Brain Research Bulletin,

Vol. 25, pp. 903-912. e PergamonPress plc. 1990.Printedin the U.S.A.

0361-92X)/90$3.00 + .oO

Muscarinic and Dopaminergic Receptor Subtypes on Striatal Cholinergic Interneurons VALINA L. DAWSON,t$’

TED M. DAWSONS2 AND JAMES K. WAMSLEY*t3

*Neuropsychiatric Research Institute, 700 First Avenue South, Fargo, ND 58103 fwestern Institute of Neuropsychiatry and the Departments of Psychiatry, Pharmacology and Toxicology University of Utah Health Sciences Center, Salt Luke City, UT 84132 $Department of Neurology, Hospital of the University of Pennsylvania, Philadelphia, PA 19104 Received 2 April 1990

DAWSON, V. L., T. M. DAWSON AND J. K. WAMSLEY. Muscarinic and dopaminergic receptor subtypes on striatal cholinBRAIN RES BULL 25(6) 903-912, 1990.-Unilateral stereotaxic injection of small amounts of the cholinotoxin, AF64A. caused minimal nonselective tissue damage and resulted in a significant loss of the presynaptic cholinergic markers [3H]hemicholinium-3 (45% reduction) and choline acetyltransferase (27% reduction). No significant change from control was observed in tyrosine hydroxylase or tryptophan hydroxylase activity; presynaptic neuronal markers for dopamine- and serotonin-contaming neurons, respectively. The AF64A lesion resulted in a significant reduction of dopamine D, receptors as evidenced by a decreasein [3H]sulpiridebinding (42% reduction) and decreaseof muscatink non-M, receptors as shown by a reduction in [3H]QNB binding in the presence of 100 nM pirenzepine (36% reduction). Saturation studies revealed that the change in [sH]sulpiride and [‘H]QNB binding was due to a change in B not K,. Intrastriatal injection of AF64A failed to alter dopamine D, or muscarinic M, receptors labeled with [“H]SCH23390 an~H]pirenzepine, respectively. In addition, no change in [3H]forskolin-labeled adenylate cyclase was observed. These results demonstrate that a subpopulation of muscarinic receptors (non-M,) are presynaptic on cholinergic intemeurons (hence, autoreceptors), and a subpopulation of dopamine D, receptors are postsynaptic on cholinergic intemeurons. Furthermore, dopamine D,, muscarinic M, and [3H]forskolin-labeled adenylate cyclase are not localized to striatal cholinergic intemeurons. ergic interneurons.

Acetylcholine AF64A Hemicholinium-3 binding Muscarinic receptor subtypes

Autoreceptors M, receptors

Cholinotoxin Non-M, receptors

THE regulation of motor control is an important function of the striatum (18-20, 77). Disorders of basal ganglia are manifest by the disruption of the control of motor movements and motor behavior as demonstrated in Parkinson’s Disease (49), Huntington’s Disease (49,58), Tourette’s Syndrome (lo), dystonia (49), and tardive dyskinesia (1). Primary and/or secondary changes in the dopaminergic system have been implicated in these disorders (1, 10, 11, 18, 30, 49, 58, 77). As such, many studies have focused on the role of striatal dopamine (DA). In addition, the ability to produce animal models of striatal dopaminergic hypofunction through selective destruction of dopaminergic cells with the neurotoxins 6-hydroxydopamine (69) and N-methyl-Q-phenyl-1,2,3,6tetrahydropyridine (MPTP) (29,39) and the development of selective and specific agonist and antagonist ligands for DA receptors, have advanced and facilitated the understanding of the role of the dopaminergic nigrostriatal pathway in control of movement. In contrast, less is known about the role of cholinergic inter-

D, receptors D, receptors Dopamine receptor subtypes

neurons in relation to striatal circuitry. Evidence indicates that striatal cholinergic intemeurons are involved in the translation of nigrostriatal dopaminergic transmission to motor patterns (41) and that there are direct synaptic contacts within the striatum between dopaminergic and cholinergic neurons as demonstrated by electron microscopic techniques (38). Biochemical studies suggest that cholinergic intemeurons are directly modulated by activity at dopamine type-2 (D2) receptors (20,63), but not dopamine type-l receptors (Dr) [dopamine receptors are classically subdivided into two groups based on the ability to activate adenylate cyclase, (Di), or the ability to attenuate the activity of this enzyme, (D2) (36,68)]. Other biochemical evidence suggests a role for non-M, muscarinic receptors, but not muscarinic type-l (M,) receptors (33), in the control of intrinsic cholinergic neurons of the striaturn. [M, receptors are defined as the muscarinic receptors which exhibit a high affinity for pirenzepine (PZ), while non-M, receptors are those which demonstrate a low affinity for this compound

