Methamidophos transiently inhibits neuronal nicotinic receptors of rat substantia nigra dopaminergic neurons via open channel block

Neuroscience Letters 369 (2004) 208–213

Methamidophos transiently inhibits neuronal nicotinic receptors of rat substantia nigra dopaminergic neurons via open channel block Silvia Di Angelantonioa,b,d,∗,1 , Giorgio Bernardia,c , Nicola B. Mercuria,c a

Laboratorio di Neurologia Sperimentale IRCCS-Fondazione Santa Lucia, Via Ardeatina 306, 00179 Roma, Italy b Dipartimento di Fisiologia Umana e Farmacologia Universit` a La Sapienza di Roma, Roma, Italy c Clinica Neurologica Universit` a di Roma-Tor Vergata, Italy d Department of Neurosciences, IRCCS NEUROMED, via Atinense, 18, 86077 Pozzilli (IS), Italy Received 25 May 2004; received in revised form 16 July 2004; accepted 20 July 2004

Abstract The use of acetylcholinesterase (AChE) inhibitors is the primary therapeutic strategy in the treatment of Alzheimer’s disease. However, these drugs have been reported to have effects beyond the simple stimulation of neuronal acetylcholine receptors (AChRs) by elevated acetylcholine (ACh), interfering directly with the nAChR. Therefore, a pure pharmacological blockade of AChE is not usually obtained. In this study, the patch–clamp technique was utilized to determine the effects of methamidophos, a pesticide that is considered a selective AChE inhibitor, on nAChRs of substantia nigra dopaminergic neurons. In spite of the fact that methamidophos has been reported to be devoid of direct nicotinic actions, our main observation was that it selectively and reversibly blocked nAChR responses, without directly affecting the holding current. Methamidophos produced a downward shift in the dose response curve for nicotine; the mechanism accounting for this non-competitive antagonism was open channel block, in view of its voltage dependence. Pre-treatment with vesamicol did not prevent the reduction of nicotine-induced currents, indicating that the effect on nAChRs was independent from the activity of methamidophos as a cholinesterase inhibitor. Our results conclude that methamidophos has a complex blocking action on neuronal nAChRs that is unlinked to the inhibition of AChE. Therefore, it should not be considered a selective AChE inhibitor and part of its toxic effects could reside in an interference with the nicotinic neurotransmission. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Acetylcholinesterase; Nicotinic receptors; Organophosphates; Alzheimer’s disease; Parkinson’s disease; Allosteric modulation

One important therapeutic approach for treating Alzheimer’s disease (AD) is the administration of AChE inhibitors with the goal of enhancing cholinergic neurotransmission [15]. However, the currently used AChE inhibitors are rather unselective drugs that interfere with the function of the nAChRs. For instance it is well-known, that galantamine facilitates nicotinic transmission [24] while donepezil reduces the effects of nicotine on central neurons [5]. Therefore, the fate ∗ Corresponding author. Present address: Dipartimento di Fisiologia Umana e Farmacologia “V. Espamer”, Universit`a La Sapienza, Piazzale Aldo Moro 5, 00185 Roma, Italy. Tel.: +39 06 49910436; fax: +39 06 49910851. E-mail addresses: [email protected], [email protected] (S. Di Angelantonio). 1 Tel.: +39 06 51501386; fax: +39 06 51501384.

0304-3940/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2004.07.074

of nicotinic receptor function with AChE inhibitors is an important issue that requires investigation. To date no definitive conclusion on the overall actions of AChE inhibition can be drawn when unselective drugs are used during the treatment of AD. In the present paper, we investigated at cellular level, the effects of methamidophos, an organophosphate (OP) compound that is considered to be a selective AChE inhibitor. This drug, currently used as an insecticide and acaricide [26], has been also utilized to examine the consequences of a pure AChE inhibition on neurotransmitter release [7,16,17]. In spite of this, there are reports that OP compounds may interact with nAChRs themselves [20,23]. Dopaminergic neurons of the substantia nigra are known to express high post-synaptic levels of the typical central

