a-Bungarotoxin-Sensitive Nicotinic Receptors Indirectly Modulate [H]Dopamine Release in Rat Striatal Slices via Glutamate Release

Nicotinic agonists elicit the release of dopamine from striatal synaptosomes by acting on presynaptic nicotinic acetylcholine receptors (nAChRs) on dopamine nerve terminals. Both a3b2* and a4b2 nAChR subtypes (but not a7* nAChRs) have been implicated. Here, we compared nAChR-evoked [H]dopamine release from rat striatal synaptosome and slice preparations by using the nicotinic agonist anatoxin-a. In the more integral slice preparation, the concentration-response curve for anatoxin-aevoked [H]dopamine release was best fitted to a two-site model, giving EC50 values of 241 nM and 5.1 mM, whereas only the higher-affinity component was observed in synaptosome preparations (EC50 5 134 nM). Responses to a high concentration of anatoxin-a (25 mM) in slices (but not in synaptosomes) were partially blocked by ionotropic glutamate receptor antagonists (kynurenic acid, 6,7-dinitroquinoxaline-2,3-dione) and by a7*-selective nAChR antagonists (a-bungarotoxin, a-conotoxin-ImI, methyllycaconitine) in a nonadditive manner. In contrast, the a3b2-selective nAChR antagonist a-conotoxin-MII partially inhibited [H]dopamine release from both slice and synaptosome preparations, stimulated with both low (1 mM) and high (25 mM) concentrations of anatoxin-a. Antagonism by a-conotoxin-MII was additive with that of a7*-selective antagonists. These data support a model in which a7* nAChRs on striatal glutamate terminals elicit glutamate release, which in turn acts at ionotropic glutamate receptors on dopamine terminals to stimulate dopamine release. In addition, non-a7* nAChRs on dopamine terminals also stimulate dopamine release. These observations have implications for the complex cholinergic modulation of inputs onto the major efferent neurons of the striatum. The dorsal striatum is concerned with the control of movement. The principal output neurons from the striatum, the GABAergic medium spiny neurons, receive glutamatergic afferents from the cortex and thalamus and dopaminergic inputs from the substantia nigra (Smith and Bolam, 1990). Cholinergic interneurons also synapse onto the medium spiny neurons. In addition to these well-established synaptic relationships, there is increasing evidence for neurochemical cross talk between the terminals of afferent neurons via presynaptic receptors. This may provide a basis for new therapeutic approaches for the treatment of movement disorders that arise from the degeneration of neuronal subpopulations (Parkinson’s and Huntington’s diseases) or as a side effect of clinical treatments (tardive dyskinesia). In vitro, glutamate stimulates the release of [H]dopamine from rat striatal slices (Roberts and Anderson, 1979), and this effect appears to be mediated by both a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate and N-methyl-D-aspartate (NMDA) receptors present on dopaminergic nerve terminals (Wang, 1991; Desce et al., 1992). Consistent with this view, in vivo infusion into the striatum via a microdialysis probe of either NMDA (Keefe et al., 1992; Morari et al., 1993; Kendrick et al., 1996) or AMPA (Kendrick et al., 1996; Smolders et al., 1996) increased local release of dopamine. There also is strong evidence for the presence on dopamine terminals of nicotinic acetylcholine receptors (nAChRs) capable of enhancing the basal release of dopamine (Wonnacott, 1997). These nAChRs appear to be heterogeneous, composed of subtypes containing a3 and b2 subunits (a3b2* nAChRs; Kulak et al., 1997; Kaiser et al., 1998) and a4 and b2 subunits (a4b2 nAChRs; Sharples et al., 2000). Furthermore, locally applied (2)-nicotine in vivo has been shown to increase striatal levels of dopamine (Marshall et al., 1997) and glutamate (Toth et al., 1993) in a mecamylaminesensitive manner. Moreover, the local application of NMDA antagonists diminished the ability of locally applied (2)This work was supported by grants from the Biological and Biotechnological Sciences Research Council and European Community. 1 Present address: Department of Biology, University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0357. ABBREVIATIONS: AMPA, a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; AnTx-a, (6)-anatoxin-a; a-Bgt, a-bungarotoxin; NMDA, Nmethyl-D-aspartate; aCtx-MII, a-conotoxin-MII; aCtx-ImI, a-conotoxin-ImI; DNQX, 6,7-dinitroquinoxaline-2,3-dione; MLA, methyllycaconitine; nAChR, nicotinic acetylcholine receptor. 