Eurdpean Jouinal of Pharmacology, 217 (1992) 173-184

173

© 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00

EJP 52530

Characterization and autoradiographic distribution of [3H]AF-DX 384 binding to putative muscarinic M 2 receptors in the rat brain Isabelle A u b e r t , D a n i e l l e C6cyre, S e r g e G a u t h i e r a a n d R 6 m i Q u i r i o n

a

Departments of Neurology and Neurosurgery, a Psychiatry and Centerfor Studies in Aging, Faculty of Medicine, McGill Uni~,ersity, Douglas Hospital Research Center, Verdun, Quebec, Canada Received 30 September 1991, revised MS received 1 April 1992, accepted 14 April 1992

The novel radioligand [3H]AF-DX 384 binds specifically and saturably to putative muscarinic M 2 receptor sites in homogenates of rat cerebral cortex. In saturation studies, [3H]AF-DX 384 appears to bind to two subpopulations of sites/states, one of high affinity (K~I = 0.28 +_0.08 nM) and another of low affinity (K~2 = 28.0 _+5.0 nM). The maximal binding capacity (Bmax) of [3H]AF-DX 384 binding sites represented 9.7 +_2.3 fmol/mg protein (Bmaxl) and 1993 + 551 fmol/mg protein (Bmax2) for the high and low affinity sites/states, respectively. The ligand selectivity profile of [3H]AF-DX 384 (at 2 nM) revealed that (-)-quinuclidinyl benzylate - atropine > 4-diphenylacetoxy-N-methylpiperidine methobromide > AQ-RA 741 > AF-DX 384 > UH-AH 371 >> methoctramine > oxotremorine-M > hexahydro-sila-defenidol >> pirenzepine > carbamylcholine >>> nicotine. This suggests that under our assay conditions [3H]AF-DX 384 binds mostly to M2-1ike muscarinic receptors in the rat central nervous system. This is further supported by the clear M2-1ike pattern of distribution observed using quantitative receptor autoradiography. High densities of specific labelling were seen in areas such as the hypoglossal nucleus, the pontine nucleus, the superior colliculus, the motor trigeminal nucleus, various thalamic nuclei and certain cortical laminae. Compared to [3H]AF-DX 116, the percentage of specific binding detected with [3H]AF-DX 384 was much higher. This is likely to be related to the greater chemical stability and affinity of [3H]AF-EX 384. In addition, autoradiograms obtained with [3H]AF-DX 384 (2 nM) are of better quality with film exposure periods five times shorter than those needed for [ 3 H ] A F - D X 116 (10 nM). Therefore, [3H]AF-DX 384 displays a good selectivity for muscarinic M2 sites and offers major advantages, including higher affinity and greater stability, over previously used ligands. [3H]AF-DX 384; Muscarinic receptors; Muscarinic M 2 receptor subtype; Receptor binding techniques; Autoradiography

I. Introduction

The existence of multiple classes of muscarinic receptors is now well established (Levine and Birdsall, 1989; Hulme et al., 1990 for reviews; see also Waelbroeck et al., 1990a,b; 1991). Five different c D N A possibly encoding for as many receptor subtypes have been cloned (Bonner, 1989a,b for reviews). However, the pharmacological characterization of these various receptor types is limited by the lack of ligands able to recognize, with high selectivity, a single receptor class. Thus far, the existence of possibly up to four ( M 1 - M 4) different classes of pharmacologically relevant muscarinic receptors have been proposed (Waelbroeck et

Correspondence to: R. Quirion, Douglas Hospital Research Centre, 6875 LaSalle Boulevard, Verdun, Quebec H4H 1R3 Canada. Tel. 1.514.7616131 ext. 23333, fax 1.514.7662503.

al., 1990a,b; 1991). The M 1 site is characterized by its high affinity for the antagonist pirenzepine and its widespread distribution in the central nervous system (CNS) (Hammer et al., 1980; H a m m e r and Giachetti, 1982; Watson et al., 1982, 1984; Potter et al., 1984; Cort6s and Palacios, 1986; Mash and Potter 1986; Spencer et al., 1986; Giraldo et al., 1987). M 2 receptors have lower affinity for pirenzepine; AF-DX 116 acts as a comparatively potent antagonist on these receptor sites which are most predominant in peripheral ganglia, heart and certain areas of the CNS such as various lower brainstem nuclei (Hammer and Giachetti, 1982; Potter et al., 1984; Watson et al., 1984; Giachetti et al., 1986; H a m m e r et al., 1986; Spencer et al., 1986; Giraldo et al., 1987; Quirion and Boksa, 1986; Quirion et al., 1989; Regenold et al., 1987, 1989; Wang et al., 1987, 1989; Araujo et al., 1989). M 3 sites are less well characterized but appear to be widely distributed and especially sensitive to H H S i D