‘Resent address: National Institute on Drug Abuse., Addiction Research Center, P.O. Box 5180, Baltimore, MD 21224. *Resent address: Department of Neuroscience, WBSB807, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205. ‘Requests for reprints should be addressed to Dr. James K. Wamsley, Neuropsychiatric Research Institute, 120 Eighth Street South, Fargo, ND 58103.

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(see below)]. Although progress has been made in the study of striatal cholinergic intemeuronal function, the cellular (i.e., preand postsynaptic) location of receptor subtypes associated with this population is not completely understood. Prior to molecular cloning experiments, Ml receptors (new classification-M,) were defined as the muscarinic receptors which exhibit a high affinity for pirenzepine (PZ), while M2 receptors (new classificationnon-M,) were those which demonstrated a low affinity for this compound (4). It is now known that there are at least 5 muscarinic receptor subtypes expressed in the brain (6, 7, 56). The anatomical distribution of the mRNA for each receptor subtype has been mapped by in situ hybridization (940) and the pharmacological profile of each receptor has been determined (8). Muscarinic receptor nomenclature has been recently changed [see (3)] in order to reflect these advances. Ml receptors are- now designated M, (its genetic counterpart is m,). M2 (non-M,) receptors are now a series of receptors designated M,, Ma, M, and M, (their genetic counterparts are m2, m3, m, and m5 respectively). Not all the genetic receptors can currently be differentiated by pharmacologic probes [see (3)]. In this study, [3H]QNB labels all muscarinic receptors, [3H]PZ preferentially labels M, receptors and [3H]QNB + 100 nM PZ preferentially labels non-M, (M,-M,) receptors. A useful tool to study the relationship of cholinergic intemeurons and receptor localization within the striatum is the neurotoxin, ethylcholine mustard aziridinium ion (AF64A), which has been shown to be a relatively selective neurotoxin for cholinergic neurons when used under precisely controlled conditions (47,48). Biochemical and histological examination of striatal tissue, following AF64A injections, have demonstrated a neurotoxic effect against cholinergic nerves while sparing gamma-aminobutyric acid-(GABA) and DA-containing neurons with selected concentrations of AF64A (62). In this study, we show a reduction in non-M, receptor populations and D, receptors, after intrastriatal injections of AF64A, that correlates with the loss of the presynaftic cholinergic markers, choline acetyltransferase (ChAT), and [ H]hemicholinium-3 ([3H]HC-3) (72). Additionally, the lesion does not result in a change in yresynaptic serotonergic and dopaminergic neuronal markers, [ Hlforskolin-labeled adenylate cyclase (AC) or D, and M, receptors (15). METHOD

Lesion Procedures AF64A was prepared according to the method of Clement and Colhoun (12). Briefly, acetylethylcholine mustard HCl (Research Biochemicals, Inc., Wayland, MA) was diluted in distilled water and brought to pH 11.5 for 30 min with 1 N NaOH. The aziridinium ion (AF64A) was formed by lowering the pH to 7 with 0.1 N HCl and adjusting to pH 7.4 with NaHCO,. Additional distilled water was added for a final concentration of 1 nM AF64A. The cyclized solution was kept cool and injected within 2 h of cyclization. The vehicle control solution was distilled water to which an equivalent amount of 1 N NaOH was added, followed by pH adjustment with HCl and NaHCO,, as with the AF64A solution. Male Sprague-Dawley rats (170-190 g) were anesthetized with chloral hydrate (150-300 mglkg) and placed in a David Kopf small animal stereotaxic instrument (Tujunga, CA). A 26-gauge needle on a O.Ol-ml Hamilton microliter syringe (Hamilton Co.; Reno, NV) was lowered through a burr hole into the striatum. Prior to injecting AF64A, methylene-blue was injected in a test animal to confii coordinates. Injections of AF64A (0.75 nmol/ 0.75 p.1) were made at 4 sites. All coordinates were referenced to