0026-895X/00/020312-07$3.00/0 MOLECULAR PHARMACOLOGY Copyright © 2000 The American Society for Pharmacology and Experimental Therapeutics MOL 58:312–318, 2000 /147/841788 312 at A PE T Jornals on A uust 4, 2017 m oharm .aspeurnals.org D ow nladed from nicotine to elicit dopamine release in vivo (Toth et al., 1992). These data lead to the hypothesis that (2)-nicotine can also act at presynaptic nAChRs on striatal glutamatergic nerve terminals to release glutamate, which in turn stimulates the release of dopamine via presynaptic ionotropic glutamate receptors on dopaminergic terminals. Recent electrophysiological recordings from striatum in situ are consistent with this argument (Garcı́a-Muñoz et al., 1996). Here, we examined the relationship between nAChRs, glutamate receptors, and dopamine release in the striatum in vitro. Comparative experiments using perfused synaptosomes (which represent isolated nerve terminals with low probability of neurochemical cross-talk) and slices (which preserve some of the anatomical integrity of the striatum) provide evidence for a component of [H]dopamine release in slices, but not in synaptosomes, that is sensitive to glutamate receptor antagonists and a7*-selective nAChR antagonists. These results are consistent with an indirect modulation of dopamine release in striatum via a7* nAChRs on striatal glutamatergic nerve terminals. Experimental Procedures Materials. Adult male Sprague-Dawley rats were obtained from the University of Bath Animal House breeding colony. [7,8-H]Dopamine (1.78 TBq/mmol) was purchased from Amersham International (Buckinghamshire, UK). a-Conotoxin-MII (aCTx-MII) was synthesized with correct disulfide bond formation as previously described (Cartier et al., 1996; Kaiser et al., 1998). (6)-Anatoxin-a (AnTx-a), 6,7-dinitroquinoxaline-2,3-dione (DNQX), and kynurenic acid were obtained from Tocris Cookson (Bristol, UK). a-ConotoxinImI (aCTx-ImI) was obtained from Calbiochem (San Diego, CA). Methyllycaconitine (MLA) and 4-aminopyridine were purchased from Research Biochemicals International (Natick, MA). a-Bungarotoxin (a-Bgt), mecamylamine, pargyline, and nomifensine were purchased from Sigma Chemical Co. (Poole, Dorset, UK). All other chemicals used were of analytical grade and were obtained from standard commercial sources. Superfusion of Rat Striatal Slices and Synaptosomes. Male Sprague-Dawley rats (approximately 250 g) were sacrificed by cervical dislocation and decapitated, and brain striata (180–240 mg tissue wet wt./rat) were rapidly dissected. P2 synaptosomes were prepared by differential centrifugation as previously described (Soliakov et al., 1995). Synaptosomes were loaded with [H]dopamine (0.1 mM, 0.132 MBq/ml) for 15 min at 37°C and superfused in open chambers (Soliakov et al., 1995). All superfusion experiments were performed in Krebs-bicarbonate buffer of the following composition: 118 mM NaCl, 2.4 mM KCl, 2.4 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 25 mM NaHCO3, and 10 mM glucose, buffered to pH 7.4 with 95% O2, 5% CO2 and supplemented with 1 mM ascorbic acid, 8 mM pargyline, and 0.5 mM nomifensine to prevent dopamine degradation and reuptake. Striatal slices (0.25 mm) were prepared as previously described (Marshall et al., 1996) using a McIlwain tissue chopper. Striatal tissue prisms were washed twice with Krebs-bicarbonate buffer and loaded with [H]dopamine (0.1 mM, 0.132 MBq/ml) for 15 min at 37°C. After two washes, slices were resuspended in Krebs’ buffer and loaded into superfusion chambers (approximately 45–50 mg of slices per chamber). Superfusion of synaptosomes (open chambers) or slices (closed chambers) was performed as previously described (Soliakov et al., 1995; Marshall et al., 1996) in a Brandel Superfusion Apparatus model SF-12 (Montreal, Quebec, Canada), using Krebsbicarbonate buffer at 37°C and a flow rate of 0.5 ml/min; 2-min fractions were collected. Chambers containing either synaptosomes or slices were washed for 20 min with Krebs-bicarbonate buffer, followed by a further 10 min with normal buffer or buffer containing antagonist (112 nM aCtx-MII, 1 mM aCtx-ImI, 50 nM MLA, 10 mM mecamylamine, 500 mM kynurenic acid, 100 mM DNQX). In the case of a-Bgt (40 nM) the preincubation time was extended to 1 h. Then, the nicotinic agonist AnTx-a or general depolarizing agent (KCl or 4-aminopyridine) was applied for 40 s in the presence or absence of antagonist. The 40-s drug pulse was separated from the bulk buffer flow by 10-s air