺/⫺ mice by dropping the pH to 5.0 or 4.0, and these pH induced

currents were further potentiated by 100 ␮M zinc. However, we cannot exclude the possibility of heteromultimeric ASIC1a/ASIC2b channels in type 3 neurons. The properties of ASICs have been extensively characterized in neurons from many regions, including the cortex (Varming, 1998; Chu et al., 2004), hippocampus (Baron et al., 2002; Askwith et al., 2004), cerebellum (Allen and Attwell, 2002), retinal ganglion (Lilley et al., 2004), spinal cord (Wu et al., 2004; Baron et al., 2008), and dorsal root ganglion (Benson et al., 2002). The results of the present study indicate that striatal neurons contain mainly homomeric ASIC1a channels. This is in contrast to other well-characterized regions where functional ASICs are composed mainly of heteromeric isoforms. For example, heteromeric ASIC1a/2a channels are the major ASIC subtype in hippocampal and spinal cord neurons (Baron et al.,

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Fig. 8. Potentiation of ASIC currents by lowering extracellular Ca2⫹ in cultured MSNs. (A) Representative traces showing the potentiation of ASIC currents by 0.5 and 0.1 mM Ca2⫹ compared to 2.0 mM Ca2⫹ in the ECF by dropping pH from 7.4 to 6.5 in the same MSN. (B) Summary data showing the potentiation of ASIC currents by 0.5 mM (n⫽9) and 0.1 mM (n⫽6) Ca2⫹. All values were normalized to the peak amplitude of the ASIC currents recorded at 2.0 mM Ca2⫹ in MSNs. * P⬍0.05 and ** P⬍0.01 compared with the 2.0 mM Ca2⫹ group.

2002, 2008; Askwith et al., 2004; Wu et al., 2004). Given recent reported genetic associations between ASICs and neurodegenerative diseases such as Parkinson’s and Huntington’s diseases (Arias et al., 2008; Wong et al., 2008), future studies to determine whether ASIC1a is differentially expressed on different types of MSN’s (e.g. dopamine receptor 1- or 2-containing neurons or PENK- or Tac-containing neurons) are being considered. Functional implications in MSNs Although we have shown that functional ASIC1a channels are highly expressed in the striatum, relatively little is known about their physiological function. Given the facts that ASIC1a channels localize to the synapses (Wemmie et al., 2002, 2003), it is reasonable to speculate that ASIC1a channels contribute to learning and memory during synaptic transmission. In support of this concept, ASIC1a channels have been shown to play a role in synaptic plasticity, learning and memory in hippocampal neu-

rons (Wemmie et al., 2002, 2003). The synaptic vesicle is acidic (pH 5.7) and the release of neurotransmitters probably ejects protons into the synaptic cleft. This might activate postsynaptic ASIC receptors, which may regulate other ion channels and receptors. For example, ASICs have been shown to modulate NMDA channels (Gao et al., 2005), and BK channels (Petroff et al., 2008). Similarly, activation of sigma-1 receptor inhibits ASIC1a-mediated membrane currents and consequent intracellular Ca2⫹ accumulation (Herrera et al., 2008). However, this is largely speculative, and further investigation is necessary to determine whether ASICs regulate synaptic transmission in MSNs. Extracellular pH decreases dramatically during both seizure activity (Simon et al., 1985; Ziemann et al., 2008) and brain ischemia (Siesjo, 1988; Xiong et al., 2004; Pignataro et al., 2007). During brain ischemia, for example, extracellular pH typically falls to 6.5 under normoglycemic conditions (Siemkowicz and Hansen, 1981) and may fall to as low as 6.0 under hyperglycemic conditions (Siemkowicz and Hansen, 1981; Kraig et al., 1985). Similarly, extracellular Ca2⫹ concentrations in the CNS fall substantially in both physiological and pathological conditions, and decreases in extracellular Ca2⫹ are known to increase neuronal excitability (Hille, 1992; Xiong et al., 1997; Chu et al., 2003). Consequently, pH decreases associated with ischemia might activate ASICs in the striatum, and this may be further potentiated by ischemia-associated drops in extracellular Ca2⫹. To explore this possibility, we performed experiments in which we dropped extracellular Ca2⫹ from 2.0 mM to levels that might occur in the pathological conditions such as brain ischemia (0.5 or 0.1 mM), to determine the effect on ASIC currents. As expected, lowering extracellular Ca2⫹ concentrations potentiated ASIC currents in MSNs. This increased ASIC activity likely exacerbates neuronal injury associated with strokes and other ischemic events. Accumulating evidence suggests that ASICs play important roles in neurodegenerative diseases. ASIC1 contributes to axonal degeneration in autoimmune inflammation of the CNS, suggesting that ASIC1 blockers have potential to provide neuroprotection in diseases such as multiple sclerosis (Friese et al., 2007). Similarly, Wong et al. (2008) demonstrated that blocking ASIC1 alleviates pathology associated with through an ubiquitin-proteasome system-dependent pathway. Thus, ASIC1a is a potential therapeutic target for the reduction of htt-polyQ aggregation in Huntington’s disease. More recently, Arias et al. (2008) indicated that in vivo treatment with amiloride, a non-specific ASIC blocker, protects the nigrostriatal dopaminergic system in an MPTP model of Parkinson’s disease, implicating ASICs in the pathogenesis of Parkinson’s disease. Given the association of ASICs in neurodegeneration of Parkinson’s disease and functional homomeric ASIC1a in the striatum, further studies are required to explore whether ASICs play any physiological and pathological roles in the striatum.

Q. Jiang et al. / Neuroscience 162 (2009) 55– 66 Acknowledgments—This work was supported in part by American Heart Association Scientist Development Grant 0735092 N and University of Missouri-Kansas City Start-up Funding (X.-P.C.). We thank Drs. John Wemmie, Margaret Price, and Michael Welsh for providing ASIC1 and -2 knockout mice.

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(Accepted 13 April 2009) (Available online 17 April 2009)