S. Di Angelantonio et al. / Neuroscience Letters 369 (2004) 208–213

nicotinic ␣4␤2 receptor [14], that can be activated and desensitized by nicotine [3,21]. Interestingly, a partial loss of nicotinic receptors occurs in Parkinson’s disease brains suggesting a protective role of nAChRs. The considerations mentioned above make the dopaminergic neurons of substantia nigra a useful model to study the interaction of methamidophos with these native nAChRs in the brain. Four to five week old Wistar rats were anaesthetized with halothane and subsequently killed by decapitation. All experiments followed international guidelines on the ethical use of animals from the European Communities Council Directive of 24 November 1986 (86/609/EEC). The brain was rapidly removed from the skull and horizontal midbrain slices (240 ␮m) were cut in cold (8–12 ◦ C) artificial cerebrospinal fluid, using a vibratome, and left to recover at 34 ◦ C for at least 1 h. Slices were separately placed in a recording chamber, on the stage of an upright microscope (Olympus BX50WI) and submerged in a continuously flowing (2.5 ml/min) solution at 34 ◦ C. Artificial cerebrospinal fluid (ACSF) composition was the following (in mm): NaCl, 126; KCl, 2.5; MgCl2 , 1.2; CaCl2 , 2.4; NaH2 PO4 , 1.2; NaHCO3 , 19; glucose, 11; saturated with 95% O2 , 5% CO2 (pH 7.4). Dopaminergic neurons were visualized with infrared Nomarski video microscopy. Patch–clamp recordings were obtained using glass electrodes (3–4 M) filled with (in mM): 115 Kmethylsulfate, 20 NaCl, 1.5 MgCl2 , 5 HEPES, 0.1 EGTA, 2 ATP, 0.5 GTP (pH 7.3, with KOH). Membrane currents were recorded with a patch–clamp amplifier (Axopatch 1D; Axon Instruments, USA), filtered at 1 kHz, digitized (10 kHz) and stored on computers using the pClamp9 software (Axon Instruments). Dopaminergic neurons were identified electrophysiologically on the basis of a prominent hyperpolarization-activated current Ih at negative voltage steps and a typical voltage sag when negative current steps were applied in current–clamp mode [18]. Methamidophos (Fluka) and Vesamicol (Tocris) were usually applied to the slice via the perfusion system. In order to minimize receptor desensitization nicotine (Sigma) was delivered by pressure application (10–20 psi) from glass micropipettes positioned over the slice in correspondence to the recorded neuron [6]. Excitatory post-synaptic currents (EPSCs) were evoked in dopaminergic cells using a bipolar Ni/Cr stimulating electrode, placed 50–100 ␮m rostral to the recording electrode. To evoke a stable EPSC each stimulus (150–300 ␮s, 20–50 mV) was delivered every 30 s. In order to block the fast GABAergic synaptic currents, picrotoxin was applied (100 ␮M). Data are presented as mean ± S.E.M. with statistical significance assessed with Wilcoxon test (for non-parametric data) or paired t-test (for normally distributed data). A value of P < 0.05 was accepted as indicative of a statistically significant difference. Dose–response curves were fitted with a standard logistic equation [25]. When nicotine was applied onto a dopaminergic neuron via a puffer pipette positioned above the slice, a rapid inward

