Acute neuropharmacologic action of chloroquine on cortical neurons in vitro

Brain Research 959 (2003) 280–286 www.elsevier.com / locate / brainres

Research report

Acute neuropharmacologic action of chloroquine on cortical neurons in vitro Thomas J. O’Shaughnessy a , Bret Zim b , Wu Ma a , Kara M. Shaffer a , David A. Stenger a , Kaveh Zamani c , Guenter W. Gross b , Joseph J. Pancrazio a , * a

Center for Bio /Molecular Science and Engineering, Code 6910, Naval Research Laboratory, Washington, DC 20375, USA Department of Biological Sciences and Center for Network Neuroscience, University of North Texas, Denton, TX 76203, USA c Department of Biology, Division of Experimental Therapeutics, Walter Reed Army Institute of Research, Silver Spring, MD 20910, USA b

Accepted 3 October 2002

Abstract Chloroquine, a common quinolone derivative used in the treatment of malaria, has been associated with neurologic side-effects including depression, psychosis and delirium. The neuropharmacologic effects of chloroquine were examined on cultured cortical neurons using microelectrode array (MEA) recording and the whole-cell patch clamp technique. Whole-cell patch clamp records under current-clamp mode also showed a chloroquine-induced depression of the firing rate of spontaneous action potentials by |40%, consistent with the observations with the MEA recording, although no changes in either the baseline membrane potential or input resistance were observed. Voltage clamp recordings of spontaneous post-synaptic currents, recorded in the presence of tetrodotoxin, revealed no obvious changes in either the amplitude or rate of occurrence of inward currents with application of chloroquine at 10 mM, suggesting that the fundamental molecular mechanisms underlying spontaneous synaptic transmission may not be affected by acute application of the drug. In contrast, a concentration-dependent inhibition of whole-cell calcium current was observed in the presence of chloroquine. These acute neuropharmacologic changes were not accompanied by cytotoxic actions of the compound, even after exposure of up to 500 mM chloroquine for 7 h. These data suggest that chloroquine can depress in vitro neuronal activity, perhaps through inhibition of membrane calcium channels. Published by Elsevier Science B.V. Theme: Neural basis of behavior Topic: Psychopharmacological agents Keywords: Aminoquinolone; Cortical neuron; Extracellular recording; Malaria; Microelectrode array; Patch clamp

1. Introduction Although initially considered to be too toxic for use in humans [8], chloroquine has been used to treat malaria for nearly 50 years. In addition, chloroquine has found utility in the treatment of rheumatic disorders such as lupus erythematosus and rheumatoid arthritis [39]. In spite of its beneficial effects, chloroquine at therapeutic dosages has been associated with the rapid onset of a variety of neurologic side-effects including psychosis, delirium and *Corresponding author. Tel.: 11-202-404-6026; fax: 11-202-7679598. E-mail address: [email protected] (J.J. Pancrazio). 0006-8993 / 02 / $ – see front matter PII: S0006-8993( 02 )03763-0

Published by Elsevier Science B.V.

depression [6,11,33]. Although few studies have probed the basis for these neurologic side-effects, electrophysiologic changes have been associated with chloroquine administration; in particular, chloroquine-induced changes in auditory evoked potentials have been reported in humans [5]. To gain insight into the acute neuropharmacologic action of chloroquine, we examined the effects of the compound on cultured cortical neurons using the wholecell patch clamp technique. Since the patch clamp method is low throughput, we initially surveyed a range of concentrations using the extracellular recording approach applied to frontal cortex cultures grown over substrateintegrated thin film microelectrode arrays (MEAs). With

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the MEA technique, the pharmacologic sensitivity of cell networks can be assessed by monitoring changes in electrical activity corresponding to action potentials (APs) [14–18,24]. With the identification of an effective in vitro concentration to focus our patch clamp studies, we confirmed the chloroquine-induced decrease in spontaneous AP firing and conducted voltage clamp measurements of membrane currents. We show that chloroquine depresses spontaneous AP firing activity in cultured cortical neurons, perhaps through inhibition of membrane calcium channels.

2. Materials and methods

2.1. Chemicals All drugs were purchased from Sigma-Aldrich (St. Louis, MO), prepared as a concentrated stock solution in water and stored in aliquots at 280 8C. Prior to use, the drugs were thawed and then diluted in the recording media to achieve the desired final concentration. Stock concentrations were 10 or 20 mM for chloroquine (CQ; diphosphate salt), 1 mM for tetrodotoxin (TTX), 10 mM for 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; disodium salt) and 5 mM for (2)-bicuculline methiodide.