174 (hexahydro-sila-difenidol) and related compounds (Mutschler and Lambrecht, 1984; Lambrecht et al., 1989) and 4-DAMP (4-diphenylacetoxy-N-methylpiperidine methobromide) (Doods et al., 1987; Fisher and Heacock, 1988) although these drugs certainly lack the appropriate selectivity to be able to recognize a single receptor class (Michel et al., 1989b; Michel and Whiting, 1990; Micheletti et al., 1990; Araujo et al., 1991; Waelbroeck et al., 1991). Finally, M 4 receptors are poorly characterized. Muscarinic receptors of NG108-15 cells and 'B' binding sites of the striatum displayed low affinities for pirenzepine and AF-DX 116 but high affinities for methoctramine, himbacine, 4-DAMP and HHSiD and were therefore tentatively classified as potential M 4 receptors (Michel et al., 1989a; Waelbroeck et al., 1990a,b,c). With regard to radioligands previously used to study muscarinic receptor subtypes in the rat brain, [3H]pirenzepine has been widely utilized for the M l receptors (Watson et al., 1982,1984; Spencer et al., 1986; Quirion et al., 1989). For the M 2 subtype, a variety of radioligands have been used including the agonists [3H]oxotremorine-M (Potter et al., 1984; Spencer et al., 1986) and [3H]acetylcholine under muscarinic conditions (Kellar et al., 1985; Quirion and Boksa, 1986; Quirion et al., 1989; Schwartz, 1986) as well as non-selective antagonists such as [3H]N-methylscopolamine ([3H]NMS) and [3H]quinuclidinyl benzylate ([3H]QNB) in the presence of pirenzepine (Potter et al., 1984; Cort6s and Palacios, 1986; Mash and Potter, 1986). The first radioligand used to directly label the M 2 muscarinic binding sites was [3H]AF-DX 116 (11-[[2-[(diethylamino)methyl]- 1-piperidinyl]acetyl]5,11-dihydro-6H-pyrido-[2,3-b][1,4]-benzodiazepin-6one) (Regenold et al., 1987, 1989; Wang et al., 1987, 1989; Araujo et al., 1989). However, its lack of stability and poor signal-to-noise ratio clearly make [3H]AF-DX 116 less than desirable for receptor binding studies. Eberlein et al. (1989) have shown that AF-DX 384, an analogue of AF-DX 116, displays comparable selectivity with higher affinity for muscarinic M a binding sites than AF-DX 116. Therefore, the recent development of [3H]AF-DX 384 (5,11-dihydro-ll-[[(2-(2-[(dipropylamino)methyl]- 1-piperidinyl)ethyl) amino]carbonyl]-6H-pyrido-[2,3-b][1,4]-benzodiazepin6-one) is of interest as a putative new radioligand to characterize M 2 receptors (Entzeroth and Mayer, 1990; Miller et al., 1991). However, it has been suggested (D6rje et al., 1991; Miller et al., 1991; present study, see discussion) that [3H]AF-DX 384 cannot discriminate between cloned m2 and m4 muscarinic receptors. We report here on the characterization and quantitative autoradiography of [3H]AF-DX 384 binding sites in rat brain. It appears that [3H]AF-DX 384 is superior to all other ligands used thus far to study M2-1ike receptor binding sites.

2. Materials and methods

2.1. Animals and chemicals

Male Sprague-Dawley rats (190-230 g) were obtained from Charles River Breeding Farms (St-Constant, Quebec, Canada). [3H]AF-DX 384 (97-101 Ci/mmol) was generously provided by Dr. Steve Hurt (New England Nuclear Co., Boston, MA, USA). Unlabeled AF-DX 384, AQ-RA 741 and UH-AH 371 were kindly given by Dr. Henry Doods (Karl Thomae, Biberach an der Riss, Germany). Pirenzepine dihydrochloride was generously supplied by Drs. M. Watson (University of Medicine and Dentistry, Newark, NJ USA) and B. Wolfe (University of Pennsylvania, PA, USA). Atropine sulfate and L-nicotine (free base) were bought from Sigma Chemical Co. (St. Louis, MO, USA). 4DAMP, methoctramine, HHSiD and carbamylcholine were purchased from Research Biochemicals Inc. (Natick, MA, USA). Bovine serum albumin (98% fatty acid free) and Ecolite scintillation cocktail were from ICN Biomedicals, Inc. (Irvine, CA, USA). Glass filters No. 32 (Schleicher & Schuell) were purchased from Zymotech (Montreal, Quebec, Canada). 3H-Hyperfilm and standards were bought from Amersham (Oakville, Ontario, Canada). Developer (D-19) and fixer (Rapid fixer) used for 3H-Hyperfilm processing were from Kodak Chemical Inc. (Montreal, Quebec, Canada). All other chemicals were of analytical grade and purchased from Fisher Scientific (Montreal, Quebec, Canada). 2.2. Binding of [3H]AF-DX 384 to rat cerebral cortex membrane homogenates Rats were decapitated and brains rapidly removed and dissected on ice. Cerebral cortex was homogenized with a Brinkmann Polytron (setting 6, 20 s) in Krebs buffer of the following composition (in mM): NaCI 120; MgSO 4 1.2; KH2PO 4 1.2; glucose 5.6; NaHCO 3 25; CaC1 e 2.5; KCI 4.7 (pH 7.4). This homogenate was centrifuged for 10 rain at 49000 × g, the supernatant discarded and membrane pellet washed twice by resuspension in fresh buffer and recentrifugation. Aliquots of this homogenate (0.15-0.65 mg protein) were incubated in Krebs buffer with various concentrations of [3H]AF-DX 384 (0.04 up to 100 nM) for 60 min at room temperature (22°C) in a total volume of 0.5 ml. Bound [3H]AF-DX 384 was separated from free ligand by rapid filtration under reduced pressure, through glass filters presoaked in 0.05% polyethyleneimine solution, using a Brandel Cell Harvester apparatus (Gaithersburg, MD, U.S.A.). Filters were then rapidly washed with ice-cold buffer (3 × 4.5 ml each) before being dried. The radioactivity on filters was determined by liquid scintillation counting using a Beckman LS7000 scintillation counter with 48% efficiency. Non-

