Presynaptic nicotinic receptors modulating dopamine release in the rat striatum

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Abstract

The modulation of striatal dopamine release by presynaptic nicotinic acetylcholine receptors is well documented for both synaptosomes and slices. Because the latter retain local anatomical integrity, we have compared [3H]dopamine release evoked by the nicotinic receptor agonists (−)-nicotine and (±)-anatoxin-a from striatal synaptosome and slice preparations in parallel. At higher agonist concentrations, mecamylamine-sensitive [3H]dopamine release was greater from slices, indicative of an additional component, and this increase was abolished by glutamate receptor antagonists. To begin to examine the localisation of specific nicotinic acetylcholine receptor subtypes in the striatum, immunogold electron microscopy was undertaken with the β2-specific monoclonal antibody 270. In striatal sections, gold particles were associated with symmetric synapses (dopaminergic) but were absent from asymmetric synapses (glutamatergic). Surface labelling of striatal synaptosomes with gold particles was also demonstrated. Taken together, these results are consistent with dopamine release mediated by β2-containing nicotinic acetylcholine receptors on dopamine terminals, while non-β2-containing nicotinic acetylcholine receptors may enhance dopamine release indirectly by releasing glutamate from neighbouring terminals.

Introduction

The widespread occurrence of nicotinic acetylcholine receptors at presynaptic locations in the central nervous system is well established (Wonnacott, 1997). The modulatory actions of presynaptic nicotinic acetylcholine receptors, by stimulating transmitter release or by enhancing synaptic efficacy (e.g., Gray et al., 1996), are considered to constitute a major role of neuronal nicotinic acetylcholine receptors (Role and Berg, 1996). The nicotinic modulation of transmitter release can be measured directly in vitro using neurochemical methods (Kaiser and Wonnacott, 1998) and dopamine release from striatal preparations has been the most widely studied example. Not only is the nicotinic stimulation of dopamine release robust, but it has merited study because of the role of dopamine in the rewarding and locomotor activating properties of nicotine. It is also pertinent to the therapeutic potential of nicotinic drugs for Parkinson's disease (Menzaghi et al., 1997).

Synaptosomes enable the direct modulation of nerve terminal function to be studied unambiguously, as anatomical connections are severed in the preparation and superfusion methodology is considered to prevent cross-talk between synaptosomes (Raiteri et al., 1974). The stimulation of [3H]dopamine release from striatal synaptosomes by nicotinic receptor agonists has been widely reported, and shown to be dose dependent and inhibited by antagonists such as dihydroβerythroidine and mecamylamine, but not by α-bungarotoxin Rapier et al., 1990, Grady et al., 1992, el-Bizri and Clarke, 1994, Soliakov et al., 1995. The nicotinic stimulation of [3H]dopamine release is Ca2+ dependent, but is also blocked by Cd2+ and by antagonists of voltage-operated Ca2+ channels (Soliakov and Wonnacott, 1996), suggesting that the mechanism of triggering transmitter release involves local depolarisation and opening of Ca2+ channels. Recent evidence suggests that nicotinic acetylcholine receptors on striatal dopamine terminals are heterogeneous, comprising one population that contains α3 and β2 subunits (that is blocked by α-conotoxin MII) and a second population that is insensitive to this toxin, possibly containing α4 and β2 subunits Kulak et al., 1997, Kaiser et al., 1998.

Slice preparations (prisms or minces) retain local anatomical integrity, enabling neuronal interactions to be examined. This may account for the differences in sensitivity to tetrodotoxin of nicotine-evoked [3H]dopamine release from synaptosomes and slices (Marshall et al., 1996). The nicotinic stimulation of dopamine release from slices is dose-dependent (Sacaan et al., 1995). Dopamine release is subject to modulation by a diversity of transmitters via heteroreceptors present on dopamine terminals (Langer, 1997). In particular, there is considerable evidence that glutamate can elicit dopamine release, by acting at AMPA and NMDA receptors on dopamine terminals in the striatum (Cheramy et al., 1996). Moreover, locally applied nicotine can provoke glutamate release, measured by in vivo microdialysis (Toth et al., 1993). In vitro, electrophysiological studies have disclosed the ability of presynaptic nicotinic acetylcholine receptors to modulate glutamate release in rat hippocampus (Gray et al., 1996) and olfactory bulb Alkondon and Albuquerque, 1995, Alkondon et al., 1996.

These various observations have prompted us to compare striatal synaptosomes and slices with respect to the release of [3H]dopamine stimulated by nicotinic receptor agonists, and to assess the effects of ionotropic glutamate receptor antagonists on nicotinic acetylcholine receptor-evoked [3H]dopamine release. This is coupled with an immunocytochemical study, at the electron microscope level, to begin to address the localisation of presynaptic nicotinic acetylcholine receptors in the rat striatum.

Section snippets

Materials

[7,8-3H]Dopamine (specific activity, 1.78 Tbq/mmol) was purchased from Amersham International, Amersham, Bucks, UK. Pargyline and (−)-nicotine ditartrate were purchased from Sigma, Poole, Dorset, UK. Nomifensine was obtained from R.B.I., Poole, Dorset, UK. and (±)anatoxin-a, 6,7-dinitroquinoxaline-2,3-dione (DNQX), and kynurenic acid were obtained from Tocris Cookson, Bristol, UK. Rat monoclonal antibody to nicotinic acetylcholine receptor β2 subunit (monoclonal antibody 270) was kindly donated

[3H]Dopamine release

(−)-Nicotine and (±)-anatoxin-a were compared for their abilities to release [3H]dopamine from rat striatal synaptosomes and slices (Fig. 1). A 40-s pulse of agonist elicited a peak of radioactivity above basal release that was almost totally abolished in the presence of 10 μM mecamylamine (Fig. 1A,B). A range of concentrations of the agonists was tested in the presence and absence of mecamylamine, and the mecamylamine-insensitive component of release was subtracted to yield dose–response

Discussion

The dose-dependant release of [3H]dopamine from striatal slices and synaptosomes is well documented Grady et al., 1992, el-Bizri and Clarke, 1994, Sacaan et al., 1995 but these preparations have rarely been compared in the same laboratory. Here we have demonstrated that higher concentrations of both nicotine and anatoxin-a evoke a relatively greater response from slices than from synaptosomes (Fig. 1C,D). As slices retain local anatomical integrity, they afford an opportunity for additional

Acknowledgements

This study was supported by grants from the Biological and Biotechnological Sciences Research Council (BBSRC) and BAT, and a studentship from the BBSRC in conjunction with SmithKline Beecham (to AM). We are grateful to Prof. Jon Lindstrom for monoclonal antibody 270 and to Prof. Paul Bolam for Lowicryl blocks of striatal tissue, and for advice.

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    Present address: Department of Biology, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0357, USA.

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