2.2. Extracellular recording The techniques used to fabricate and prepare MEAs have been described elsewhere [12,13,15,31]. Frontal cortical tissues were harvested from embryonic day 16 HSD:IRC mice (Charles River Laboratories, Wilmington, MA), dissociated enzymatically and mechanically, and plated as described previously [31]. Activity was recorded by a multiamplifier system (Plexon, Dallas, TX) and microelectrode sites showing activity from one or more units were distinguished using the Plexon multineuron acquisition processor. For pharmacologic evaluation, increasing concentrations of chloroquine were added to the recording media; in the absence of drug application, network activity remains constant over periods that span the typical durations of the experiments. These experiments were conducted at the University of North Texas in Denton, TX.

2.3. Cortical culture for whole-cell patch clamp recording As described previously, cortical tissue was dissected from embryonic day 18 rats and cells were dissociated, plated on poly-D-lysine coated 35 mm-diameter petri dishes, and cultured as detailed earlier [31]. Neurons were cultured for 11–15 days prior to use in patch clamp experiments to ensure mature morphologic and electrophysiologic properties, as we have demonstrated previously [1,23,31].

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2.4. Whole-cell patch clamp recordings Whole-cell patch clamp methods were employed as described by Hamill and colleagues [20]. Technical aspects of the whole-cell patch clamp work were described previously [31]. For current clamp measurements, the patch pipette was filled with an intracellular solution containing (in mM): 50 KCl, 90 K-aspartate, 5 ethylene glycol-bis(baminoethyl ether)-N,N,N9,N9-tetraacetic acid (EGTA)KOH, 5 HEPES, 5 MgATP, pH to 7.4 with KOH. The extracellular bathing solution contained (in mM): 140 NaCl, 5 KCl, 1.8 CaCl 2 , 0.8 MgCl 2 , 10 glucose, and 10 N-[2-hydroxyethyl] piperazine-N9-[2-ethanesulfonic acid] (HEPES), pH to 7.4 with NaOH. Action potential duration was estimated by calculating the time difference between threshold crossing, where the threshold was set at 50% of the action potential amplitude relative to the baseline membrane potential. For calcium current measurements, the patch electrode solution contained (in mM): 120 CsCl, 20 tetraethylammonium chloride (TEA-Cl), 1 CaCl 2 , 11 EGTA-CsOH, 10 HEPES, 5 MgATP, pH to 7.4 with CsOH while the extracellular bathing solution contained (in mM): 145 TEA-Cl, 2 CaCl 2 , 0.8 MgCl 2 , 10 glucose, 10 HEPES, pH to 7.4 with CsOH. In addition, the external solution was supplemented with 1 mM TTX, 10 mM CNQX and 10 mM bicuculline. All patch clamp experiments were conducted at room temperature at the Naval Research Laboratory in Washington, DC.

2.5. Determination of cytotoxicity To assess the potential neurotoxicity of chloroquine, cells were exposed to various concentrations of chloroquine for 7 h and then tested for cell survival. This exposure duration was chosen to be greater than or equal to the maximum exposure duration for chloroquine in any of the electrophysiologic studies performed in the present work. Testing was done using the LIVE / DEAD Viability / Cytotoxicity kit from Molecular Probes (Eugene, OR) using the manufacturer’s suggested protocol.

2.6. Statistics Where appropriate, data are presented as mean6S.E.M. and the number of cells / experiments performed (n). Statistical significance was determined using Student’s t-test with P,0.05 considered significant. For analysis of the spontaneous synaptic events, only events that had no additional superimposed event during the decay phase were chosen for curve fitting analysis of the decay phase. Cumulative histograms, a conventional approach for the analysis of spontaneous inhibitory post-synaptic currents (sIPSCs) [2,35,38], were statistically compared using the Kolmogorov-Smirnov test where P,0.05 was considered to yield a significant difference.