175 specific binding was defined in the presence of 1 /xM atropine sulfate. Specific binding was calculated as the difference between the radioactivity bound in the absence (total binding) and in the presence (nonspecific binding) of 1 /zM atropine and represented approximately 80% of total binding at a concentration of radioligand close to 2 nM. In a series of preliminary experiments, we c o m p a r e d various incubation temperatures (4, 22 and 37°C), time (5-120 min) and protein contents (0.15-4 mg protein). The best signal-to-noise ratio was obtained at 22°C for 1 h with a concentration of protein ranging between 0.15-0.65 mg (data not shown). U n d e r these conditions, equilibrium was reached within 45 min and was stable for at least 120 min. Therefore, all subsequent experiments were performed at 22°C using 1 h incubation as optimal time to reach equilibrium. The ligand selectivity profile of [3H]AF-DX 384 binding was determined in homogenates of rat brain cortices incubated in the presence of 2 nM [3H]AF-DX 384 and a broad range of concentrations ( 1 0 - u - 1 0 -5 M) of various cholinergic drugs. Protein was measured by the method of Lowry et al. (1951) using bovine serum albumin as the standard. All m e m b r a n e binding data were analyzed by the computerized method ( L I G A N D ) of Munson and Rodbard (1980) as modified by G.A. McPherson (1983).

from the skull and immersed in 2-methylbutane at - 4 0 ° C for several seconds and then stored at - 8 0 ° C until used. Coronal and sagittal frozen sections (20 txm thick) were cut at - 1 8 ° C , thaw-mounted on gelatin coated slides (two sections per slide), desiccated overnight at 4°C and then stored at - 8 0 ° C until used. On the day of the experiment, sections were brought back to room t e m p e r a t u r e and preincubated in Krebs buffer (same composition as above, see 2.2.) for 15 min before being incubated at 22°C for 1 h in fresh buffer containing 2 nM [3H]AF-DX 384, with or without 1 /zM atropine to determine the proportion of specific binding. After this incubation period, slides were transferred sequentially through three rinses (4 min each) in Tris HCI buffer (50 mM, p H 7.4) at 4°C, followed by a rapid dip in cold distilled water. Sections were then dried under a steam of cold air and tightly juxtaposed to tritium-sensitive film for 15 days. Films were developed as described before (Quirion et al., 1981) and [3H]AF-DX 384 binding was quantified using tritiumlabelled standards and computerized image analysis ( M C I D System, Image Research Inc., St-Catharines, Ontario, Canada). U n d e r these conditions, specifically bound [3H]AF-DX 384 represented close to the totality of bound ligand.

3. Results

2.3. Autoradiographic distribution of [3H]AF-DX 384 binding sites in rat brain R a t brain sections were p r e p a r e d as described before (Quirion et al., 1981). Briefly, rats were killed by decapitation and their brains were rapidly removed

3.1. Characteristics of [3H]AF-DX 384 binding sites to rat brain homogenates U n d e r the present assay conditions, the results have shown (fig. 1A, B; table 1) that, in the cerebral cortex,

A

3000 40

0 ,-

,

,

80-

1500

0

20

40

60

[3H]AF-DX384 (riM)

80 500 1000 [3H]AF-DX384 BOUND(fmol/rag protein)

Fig. 1. Saturation curves of [3H]AF-DX 384 binding to rat cerebral cortex homogenates. In (A): total (©), specific (e) and nonspecific (t:3) [3H]AF-DX 384 binding vs. free concentrations of [3H]AF-DX 384. Nonspecific binding was defined in the presence of 1 /~M atropine. In (B): Scatchard plot derived from full saturation analysis of [3H]AF-DX 384 revealed the existence of two binding sites/affinity states, one with low capacity (Bmaxl) and high affinity (Kal) and the other with a higher capacity (Bmax2)and lower affinity (Ka2). The inset is a blow-up of the data obtained at low concentrations of [3H]AF-DX 384. Data are representative of five separate experiments; each concentration was tested in duplicate.

176 TABLE 1 Binding parameters of [3H]AF-DX 384 to homogenates of rat cerebral cortex. Kdl and Ka2 represent the apparent affinity constants for the high and low affinity binding sites/states, respectively. Similarly, Bmaxl and Bma×2 represent the apparent maximal binding capacities and nil1 , nil2 are the Hill coefficient values for the high and low affinity binding components. Values are the means + S.E.M. of five separate experiments; each concentration of [3HIAF-DX 384 was tested in duplicate.