DAWSON AND WAMSLEY

lambda and are as follows: 1) rostral +7.2 mm, lateral +2.6 mm, ventral -5.0 mm, 2) rostra1 +7.2 mm, lateral +2.6 mm, ventral -6.0 mm, 3) rostra1 +7.2 mm, lateral +2.4 mm, ventral -5.0 mm, 4) rostra1 +7.2 mm, lateral +2.4 mm, ventral -6.0 mm. These coordinates place the injection site in the central caudate-putamen. Injections were made over two minutes at each of the four sites. After each injection, the needle was left in place for 5 min to minimize diffusion of neurotoxin along the needle tract. Control animals received injections of an equal volume of vehicle alone. The animals were placed in separate cages, given free access to standard laboratory feed and water, and housed for 14 days in a room with lighting controlled on a 12-h light/dark cycle. The animals were then deeply anesthetized with chloroform and given an intracardial perfusion with ice-cold saline. For receptor autoradiography, the brain was immediately removed and frozen by slow immersion into - 70°C isopentane. Serial coronal sections of tissue, 10 microns thick, were cut on a cryostat (maintained at - 16°C) and then thaw-mounted onto cold, chrome-alum/gelatin-coated microscope slides. The slides were stored in slide boxes in a -70°C freezer until used. For enzyme analysis, the animals were handled the same but they were not perfused. The brain was immediately removed and the caudate-putamen in the region of the needle track in both treated and control brains was microdissected over ice and stored at - 70°C until analyzed. Receptor Binding Conditions Techniques for labeling tissue sections with [3H]SCH23390 (13), [3H]sulpiride (25), [3H]QNB (quinuclidinyl benzilate) (75), [3H]pirenzepine (PZ) (78), [3H]hemicholinium-3 ([3H]HC-3) (72) and [3H]forskolin (24) have been described. Briefly, D, receptors were labeled by incubating tissue sections for 30 min at 25°C in a 50 mM Tris buffer (120 mM NaCl, 5 mM KCl, 2 mM CaCl,, and 1 mM MgCl, at pH 7.4) containing 1 nM [3H]SCH23390 (80.0 Ci/mmol, Dupont NEN; Boston, MA). Nonspecific binding was defined by the addition of 1 pM [SCH23390] (ScheringPlough Research; Bloomfield, NJ). The slides were rinsed in fresh, ice-cold buffer (2 x 5 min) before drying. Other tissue sections were incubated with 20 nM [3H]sulpiride (76.0 Ci/mmol, Dupont NEN; Boston, MA) in a 0.17 M Tris buffer (120 mM NaCl, 5 mM KCl, 2 mM CaCl,, 1 mM MgCl,, and 0.001% ascorbate at pH 7.7) for 20 min in order to label D, receptors. A 1 pM concentration of haloperidol was used to determine nonspecific binding. Tissue sections were rinsed in fresh buffer at 4°C (4 x 1 min) before drying. Saturation studies with [3H]sulpiride were performed under similar conditions in which the concentration of [3H]sulpiride was varied from 10 nM to 40 nM with at least 7 different concentrations. Muscarinic receptors were labeled by incubating with 1 nM [3H]QNB (34.7 Ci/mmol, DuPont NEN; Boston, MA), in Kreb’s phosphate buffer (pH 7.4) at 25°C for 60 min, and non-M, receptor populations were defined by 1 nM [3H]QNB plus 100 nM PZ. This concentration of PZ was chosen to occupy all M, sites while leaving the majority of the non-M, sites free for labeling with the radioactive compound (52). Nonspecific binding was defined by the addition of 1 p,M atropine (Sigma, St. Louis, MO) to the incubation bath. The slides were then rinsed (2 X 5 min) in fresh, ice-cold buffer before drying. Saturation experiments with [3H]QNB involved the same conditions, but the concentration of radiolabeled QNB in the incubation bath was varied from 0.1 nM to 5.0 nM with at least 7 different concentrations. M, receptors were labeled directly by incubating tissue sections with 20 nM [3H]PZ (76.0 Ci/mmol, Dupont NE.N; Boston, MA) for 60 min, at room temperature, in Kreb’s phosphate buffer (pH 7.4) following a 30-r& preincubation at room temperature in buffer alone.

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TPH FIG. 1. Choline acetyltransferase (ChAT), tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH) enzyme activity in the caudate-putamen of animals receiving intrastriatal injection of the cholinergic neurotoxin, AF64A, or vehicle (see text for details). The tissue was microdissected from the caudate-putamen in the immediate region of the lesion for all enzymatic assays. The data represent the meant S.E.M. from three experimental and three control animals. *p