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current developed mediated by the activation of post-synaptic nAChRs. The current was indeed blocked by the nAChRs antagonist dihydro-␤-erythroidine, and was left unchanged by applying tetrodotoxin (1 ␮M). To test if the nicotine induced current was mediated only by post-synaptic nAChRs, or it was also due to an activation of pre-synaptic nAChRs, that in turn facilitated the release of other transmitters such as glutamate, we bath applied a mixture of antagonists to block glutamate and GABAA receptors. The whole cell current elicited by puffer pulses of nicotine was left unchanged when AP5 10 ␮M, CNQX 50 and 20 ␮M bicuculline were added (98.7 ± 0.6%, n = 5), indicating that the recorded nicotine currents were mediated by post-synaptic nAChRs. Fig. 1A shows inward currents generated by nicotine applied on a dopaminergic neuron, via brief (100 ms) pressure pulses from a pipette (final dilution 1 mM in ACSF), to minimize rapid desensitization [6,12] (Fig. 1A, left). When the same pulse was delivered in the presence of the acetylcholinesterase inhibitor methamidophos (100 ␮M; bath applied for 5 min), the inward current was reduced (63%; Fig. 1A, middle), without any direct action of methamidophos on resting conductance or holding current. The residual current was on average 53 ± 7%, the current decay was not reduced, as depicted when the two currents were scaled (insert in Fig. 1A), and the depression was reversible after 3 min of methamidophos washout. The latter observation makes it unlikely that the nicotine current depression was due to an increased level of free ACh in the tissue, because of irreversible binding of methamidophos to AChE in our time scales [4]. Fig. 1B shows the time course of methamidophos induced depression for six neurons. It is noteworthy that the extent of depression increased progressively during 200 ␮M methamidophos application, indicating use dependence of the block, and that recovery was achieved 3 min after drug washout during repeated agonist applications, indicating use dependence. The steady state of the block was reached after 3–4 min of methamidophos application (arrowhead) and the residual current was 37 ± 8% of the control one (n = 6, P < 0.01). The slow onset of inhibition by methamidophos could be due to a slow action of methamidophos or an action that requires the presence of agonist. We tested these two possibilities by incubating the slice with methamidophos for up to 10 min without applying nicotine. After pre-incubation nicotine was applied as in control condition and the first current was not different respect to the control one. Steady state of the block was obtained after 3–4 min as without pre-application of the drug, indicating use dependence of the block. However, the kinetics of the block of the nicotine-induced current seemed to be faster than those observed for the very slowly reversible action of methamidophos on acetylcholinesterases [4]. These observations lead to the hypothesis that methamidophos directly interacted with the nicotinic receptors, likely involving a certain degree of channel block.

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Fig. 1. Block of nicotine-induced responses by the organophosphoric methamidophos. (A) Current records obtained with 100 ms nicotine (1 mM pipette concentration; left), 5 min after starting bath application of methamidophos (100 ␮M; middle) and 3 min after washout. Note reversible reduction in nicotine current amplitude. The insert shows, in a different time scale, that at the steady state of the block, when the current blocked by methamidophos (b) was scaled to the control one (a), the deactivation time of the current was left unchanged. (B) Time course of methamidophos block on nicotine induced currents. The arrowhead indicates steady state of the block. Note significant use dependence. (C) Plot of the fractional reduction in current amplitude against different log concentrations of methamidophos (ranging from 10 to 500 ␮M). The test pulse (50 ms, 1 mM) of nicotine was the same for all concentration of methamidophos (n = 7–12). The calculated IC50 value for methamidophos was 71 ± 15 ␮M. (D) Plot of nicotine current amplitude vs. increasing duration of nicotine pressure pulses in control solution and in the presence of methamidophos. Ordinate, current amplitude normalized with respect to the response evoked by 50 ms in control solution for each neuron. Abscissa, pulse duration of nicotine (1 mM) applications. Methamidophos (100 ␮M) was applied for ∼5 min (n = 12). Note downward shift of the curve in the presence of methamidophos.