5O •

[3H]AF-DX 384 binding parameters

4-DAMP

• Ar-ox~

\ N\ \

%,

Apparent affinity (nM) Kdl Kd2

0.28 +_ 0,08 28.0 _+ 5.0

"2_ -11

Maximal capacity (fmol / mg protein) Bmaxl Bmax2

9.7 1993

+ 2.3 "+551

Hill coefficient n H1 n H2

0.986_+ 1.005 +

0.007 0.009

[3H]AF-DX 384 binds saturably, specifically and with high affinity to two populations of sites/states as revealed by the biphasic nature of the Scatchard plot. The apparent affinity (K d) is 100-fold greater for the high affinity (Kal) than for the low affinity (Kd2) site/states while maximal binding capacity (Bmax) is significantly greater (200 times) for this second site/ state (Bmax2) as compared to the first one (Bmaxl) (table 1). Hill coefficient values are close to unity for both sites (table 1). Using the LIGAND program (Munson and Rodbard, 1980; McPherson, 1983) to analyze the data, all saturation curves were best fitted (P < 0.001) with the two sites rather than the one-site model.

3.2. Ligand selectiuity profile of [3H]AF-DX 384 binding sites in rat brain homogenates As shown in fig. 2 and table 2, the ligand selectivity profile of [3H]AF-DX 384 binding at 2 nM suggests the labelling of the muscarinic M 2 receptors in the rat cerebral cortex. AF-DX 384 itself and the M z selective drug AQ-RA 741 (ll-[[4-[4-(diethylamino)butyl]-lpiperidinyl]acetyl]-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepin-6-one) (Eberlein et al., 1989; Doods et al., 1991), are potent competitors of [3H]AF-DX 384 binding as well as various non-selective drugs such as ( - ) - Q N B and atropine. The poorly selective antagonist 4-DAMP is also a potent inhibitor of [3H]AF-DX 384 binding while UH-AH 371 (6-chloro-5,10-dihydro5-[(1-methyl-4-piperidinyl)acetyl]-llH-dibenzo-[b,e][1, 4]-diazepine-ll-one hydrochloride), the ( + ) enantiomer of UH-AH 37, a pirenzepine-type compound (Eberlein et al., 1989), displays intermediate affinity. Methoctramine, a purported cardiac M 2 blocker (Giraldo et al., 1988; Melchiorre et al., 1987), ox-

:, -5

-9 -7 LOG [DRUG] (M)

Fig. 2. Comparative potencies of various cholinergic drugs for [3H]AF-DX 384 (2 nM) binding sites in rat cerebral cortex membrane homogenates. Results of selected cholinergic drugs (see table 1 for a more exhaustive list) are expressed as percent control and are representative of 4-13 experiments, in which each concentration (10-11-10 -5 M) of competing drugs was tested in duplicate. Nonspecific binding was determined in the presence of 1 ~M atropine and represents less than 25% of total binding under our assay conditions.

otremorine-M, a putative M 2 agonist, and the relatively selective M 3 antagonist HHSiD display moderate affinities for [3H]AF-DX 384 binding sites. Pirenzepine, a rather specific M t antagonist (Watson et al., 1982), is a weak competitor of [3H]AF-DX 384 binding sites. The agonist carbamylcholine also displays low affinity for [3H]AF-DX 384 binding sites. Finally, nicotine is unable to act on [3H]AF-DX 384 binding sites revealing their muscarinic nature.

TABLE 2 Comparative potency of various cholinergic drugs for [3H]AF-DX 384 binding sites in rat cerebral cortex. Homogenates were prepared as described in the text and incubated with 2 nM [3H]AF-DX 384 in the presence of a broad range of concentrations (10-H-10 5 M) of various cholinergic drugs, each being tested in duplicate in 3-13 different experiments (N). ICs0 represents the concentration (nM) of drug required to inhibit 50% of specifically bound [3H]AF-DX 384 (2 nM). Nonspecific binding was determined in the presence of 1 ~M atropine and represents less then 25% of total binding. Cholinergic drugs

IC50 (nM)

N

( - )-QNB Atropine 4-DAMP AQ-RA741 AF-DX 384 UH-AH 371 Methoctramine Oxotremorine-M HHSiD Pirenzepine Carbamylcholine Nicotine

0.47 _+0.01 0.55 + 0.08 2.2 _+ 0.5 4.1 _+ 0.9 7.2 -+ 0.6 14.9 +_ 3.5 73.5 __+ 5.2 127 -+ 14 128 _+ 3 220 + 22 250 + 37 > 10 000