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3. Results

3.1. Extracellular recordings Frontal cortex cultures grown on MEAs were used to survey the in vitro concentration-dependence of the effects of chloroquine on electrophysiologic function; these initial data were used to identify an effective concentration for our primary efforts with the whole-cell patch clamp method. The MEA data described in the present work consist of discriminated extracellular spikes or single units; for pharmacologic analysis, rates have been averaged across the entire network. Among cultured frontal cortex networks, typical spontaneous baseline spike rates, averaged across the units recorded, ranged from 2.5 to 6.0 spikes per second. Application of chloroquine resulted in a depression of spike rate with no changes in spike amplitude. A chloroquine titration from 2 to 31 mM resulted in a concentration-dependent inhibition of spike rate (Fig. 1A). A statistically significant decrease in mean spike rate to 7964% (mean6S.E.M., n54 networks) of control levels was observed with administration of 8 mM chloroquine. Concentration–response curves were generated for each culture tested by expressing the results as the percent of the mean reference spike rate (Fig. 1B), yielding an IC 50 of 1462 mM (n54 cultures).

3.2. Whole-cell patch clamp recordings After establishing effective concentration ranges via the MEA experiments, patch clamp measurements were used to gain insight into the mechanisms underlying chloroquine-induced inhibition of neuronal activity. First, we examined the effects of 10 mM chloroquine on cultured cortical neurons measured under current-clamp conditions. Under control conditions, from a baseline membrane potential of 26062 mV (mean6S.D., n59), neurons

generated spontaneous APs at a rate of 1.360.5 Hz (mean6S.D., n59). Cortical neurons measured under patch clamp conditions did exhibit bursting at a rate of 0.3460.21 Hz (mean6S.D., n59) with each burst consisting of 2.561.2 APs (mean6S.D., n58). As expected based on the MEA experiments, administration of chloroquine (10 mM) reversibly inhibited the spontaneous AP firing rate to 57611% (mean6S.E.M., n59, P50.002) of control levels, with no significant change in the baseline membrane potential (Fig. 2). With regard to bursts, both the frequency and the number of spikes per burst fell to 48619% (mean6S.E.M., n59 cells) and 7469% (mean6S.E.M., n59 cells) of control levels, respectively. No statistically significant changes in AP amplitude or baseline membrane potential were observed. These changes were reversible. Chloroquine produced no change in the input resistance, R IN , of 214645 MV (mean6S.D., n54 cells) as measured from evoked potentials in quiescent cells. To evaluate the extent to which chloroquine affects synaptic function in cortical neurons, voltage clamp experiments were conducted to measure spontaneous synaptic events and calcium currents. The rate and amplitude of spontaneous miniature post-synaptic currents (PSCs), which reflect the status of the presynaptic and postsynaptic processes, respectively [23], were measured before and after exposure to chloroquine. Under voltage clamp conditions neurons were maintained at a holding potential of 270 mV and in the presence of 1 mM TTX, spontaneous inward currents were observed (Fig. 3). Two classes of spontaneous events could be discerned: slowly decaying bicuculline-sensitive and rapidly decaying, CNQX-sensitive events that correspond to GABAA - and AMPA-mediated spontaneous release events, respectively (data not shown). For a representative data set of |3 min in duration and under control conditions, fast and slow decay events exhibited time constants of decay of 3.060.1 ms

Fig. 1. Effect of chloroquine on the extracellular potentials derived from spontaneously active frontal cortex cultures grown over microelectrode arrays (MEAs). The MEA data described in the present work consist of discriminated extracellular spikes or single units; for pharmacologic analysis, rates have been averaged across the entire network. (A) Average spike rate calculated in 60-s bins derived from 23 discriminated units for a representative culture before and after exposure to increasing concentrations of chloroquine. (B) Concentration–response curves derived from four titration experiments; non-linear curve fitting to a logistic curve function yielded IC 50 s of 14 mM (closed circle), 17 mM (open circle), 11 mM (closed triangle) and 13 mM (open triangle). The data shown in A are summarized as the closed triangles.

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Fig. 2. Effect of chloroquine on action potentials recorded from cortical neurons in the whole-cell patch clamp configuration. Representative 100-s long traces from a patched neuron showing AP firing before and after the addition of 10 mM chloroquine demonstrating a marked reduction in the spontaneous AP firing rate.