Fig. 1C shows a plot of the fractional reduction in current amplitude against different log concentrations of methamidophos. Methamidophos concentrations (ranging from 10 to 500 ␮M) were tested on responses evoked by the same pulse duration of nicotine (50 ms, 1 mM; n = 7–12). Data were plotted on a semi-logarithmic scale and fitted with a logistic equation giving an IC50 value of 71 ± 15 ␮M. Further tests were performed to characterize the mechanism underlying the depression of nicotine evoked currents exerted by methamidophos. Fig. 1D shows that on eight dopaminergic neurons, increasing the duration (20–1000 ms) of 1 mM (pipette concentration) nicotine pulses, yielded progressively larger currents with apparent saturation at 500 ms pulses. When same pulse duration of nicotine applications were repeated in the presence of 100 ␮M methamidophos (5 min bath application), all currents induced by 20–1000 ms nicotine pulses were blocked. Thus, the plot was downward shifted by the application of methamidophos. Taking average responses at approximately the midpoint of the curve (100 ms) before and after methamidophos application gave a 52 ± 12% depression with the 100 ␮M methamidophos dose (n = 12). This pattern of antagonism was typical of a

non-competitive antagonist of nAChRs. Therefore, methamidophos may either act as an allosteric modulator, or as an open channel blocker. In order to clarify the action of methamidophos on desensitized responses, the decay phase of nicotine induced currents was examined using a 1 s pulse of nicotine (1 mM pipette concentration), in control and in the presence of methamidophos. In control condition a 1 s pulse evoked a desensitized response with a decay constant, during agonist application, τ equal to 908 ± 91 ms. In the presence of 100 ␮M methamidophos, the same pulse was applied on the same set of neurons, without any prior nicotine application. Under this condition, the peak of the current wash slightly reduced, but the fading of the current was accelerated, and the corresponding τ-value was 625 ± 78 ms (n = 5, P < 0.05) (not shown); after washout of methamidophos the current decay was as in control condition. This phenomenon could be explained as open channel block, in fact the antagonist could enter the channel opened by the agonist causing a more pronounced decay. However, we still cannot exclude an allosteric modulation of the receptor channel complex.

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Fig. 2. Treatment with vesamicol did dot prevent methamidophos reduction of nicotine currents. (A) Representative experiments showing the effect of blocking AChE with methamidophos on EPSCs recorded in control condition and in the presence of 5 ␮M vesamicol. (a) When methamidophos was applied in control condition, higher levels of free AChE produce reduction of EPSCs acting via pre-synaptic muscarinic receptors. (b) When the same protocol was applied in the presence of the ACh-depleting drug vesamicol (5 ␮M, pre-applied for 15 min), methamidophos application left EPSCs amplitude unchanged. (B) Averaged EPSCs amplitude in control (dark bars), and in the presence of 200 ␮M methamidophos (light bars) before and during treatment with 5 ␮M vesamicol (n = 5). (C) Current records obtained with 50 ms pulses of 1 mM nicotine. Methamidophos depressed nicotine-evoked current in control condition and in the absence of free ACh. (D) Average value of peak amplitude reduction obtained with methamidophos in control conditions (dark bars) and in the presence of 5 ␮M vesamicol (light bars). Note that the extent of depression was different after ACh depletion; data are from six neurons.

To further analyze if the methamidophos-induced facilitation of nAChR desensitization was due either to an increased level of free ACh in the tissue or to a direct interaction with the nicotinic receptor itself, slices were depleted of ACh by a treatment with vesamicol (5 ␮M) [27,28]. It has been previously shown that AChE inhibition leads to a reduction of evoked EPSCs in dopaminergic neurons of the substantia nigra pars compacta (SNc), due to the activation of pre-synaptic muscarinic receptors by the increased level of ACh [10]. Accordingly, methamidophos 200 ␮M reversibly depressed EPSCs to 73 ± 6% (n = 5, P < 0.05) of control. Fig. 2 A(b) shows that on a different cell, in another preparation, after 15 min incubation with 5 ␮M vesamicol (dashed line), the same application of methamidophos did not affect EPSCs, indicating that no tonic ACh was released under vesamicol (99 ± 4%, n = 5, P > 0.05), thus confirming that 5 ␮M vesamicol was effective in depleting ACh. Conversely in the presence of 5 ␮M vesamicol, methamidophos was still effective in reducing nicotine induced currents (Fig. 2C). Moreover the extent of depression for all methamidophos concentrations tested after vesamicol application was smaller with respect to the one observed in control conditions, as shown by histograms in Fig. 2D. The latter observation might suggest that, in control conditions, increased