4 4 4 4 13 4 3 4 3 4 8 4

177

3.3. Autoradiographic distribution of [3H]AF-DX 384 binding sites in rat brain The distribution of [3H]AF-DX 384 binding sites was investigated using in vitro receptor autoradiography on sagittal (fig. 3) and coronal (figs. 4-6) rat brain sections. Quantification of some of the regions analyzed is given in table 3. In the text, site densities are quantified as highest (190-115), high (114-80), moderate (79-40), low (39-25) and very low (~< 25 f m o l / m g wet tissue). The highest densities of [3H]AF-DX 384 binding sites are found in the external plexiform layer of the olfactory bulb (fig. 4A,B), the lateroposterior nucleus of the thalamus (fig. 5D), the superior colliculus (figs. 3C, 5E), the pontine nucleus (fig. 6A,B), and the hypoglossal nucleus (figs. 3C, 6F,G). High densities of [3H]AF-DX 384 binding sites are located in the olfactory tubercule (figs. 3A, 4C,D), the core and shell of the nucleus accumbens (fig. 4D), the caudate-putamen (figs. 3A, 4D,E), the anteroventral nucleus of the thalamus (figs. 3B, 5A) and the motor trigeminal nucleus (figs. 3B, 6C). Moderate densities of [3H]AF-DX 384 binding sites are present in the internal plexiform layer of the olfactory bulb (fig. 4A), the frontal cortex, the medial laminae being more heavily labelled than the superficial

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Hi

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[•

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and deep layers (figs. 3A, 4B,C), the parietal cortex (fig. 4D,G) and the occipital cortex (figs. 3C, 5E,F,G) in which the medial laminae is also enriched in [3H]AF-DX 384 labelling. The lateral septal nucleus, its ventral portion being more intensely labelled than the intermediate and dorsal parts (figs. 3C, 4E,F), the medial septal nucleus (figs. 3C, 4F), the vertical limb of the diagonal band nucleus (figs. 3C, 4F) and the laterodorsal and mediodorsal nuclei of the thalamus (fig. 5B), the ventrolateral and dorsolateral parts of the geniculate nucleus (fig. 5D) are also moderately labelled. The pyramidal layer of the hippocampal formation is enriched with [3H]AF-DX 384 binding sites followed by the stratum radiatum and oriens layers of the CA1 and CA3 fields of the Ammon's horn. Similarly, in the dentate gyrus, the granular layer is more enriched in [3H]AF-DX 384 binding sites than the molecular layer (figs. 3A,B, 5B,C,D,E). Moderate densities of [3H]AF-DX 384 binding sites are also found in the amygdala (fig. 5B), the inferior colliculus (figs. 3C, 5G, 6A), the interpeduncular nucleus (fig. 5E), the central gray matter (fig. 5F), the locus coeruleus (fig. 6C) and the facial nucleus (figs. 3B, 6D,E). Low densities of [3H]AF-DX 384 binding sites are observed in the ventral posteriomedial nucleus of the thalamus (fig. 3A) and in the medial part of the geniculate nucleus (fig. 5E) while very low labelling is seen in

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.... VDB

Fig. 3. Photomicrographsof the autoradiographicdistribution of [3H]AF-DX 384 binding sites in sagittal sections of the brain. Sections were incubated with 2 nM [3H]AF-DX 384 and nonspecificbinding, as represented in (D), was determined in presence of 1 p.M atropine. Anatomical identification is based on Paxinos and Watson's atlas (1982). Abbreviations used: AO, anterior olfactorynucleus; AV, anteroventral thalamic nucleus; CA1-3, fields CA1-3 of Ammon's horn of the hippocampus; cc, corpus callosum;Ce, cerebellum; CPu, caudate-putamen; DG, dentate gyrus of the hippocampus; Fr, frontal cortex; Hi, hippocampus; IC, inferior colliculus;LS, lateral septal nucleus;Mo5, motor trigeminalnucleus; MS, medial septal nucleus; nul2, hypoglossalnucleus; nu7, facial nucleus; OB, olfactorybulb; Oc, occipital cortex; SC, superior colliculus;Tu, olfactorytubercule; VDB, vertical limb of the diagonalband nucleus;VPM, ventral posteromedialthalamicnucleus.

178 the substantia nigra (fig. 5E) and in the cerebellum (figs. 3A,B,C 6B,C,D,E,F). Nonspecific labelling is undetectable in all brain regions (figs. 3D, 4H, 5H, 6H).

4. Discussion T h e present series of experiments indicate that [ 3 H ] A F - D X 384 is a selective radioligand suitable for the characterization of muscarinic M e receptors in rat

brain m e m b r a n e preparations as well as in quantitative receptor autoradiography. It offers major advantages over previously used radioligands, such as [3H]acetylcholine (Kellar et al., 1985; Schwartz et al., 1986; Quirion et al., 1989) and [ 3 H ] A F - D X 116 ( R e g e n o l d et al., 1987, 1989; W a n g et al., 1987, 1989; A r a u j o et al., 1989) especially in regard to its high affinity for M 2 binding sites and its chemical stability. T h e selectivity of [ 3 H ] A F - D X 384 and [ 3 H ] A F - D X 116 are comparable for muscarinic M 2 over M~ sites ( W a n g et al., 1987; A r a u j o et al., 1989; see also Eberlein et al., 1989; and present study) but the stability of the f o r m e r ensures a