(mean6S.D., n5202 events) and 30.160.9 ms (mean6S.D., n5214), respectively, with average amplitudes of 15.460.5 pA (mean6S.D., n5202) and 21.461.1 pA (mean6S.D., n5214), respectively. The ranges for the rates of fast and slow decay events under control conditions were 0.6–0.9 and 1.1–2.7 Hz, respectively. Distributions of fast and slow decay events were

assembled in the absence and presence of chloroquine and the Kolmogorov-Smirnov test was applied to determine any statistically significant effects for paired analysis. No change in the rate or amplitude of either fast or slow decay spontaneous events could be discerned with 10 mM chloroquine (n52 cells; Fig. 3). Therefore, alterations in action potential firing rate and extracellular spikes were

Fig. 3. Lack of marked effect of chloroquine on spontaneous neurotransmitter release derived from whole-cell recordings from cortical neurons. (A) Representative records from a post-synaptic neuron voltage clamped at 270 mV before and after exposure to chloroquine. Two classes of spontaneous PSCs are evident: fast-decay events, which are CNQX-sensitive (glutamatergic transmission) and denoted by ‘?’, and slow-decay events, which are bicuculline-sensitive (GABAergic transmission). For the representative neuron, a detailed examination of amplitude cumulative histograms showed that neither the slow decay events (B) nor fast-decay events (C) were statistically altered by 10 mM chloroquine.

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evident at chloroquine concentrations below which any changes in spontaneous PSCs could be distinguished. Whole-cell calcium current (ICa ) was assessed in the presence of bicuculline (10 mM) and CNQX (10 mM), to block synaptic transmission, and with extracellular Na 1 replaced with tetraethylammonium and supplemented with 1 mM TTX, to suppress sodium and potassium channel

mediated current. Under control conditions, the inward current was activated from a holding potential of 280 mV to depolarizing potentials from 260 to 60 mV, reaching a peak of 2854694 pA (mean6S.D., n510 cells) at 210 to 0 mV. During whole-cell recording, only a modest degree of calcium channel rundown was observed; after 4 min, the peak calcium current fell to 8964% (mean6S.E.M., n510) of the initial control levels (Fig. 4A). Application of chloroquine resulted in a partially reversible inhibition of peak ICa ; 10 mM chloroquine depressed peak ICa to 7563% (mean6S.E.M., n59; P5 0.008) of control levels (Fig. 4A–C). The effect of chloroquine was concentration-dependent since 30 mM chloroquine depressed peak ICa to 6163% (mean6S.E.M., n57; P,0.001) of control levels (Fig. 4A).

3.3. Lack of acute chloroquine-induced cytotoxicity Cortical neurons, in culture for 15 days, were exposed to 30- and 500-mM concentrations of chloroquine for 7 h before testing for cell survival with a fluorescent live / dead stain. Two cultures at each concentration were used to evaluate possible cytotoxicity. Experiments, which were performed in triplicate, showed no difference in cell survival between control and chloroquine treated cells (data not shown).

4. Discussion

Fig. 4. Effect of chloroquine on ICa . Whole cell calcium currents were measured using 75-ms step depolarizations from a holding potential of 280 mV. Ohmic leak currents were subtracted using the P/ N4 leak subtraction and the patch amplifier’s series resistance compensation circuit was used at a 90% level to improve the voltage clamp response time in the large neuronal cells. (A) Graph showing the percent of control peak ICa 4 min after the addition of 0 (control), 10 or 30 mM chloroquine. The small reduction in the control case represents calcium current rundown (* indicates significant difference from control; P,0.05). (B) Current–voltage (I–V ) curves for a representative neuron in the wholecell voltage clamp configuration before and after the addition of 10 mM chloroquine. (C) Current traces from the same cell as in B showing the effect of chloroquine on ICa . Currents in this plot were elicited from a holding potential of 280 mV by a 75-ms depolarization to 0 mV.

To our knowledge, this study is the first to examine in detail the effects of chloroquine on the electrical activity of cultured neurons. Previous cytotoxicity experiments using chick embryonic neurons showed that long-term exposure (7 days) to chloroquine diminished viability at concentrations consistent with in vivo serum lethal levels of 14–16 mM [7]. The lack of cytotoxicity with acute exposure shown in the present study suggests that the neuropharmacologic effects are distinct from the diminished viability reported previously. The inhibitory effects on cultured neuronal networks that we show in the present work become statistically significant at 8 mM with IC 50 values of 14 mM, concentrations at or below previous in vitro studies with chloroquine. Biochemical assays showed low to moderate competitive binding of chloroquine to neuronal benzodiazepine, GABA and opiate receptors with Ki values of 7–50 mM [27]. Quinolone derivatives, including chloroquine, have been reported to inhibit neuromuscular transmission in mice via both pre- and postsynaptic sites, where 50 mM induced 40–50% reductions in quantal content [36]. Chloroquine blocks background potassium currents modulated by muscarinic receptors expressed in cardiac myocytes [4]. Inhibition of various potassium channel subtypes has also been reported for the related quinolone, quinine [22,26]; however, an effect of chloroquine on neuronal potassium channels cannot readily