levels of free ACh may partially desensitize nAChRs and then reduce the inward currents upon nicotine appplication. However, nicotine current reduction was mostly due to a direct interaction of methamidophos with nAChRs, via a mechanism different and independent from AChE block. Several nAChR blockers are known to display a variable degree of voltage dependence [1,2,8,11,19]. The present experiments were performed to examine if the effect of methamidophos on dopaminergic neurons was also voltage dependent. Fig. 3A shows the extent of methamidophos block for three holding potentials; when 100 ␮M methamidophos was tested on a set of neurons held at −30 mV, the depression of nicotine currents (50 ms, 1 mM pipette concentration) was relatively weak. Conversely, a much stronger block was observed when cells were held at −60 or −90 mV (n = 6). The rectification properties of nAChRs precluded tests of methamidophos block at positive holding potentials, however, we tested if the methamidophos block could be relieved by a protocol combining membrane depolarization with a nicotine pulse. In the continuous presence of 100 ␮M methamidophos, after achieving steady state of nicotine current depression, the membrane potential was shifted to +30 mV for 15 s during which a single nicotine pulse was applied. At this potential nicotine did not elicit a response;

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Fig. 3. Methamidophos selectively antagonizes nAChR via an open channel block. (A) Bar charts showing significant voltage dependence of methamidophos block. Average of nicotine currents obtained at three different hyperpolarized holding potentials (−30, −60, and −90 mV, n = 6) in control (light bars) and in the presence of methamidophos (dark shaded bars). (B) Current records obtained with 500 ms AMPA (10 ␮M pipette concentration) in control and 2 min after starting bath application of methamidophos (200 ␮M). Note that methamidophos did not produce any depression of these currents (n = 5).

on return to standard holding potential (−70 mV), nicotine evoked a current that was not different from the one observed in the presence of methamidophos. These results indicated a voltage dependence of the channel block, which was not relieved by rapid depolarization. We examined the issue of specificity by determining whether methamidophos could modify responses to another fast-acting receptor–channel agonist such as AMPA [9]. When AMPA was delivered via puffer application onto four dopaminergic neurons (10 ␮M; pipette concentration, 500 ms), inward currents were recorded. Bath-application of 200 ␮M methamidophos did not produce any depression of these currents (98 ± 5%, n = 5) as exemplified in Fig. 3B. The principal finding of the present study is that methamidophos causes a non-competitive antagonism on native nAChRs expressed by SNc dopaminergic neurons. This was manifested as a rapid onset, use dependent, and agonistinsurmountable block of inward currents evoked by pulse applications of nicotine. Such a phenomenon was not dependent on the cleavage of AChE exerted by methamidophos in view of its reversibility, and its persistence even in the absence of free ACh. When observing the effect of methamidophos application on the dose response curve for nicotine, it appeared that the block was slightly dependent on agonist concentration and that maximal responses were blocked; moreover, on desensitized responses, methamidophos application accelerates the decay phase of the current during agonist application. These characteristics of the block make plausible hypothesis of a non-competitive inhibition of the receptor–channel complex; methamidophos may act either as an allosteric modulator of the nAChRs, such as for instance donepezil [5], or as an open channel blocker. However, the use and voltage dependence of methemidophos block indicates that the effect of methamidophos can be classified as “uncompetitive” antagonism. This type of block implies that the blocker can interact if the receptor has been activated. This form of antagonism,