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Fig. 4. Photomicrographs of the autoradiographic distribution of [3H]AF-DX 384 binding sites in coronal sections of the rat forebrain. Sections were incubated with 2 nM of [3H]AF-DX 384 and nonspecific binding, as represented in (H), was defined in the presence of 1 #M atropine. Anatomic identification is based on Paxinos and Watson's atlas (1982). Abbreviations used; ac, anterior commisura; aca, anterior commisura, anterior part; Acb, nucleus accumbens; aci, anterior commisura, intrabulbar part; AO, anterior olfactory nucleus; cc, corpus callosum; Cg, cingulate cortex; CPu, caudate-putamen, EPI, external plexiform layer of the olfactory bulb; fmi, forceps minor of the corpus callosum; Fr, frontal cortex; IGr, internal granular layer of the olfactory bulb; IPI, internal plexiform layer of the olfactory bulb; LSD, lateral septal nucleus, dorsal; LSI, lateral septal nucleus, intermediate; MS, medial septal nucleus; ON, olfactory nerve layer; Par, parietal cortex; Tu, olfactory tubercule; VDB, vertical limb of the diagonal band nucleus.

179

higher percentage of specific binding and better data reproducibility. Saturation analysis revealed that [3H]AF-DX 384 apparently binds to two classes of sites/affinity states in the rat cerebral cortex. Araujo et al. (1989) reported similar data using [3H]AF-DX 116. However, the apparent affinity values are significantly lower (higher affinity) for [3H]AF-DX 384. It is still unclear if these

[]

AV

vhc

two affinity sites represent specific receptor subtypes or different affinity states of a single class of receptors. Experiments to investigate the effects of GTP-related analogues and divalent cations should be of interest in this regard and may reveal if these two sites represent genuine receptor subtypes (Araujo et al., 1989; Spencer et al., 1988a,b; McKinney and Richelson, 1984; Hoss and Ellis, 1985). On the basis of the ligand selectivity

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nt Py Fig. 5. Photomicrographs of the autoradiographic distribution of [3H]AF-DX 384 binding sites in coronal sections of the rat forebrain and hindbrain. Sections were incubated with 2 nM [3H]AF-DX 384 and nonspecific binding, as represented in (H), was determined in the presence of 1 p.M atropine. Anatomical identification is based on Paxinos and Watson's atlas (1982). Abbreviations used: AMG, amygdala; AV, anteroventral thalamic nucleus; CA1-3, fields CA1-3 of Ammon's horn of the hippocampus; Ce, cerebellum; CG, central gray; CGPn, central gray of the pons; CPu, caudate-putamen; DG, dentate gyrus of the hippocampus; Ent, entorhinal cortex; GrDG, Granular layer of the dentate gyrus; Hi, hippocampus; IC, inferior colliculus; IP, interpeduncular nucleus; LD, laterodorsal thalarnic nucleus; LG, lateral geniculate nucleus; LP, lateroposterior thalamic nucleus; MD, mediodorsal thalamic nucleus; MG, medial geniculate nucleus; Mol, molecular layer of the dentate gyrus; Oc, occipital cortex; Or, oriens layer of the hippocampus; Par, parietal cortex; Py, pyramidal layer of the hippocampus; py, pyramidal tract; Rad, stratum radiatum layer of the hippocampus; Rt, reticular thalamic nucleus; SC, superior colliculus; SN, substantia nigra; Te, temporal cortex; vhc, ventral hippocampal commisura; VL, ventrolateral thalamic nucleus.

180 profile and Bmax values reported here, it would a p p e a r more likely that they represent two affinity states of a single receptor class. Entzeroth and Mayer (1990) have reported on the characterization of [3H]AF-DX 384 binding sites in the rat heart. While the ligand selectivity profile was similar to that observed in the present study, these authors reported the existence of a single class of high affinity sites in their preparation. This apparent discrepancy is probably related to the use of a different tissue preparation, the heart containing a more homogeneous population of M 2 binding sites, or to the limited range

(0.1-20 nM) of radioligand concentrations tested. More recently, Miller et al. (1991) have also reported that [3H]AF-DX 384 (0.3-30 riM) binds to a single population of sites in the rat heart. However, supporting our data, two population of sites or affinity states were detected in rat cortical m e m b r a n e homogenates (Miller et al., 1991). The ligand selectivity pattern of [3H]AF-DX 384 binding sites in rat brain cortex provides clear evidence for the specific labelling of muscarinic M2-1ike receptors. Various rather selective M 2 receptor antagonists including unlabeled A F - D X 384 (Eberlein et al., 1989)

Pn

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~ Pn

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pyx

Fig. 6. Phot'omicrographsof the autoradiographic distribution of [3H]AF-DX 384 binding sites in coronal sections of the rat hindbrain. Sections were incubated with 2 nM of [3H]AF-DX 384 and nonspecific binding, as represented in (H), was determined in the presence of 1/zM atropine. Anatomical identification is based on Paxinos and Watson's atlas (1982). Abbreviations used; Ce, cerebellum; IC, inferior colliculus; LC, locus coeruleus; lfp, longitudinal fasciculus pons; Mo5, motor trigeminal nucleus; nul2, hypoglossal nucleus; nu7, facial nucleus; PB, parabranchial nucleus; Pn, pontine nucleus; pyx, pyramidal decussation; Sol, nucleus of the solitary tract; SP5, spinal trigeminal nucleus; Ve, vestibular nucleus.