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explain the present findings. Interestingly, antimalarial compounds including chloroquine have been shown to inhibit calcium-dependent backward swimming in paramecium over a concentration range of 0.01–30 mM [3]. Our whole-cell patch clamp work demonstrates that at a concentration that can markedly inhibit spontaneous AP firing (10 mM), no changes in the resting membrane properties could be detected. In addition, the lack of effect on spontaneous PSCs in the presence of TTX suggests that the neither basal synaptic release nor post-synaptic receptors were affected by chloroquine at this concentration. Since AP frequency was reduced, we considered the possibility that pre-synaptic calcium current may be affected by chloroquine, perhaps resulting in diminished synaptic transmission and consequent decrease in postsynaptic neuronal firing. A statistically significant chloroquine-induced inhibition of voltage-gated calcium current was observed; this effect may, at least in part, explain the observed decrease in the spontaneous AP firing. Given a fourth order relationship between neurotransmitter release and calcium influx in cultured cortical neurons [34], a 25% inhibition of ICa may result in up to a 70% reduction in neurotransmission. In addition, depression of high-voltage activated calcium current by an antidepressant has shown a marked reduction in neuronal bursting [9], an observation consistent with the present findings. Still, we cannot exclude a role for other cation channels for the effects of chloroquine, such as slowly inactivating or persistent sodium currents that have been reported to affect bursting in neocortical neurons [19]. Future work will be necessary to examine these other possible targets and explore the degree of selectivity of chloroquine for classes of calcium channels expressed in cortical neurons [40]. Therapeutic doses of chloroquine for rheumatoid patients can reach serum concentrations of 1–2 mM [7]. Likewise, intramuscular injection of chloroquine to treat malaria patients results in peak plasma concentrations of 0.9–1.7 mM [32]. Intravenous injections of high dose chloroquine for treatment of severe malaria can yield free concentrations as large as 4 mM [41]. Lethal overdose levels of chloroquine are reached at serum levels of 14–16 mM [7]. It is reasonable to suppose that the lack of permeability of the blood–brain barrier to chloroquine largely explains why it has been an effective therapeutic. While neurologic side-effects of chloroquine are rare, the present findings suggest that a compromise to the blood– brain barrier could result in effects on neuronal function. It is reasonable to ask whether or not the known properties of chloroquine, particularly with respect to its antimalarial effects, could play a role in the neuropharmacologic effects we have observed. There are several mechanisms currently proposed to explain how chloroquine effectively treats infection by the malaria parasite. The mechanism that is believed to primarily account for the effectiveness of chloroquine involves the disruption of

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the parasite’s ability to digest hemoglobin. It is well known that chloroquine is a weak base accumulated in the acidic vesicles of the malaria parasite. However, weak bases capable of collapsing the pH gradient in synaptic vesicles have been associated with an increase in exocytosis [28,37], an effect that contrasts with the present findings. It has been proposed that the raising of vesicular pH alone is sufficient to inhibit the proteases involved in hemoglobin breakdown [21,25], though other evidence suggests that chloroquine forms a complex with heme that interferes with the protease breakdown of hemoglobin [10]. Other hypotheses involve chloroquine inhibiting protein, DNA or RNA synthesis or one or more proteases or lipases involved in hemoglobin degradation (see Ref. [29] for a brief review). Clearly, mechanisms involving hemoglobin breakdown and inhibition of nucleic acid synthesis are unlikely to account for the acute neuropharmacologic effects of chloroquine, thus it is likely that the effects observed in the present study may be due to a novel action of the compound. Electrophysiologic methods offer the ability to evaluate putative neuropharmacological agents with metrics based on physiologic function [30,31]. Single microelectrode techniques such as patch clamp yield detailed information concerning molecular mechanisms of action, but suffer with regard to throughput. The use of MEAs presents the capability of screening compounds for neuropharmacologic action in a comparatively rapid manner, and identifying relevant concentrations for more detailed patch clamp investigation.

Acknowledgements This work was funded by the Defense Advanced Research Projects Agency and the Office of Naval Research. T.J.O. was supported by a postdoctoral associateship from the National Research Council. The opinions and assertions contained herein are strictly those of the authors and are not to be construed as official or reflecting the views of the Department of the Navy or the military at large.

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