similar to the one observed for mecamylamine [8], is therefore, dissimilar from conventional non-competitive antagonism when the blocker can interacts with resting as well as activated receptors. This blocking behavior is different from that of the other AChE inhibitor, donepezil, that is instead voltage-independent [5]. These results also point out that the organophosphate compound, methamidophos, could not be considered a pharmacological tool for investigating cholinergic functions after cholinesterase inhibition [7,16,17], but it also affects the function of nAChRs in the central nervous system. In a previous study, Camara et al. [4] found that in cultured hippocampal neurons, methamidophos did not affect the whole-cell currents induced by application of acetylcholine, glutamate or GABA; on the other hand our present paper describes a direct effect of methamidophos on nAChRs. This discrepancy could be due to a different nAChR subunit composition; in fact hippocampal cultured neurons mainly express homomeric ␣7 nAChRs that are blocked by low concentration of methyllicaconitine (MLA) [4], while postsynaptic receptors of substantia nigra dopaminergic neurons are mainly constituted by ␣4␤2 nAChRs [14]. Interestingly, other AChE inhibitors, such as physostigmine, galanthamine, donepezil and tacrine that are currently used in the treatment of AD [5,15,22] interfere with nAChRs. A reduced nAChR function in the dopaminergic cells could be partly associated with either symptoms of intoxication or a decreased resistance of these cells to endogenous and exogenous neurotoxins. Therefore, our data could also have clinical implications, if we consider the pattern of human exposure to methamidophos when it is applied as a pesticide.

References [1] P. Ascher, W.A. Large, H.P. Rang, Studies on the mechanism of action of acetylcholine antagonists on rat parasympathetic ganglion cells, J. Physiol. 295 (1979) 139–170.

S. Di Angelantonio et al. / Neuroscience Letters 369 (2004) 208–213 [2] B. Buisson, D. Bertrand, Open-channel blockers at the human alpha4beta2 neuronal nicotinic acetylcholine receptor, Mol. Pharmacol. 53 (1998) 555–563. [3] P. Calabresi, M.G. Lacey, R.A. North, Nicotinic excitation of rat ventral tegmental neurones in vitro studied by intracellular recording, Br. J. Pharmacol. 98 (1989) 135–140. [4] A.L. Camara, M.F. Braga, E.S. Rocha, M.D. Santos, W.S. Cortes, W.M. Cintra, Y. Aracava, A. Maelicke, E.X. Albuguergue, Methamidophos: an anticholinesterase without significant effects on postsynaptic receptors or transmitter release, Neurotoxicology 18 (1997) 589–602. [5] S. Di Angelantonio, G. Bernardi, N.B. Mercuri, Donepezil modulates nicotinic receptors of substantia nigra dopaminergic neurones, Br. J. Pharmacol. 141 (2004) 644–652. [6] S. Di Angelantonio, A. Nistri, Calibration of agonist concentrations applied by pressure pulses or via rapid solution exchanger, J. Neurosci. Methods 110 (2001) 155–161. [7] J.R. Genzen, D.S. McGehee, Short- and long-term enhancement of excitatory transmission in the spinal cord dorsal horn by nicotinic acetylcholine receptors, Proc. Natl. Acad. Sci. U.S.A. 100 (2003) 6807–6812. [8] R.A. Giniatullin, E.M. Sokolova, S. Di Angelantonio, A. Skorinkin, M.V. Talantova, A. Nistri, Rapid relief of block by mecamylamine of neuronal nicotinic acetylcholine receptors of rat chromaffin cells in vitro: an electrophysiological and modeling study, Mol. Pharmacol. 58 (2000) 778–787. [9] T. Gotz, U. Kraushaar, J. Geiger, J. Lubke, T. Berger, P. Jonas, Functional properties of AMPA and NMDA receptors expressed in identified types of basal ganglia neurons, J. Neurosci. 17 (1997) 204–215. [10] P. Grillner, A. Bonci, T.H. Svensson, G. Bernardi, N.B. Mercuri, Presynaptic muscarinic (M3) receptors reduce excitatory transmission in dopamine neurons of the rat mesencephalon, Neuroscience 91 (1999) 557–565. [11] A.M. Gurney, H.P. Rang, The channel-blocking action of methonium compounds on rat submandibular ganglion cells, Br. J. Pharmacol. 82 (1984) 623–642. [12] L. Khiroug, R. Giniatullin, E. Sokolova, M. Talantova, A. Nistri, Imaging of intracellular calcium during desensitization of nicotinic acetylcholine receptors of rat chromaffin cells, Br. J. Pharmacol. 122 (1997) 1323–1332. [14] R. Klink, A. de Kerchove d’Exaerde, M. Zoli, J.P. Changeux, Molecular and physiological diversity of nicotinic acetylcholine receptors in the midbrain dopaminergic nuclei, J. Neurosci. 21 (2001) 1452–1463.