181 TABLE 3 Quantitative distribution of [3H]AF-DX 384 binding sites in selected areas of the rat brain. Sections were incubated with 2 nM [3H]AF-DX 384 and film autoradiograms analyzed using computerized densitometry as described in the text. Results are expressed in fmol/mg wet tissue weight and represent the means 5: S.E.M. of four experiments. Nonspecific binding of [3H]AF-DX 384 was below detection level in the presence of 1 /xM atropine. Brain area Olfactory bulb External plexiform layer Internal plexiform layer Olfactory tubercule Frontal cortex Superficial laminae Medial laminae Deep laminae Accumbens nucleus Core Shell Caudate-putamen Parietal cortex Superficial laminae Medial laminae Deep laminae Lateral septal nucleus Dorsal Intermediate Ventral Medial septal nucleus Vertical limb of the diagonal band Hippocampus CAl-oriens layer CAl-pyramidal layer CAl-stratum radiatum layer CA3-oriens layer CA3-pyramidal layer CA3-radiatum layer Dentate gyrus-granular layer Dentate gyrus-molecular layer Thalamus Anteroventral nucleus Ventral posteriomedial nucleus Laterodorsal nucleus Mediodorsal nucleus Lateroposterior nucleus Amygdala Geniculate nucleus Ventrolateral Dorsolateral Medial Superior colliculus (superficial graylayer) Substantia nigra Interpeduncular nucleus Occipital cortex Superficial laminae Medial laminae Deep laminae Central gray matter Inferior colliculus Pontine nucleus Cerebellum Locus coeruleus Motor trigeminal nucleus Facial nucleus Hypoglossal nucleus

[3H]AF-DX 384 binding (fmol/mg wet tissue) 190.2 +_10.2 73.2+ 7.2 100.4+ 2.8 63.15:6.7 75.5 + 6.4 70.8+_ 7.4 91.1 + 3.5 103.3+ 4,8 96.9 +_ 4.1 62.7+_ 2.7 69.6+_ 4.3 61,9 5:3.5 53.1 + 10.2 61.4+ 4.6 62.65:1.5 60.85:7.9 69.7 J: 2.9 55.1+ 2.3 72.45:4.1 60.15:2.5 43.2_+ 2.1 67.85:2.2 43.0+ 2.8 63.05:1.4 47.65:2.3 88.8+ 8.7 35.2+ 5.5 61.2_+ 5.8 56.45:6.8 118.2 + 25.3 52.4 + 7.0 41.5+ 6.8 55.6+ 1.1 39.45:2.1 116.1+_ 6.0 20.0+ 0.6 68.35:7.8 65.3_+ 4.4 77.3+_ 1.8 70.5+ 0.8 72.7 + 6.7 46.05:2.8 119.2 +_20.0 23.1 5:1.5 58.7 5:1.3 99.5 5:7,4 77.6_+ 7.4 125.5 5:23.5

182 and AQ-RA 741 (Eberlein et al., 1989; Doods et al., 1991) are potent competitors for [3H]AF-DX 384 binding sites, in contrast to the weak activity of the relatively selective M 1 antagonist pirenzepine (Birdsall et al., 1984; Messer and Hoss, 1987; Watson et al., 1982). This clearly reveals that under the present assay conditions, [3H]AF-DX 384 did not bind efficiently to M~ receptor sites. As expected, non-selective muscarinic antagonists such as atropine and ( - ) - Q N B also potently inhibited [3H]AF-DX 384 binding, while agonists like oxotremorine-M and carbamylcholine exhibited moderate affinities for [3H]AF-DX 384 binding sites. In contrast, the rather high potencies of the purported M 3 antagonist 4-DAMP (Doods et al., 1987; Fisher and Heacock, 1988) and of UH-AH 371 (the ( + ) enantiomer of the racemate UH-AH 37) (Eberlein et al., 1989) for [3H]AF-DX 384 binding sites is, at first, surprising. However, it is now clear that these compounds display a poor selectivity for most muscarinic receptor subtypes (Araujo et al., 1991; Micheletti et al., 1990; D6rje et al., 1991; Wess et al., 1991). For example, recent studies in CHO-K1 cell lines transfected with each of the five human muscarinic receptor genes (ml-m5) have shown that both 4-DAMP and UH-AH 37 have similar affinity for ml, m3, m4 and m5 receptors and lower affinity for m2 receptors (although still in the nanomolar range). It is thus now evident that 4-DAMP and UH-AH 37 may not be suitable tools to distinguish between most of the muscarinic receptor subtypes characterized thus far. The fact that HHSiD was found to be a rather weak competitor of [3H]AFDX 384 binding sites suggests the apparent lack of labelling of the M 3 receptor subtype by [3H]AF-DX 384. Finally, methoctramine displays an intermediate potency for [3H]AF-DX 384 binding sites. However, the similar affinities of this compound, as well as of AF-DX 384 and AQ-RA 741 for cloned m2 and m4 receptors expressed in CHO-K1 cells (D6rje et al., 1991; Miller et al., 1991) do not allow for the possible exclusion of labelling of the m4 receptor subtype by [3H]AF-DX 384. This could also relate to the relatively high potency of 4-DAMP and UH-AH 371 on [3H]AFDX 384 binding sites in our assay since both bind to cloned m4 receptor sites with high affinity (D6rje et al., 1991; Wess et al., 1991). It may be that a certain proportion of the sites labelled by [3H]AF-DX 384 represents a pharmacologically relevant m4 receptor present in rat brain. More definite evidence in support of this hypothesis will await the development of highly selective muscarinic m4 receptor ligands. The overall autoradiographic distribution of [3H] AF-DX 384 binding sites in the rat brain resembles that seen using other putative muscarinic M 2 ligands such as [3H]AF-DX 116 (Regenold et al., 1987, 1989; Wang et al., 1989), [3H]acetylcholine under muscarinic conditions (Quirion and Boksa, 1986; Schwartz et al.,

1986; Quirion et al., 1989), [3H]oxotremorine-M (Spencer et al., 1986), [3H]N-methylscopolamine (Cortes and Palacios, 1986) and [3H]QNB in the presence of pirenzepine (Mash and Potter, 1986). However, the higher percentage of specific labelling obtained with [3H]AF-DX 384 is certainly a major advantage over previous used ligands especially for autoradiographic studies. As expected if labelling M 2 receptor sites, high densities of [3H]AF-DX binding sites were visualized in various brainstem areas, the superior colliculus and certain thalamic nuclei. As compared to M~ sites (Cortes and Palacios, 1986; Mash and Potter, 1986; Spencer et al., 1986; Quirion et al., 1989), lower densities of labelling were detected in most cortical (especially in superficial laminae) and hippocampal areas, such as the oriens and stratum radiatum layers of the CA1 field of Ammon's horn and the molecular layer of the dentate gyrus. In the hippocampal formation, [3H]AF-DX 384 and [3H]AF-DX 116 (Regenold et al., 1987, 1989; Wang et al., 1989) labelling profiles are similar although the laminar definition obtained with [3H]AF-DX 384 is clearer. This is probably related to the higher affinity of this radioligand for M 2 sites. However, the distribution of [3H]AF-DX 116 and [3H]AF-DX 384 binding sites is different from those reported using either [3H]acetylcholine (Schwartz et al., 1986; Quirion et al., 1989), [3H]oxotremorine-M (Spencer et al., 1986) or [3H]QNB in the presence of pirenzepine (Mash and Potter, 1986). Globally, the localization of M 2 receptor sites observed with the later ligands is closer to that of choline acetyltransferase (CHAT) immunoreactivity with a very discrete pattern of labelling, especially of pyramidal cells (Fibiger, 1982). To explain this difference, we have previously suggested that [3H]AF-DX 116 (Regenold et al., 1989) and now possibly [3H]AFDX 384, may recognize an additional subtype of M 2 receptors in the hippocampal formation. The mRNA for the m2 receptor subtype is apparently very poorly expressed in the basal ganglia (including the caudate-putamen, nucleus accumbens and olfactory tubercule) and it as been suggested that labelling detected in this area with most M 2 ligands (including [3H]AF-DX 384) relates to their capacity to bind to m4 receptors which mRNA is heavily expressed in this area (Brann et al., 1988; Buckley et al., 1988; Vilar6 et al., 1991a, in press; Weiner et al., 1991). However, in most other brain regions, very good correlations are generally observed between the distribution of M 2 receptor binding sites and m2 mRNA (Vilarb et al., in press), this being especially evident when using [3H]AF-DX 384 as receptor probe (this study). In addition, the distribution of M 2 receptors binding sites labelled with [3H]AF-DX 384 correlates rather well with the immunocytochemical m2 receptors localization, particularly in various cortical laminae, in the

183

superior colliculus and in the pontine, the motor trigeminal and the facial nuclei (Levey et al., 1991). Finally, the presence of [3H]AF-DX 384 binding sites in various areas highly enriched with ChAT immunoreactive cells (Fibiger, 1982) supports their purported role as autoreceptors (Raiteri et al., 1984; Mash et al., 1985; Lapchak et al., 1989; Weiler, 1989) although it certainly does not exclude that a proportion of them are also postsynaptically located on cholinoceptive cells as well as in other cell types (Levey et al., 1991; Vilar6 et al., 1991b). In summary, [3H]AF-DX 384 appears to be a rather selective muscarinic Mz-like receptor ligand which possesses high affinity and good chemical stability ensuring a better signal-to-noise ratio than most of the previously used radioligands. It should thus be a most useful molecule in the further characterization of the M2-1ike receptors in various tissues.

Acknowledgements This work was supported by a grant from the Medical Research Council of Canada (MRCC) to R6mi Quirion, a 'Chercheur-Boursier' of the 'Fonds de la Recherche en Sant6 du Quebec' (FRSQ). lsabelle Aubert is the holder of a studentship from the Alzheimer Society of Canada.

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