213

[15] A. Maelicke, M. Samochocki, R. Jostock, A. Fehrenbacher, J. Ludwig, E.X. Albuquerque, M. Zerlin, Allosteric sensitization of nicotinic receptors by galantamine, a new treatment strategy for Alzheimer’s disease, Biol. Psychiatry 49 (2001) 279–288. [16] H.D. Mansvelder, D.S. McGehee, Cellular and synaptic mechanisms of nicotine addiction, J. Neurobiol. 53 (2002) 606–617. [17] H.D. Mansvelder, J.R. Keath, D.S. McGehee, Synaptic mechanisms underlie nicotine-induced excitability of brain reward areas, Neuron 33 (2002) 905–919. [18] N.B. Mercuri, A. Bonci, P. Calabresi, A. Stefani, G. Bernardi, Properties of the hyperpolarization-activated cation current Ih in rat midbrain dopaminergic neurons, Eur. J. Neurosci. 7 (1995) 462– 469. [19] E. Neher, J.H. Steinbach, Local anaesthetics transiently block currents through single acetylcholine-receptor channels, J. Physiol. 277 (1978) 153–176. [20] D. Paterson, A. Nordberg, Neuronal nicotinic receptors in the human brain, Prog. Neurobiol. 61 (2000) 75–111. [21] V.I. Pidoplichko, M. DeBiasi, J.T. Williams, J.A. Dani, Nicotine activates and desensitizes midbrain dopamine neurons, Nature 390 (1997) 401–404. [22] R.J. Prince, R.A. Pennington, S.M. Sine, Mechanism of tacrine block at adult human muscle nicotinic acetylcholine receptors, J. Gen. Physiol. 120 (2002) 369–393. [23] M. Quik, J.M. Kulak, Nicotine and nicotinic receptors; relevance to Parkinson’s disease, Neurotoxicology 23 (2002) 581–594. [24] M.D. Santos, M. Alkondon, E.F. Pereira, Y. Aracava, H.M. Eisenberg, A. Maelicke, E.X. Albuquerque, The nicotinic allosteric potentiating ligand galantamine facilitates synaptic transmission in the mammalian central nervous system, Mol. Pharmacol. 61 (2002) 1222–1234. [25] E. Sokolova, A. Nistri, R. Giniatullin, Negative cross talk between anionic GABAA and cationic P2X ionotropic receptors of rat dorsal root ganglion neurons, J. Neurosci. 21 (2001) 4958– 4968. [26] C.M. Thompson, Preparation, analysis, and toxicity of phosphotiolates, in: J.E. Chambers, P.E. Levi (Eds.), Organophosphates, Chemistry, Fate and Effects, Academic Press, San Diego, 1992, pp. 79–105. [27] F.M. Zhou, Y. Liang, J.A. Dani, Endogenous nicotinic cholinergic activity regulates dopamine release in the striatum, Nat. Neurosci. 4 (2001) 1224–1229. [28] F.M. Zhou, C.J. Wilson, J.A. Dani, Cholinergic interneuron characteristics and nicotinic properties in the striatum, J. Neurobiol. 53 (2002) 590–605.