SYNAPSE 9:165-176 (1991)

Heterogeneous Binding of r3H]4-DAMP to Muscarinic Cholinergic Sites in the Rat Brain: Evidence From Membrane Binding and Autoradiographic Studies -

DALIA M. ARAUJO, PAUL A. LAPCHAK, AND REMI QUIRION Douglas Hospital Research Center and Departments of Psychiatry and Pharmacology, Faculty of Medicine, McGill Uniuersity, Montreal, Quebec, Canada

KEY WORDS

4-DAMP, Muscarinic receptor subtypes, Autoradiographic localization, Membrane binding techniques

ABSTRACT The present study shows that t3H14-DAMPbinds specifically, saturably, and with high affinity to muscarinic receptor sites in the rat brain. In homogenates of hippocampus, cerebral cortex, striatum, and thalamus, L3H14-DAMPappears to bind two sub-populations of muscarinic sites: one class of high-affinity, low capacity sites (I(d < 1 nM; B, = 45-152 fmoYmg protein) and a second class of lower-affinity, high capacity = 263-929 fmoVmg protein). In cerebellar homogenates, the B, sites (& > 50 nM; B, of L3H14-DAMPbinding sites was 20 2 and 141 2 21 fmoYmg protein for the high- and the lower-affinity site, respectively. The ligand selectivity profile for 13H14-DAMPbinding t o its sites was similar for both the high- and lower-affinity sites; atropine = (- )QNB = 4DAMP >> pirenzepine > AF-DX 116, although pirenzepine was more potent (16-fold)at the lower- than at the high-affinity sites. The autoradiographic distribution of [3H]4DAMP sites revealed a discrete pattern of labeling in the rat brain, with the highest densities of [3H14-DAMP sites present in the CA, sub-field of Ammon’s horn of the hippocampus, the dentate gyrus, the olfactory tubercle, the external plexiform layer of the olfactory bulb and layers 1-11 of the frontoparietal cortex. Although the distribution of [3H]pirenzepine sites was similar to that of [3H]4-DAMPsites in many brain regions, significant distinctions were apparent. Thus, both the ligand selectivity pattern of L3H]4-DAMPbinding and the autoradiographic distribution of sites suggest that although the high-affinity c3H14-DAMPsites may consist primarily of muscarinic-M, receptors, the lower-affinity [3H]4-DAMPsites may be composed of a large proportion of muscarinic-M, receptors.

*

INTRODUCTION The heterogeneous nature of the muscarinic acetylcholine (ACh) receptor has been decisively demonstrated with the recent cloning of five different muscarinic receptors designated m l , m2, m3, m4, and m5 (Bonner et al., 1987,1988; for review, see Hulme et al., 1990). While there is a distinct distribution of each subtype in peripheral and central nervous tissues, all five subtypes appear to be expressed in both the periphery and brain (Bonner et al., 1987,1988; Buckley et al., 1988; Peralta et al., 1987; Weiner et al., 1990). Conventional pharmacological studies have identified at least three subtypes of muscarinic receptors subclassified as M,, M,, and M3 (Bloom et al., 1987; Doods et al., 1987; Duckles et al., 1987; Waelbroeck et al., 1986, 19871, but a clear’distinction between subtypes has been hampered in part by conflicting nomen0 1991 WILEY-LISS,INC.

clature (see Eglen and Whiting, 1986,1987; Waelbroeck et al., 1986,1987) and by the lack of drugs selective for one subtype over another. The MI subtype of muscarinic receptor can be directly and selectively labeled using I3H1pirenzepineas ligand (Araujo et al., 1988,1990; Quirion et al., 1989b; Watson et al., 1982). Similarly, selective ligands for M,-muscarinic receptors include L3H1ACh, in the presence of nicotine, (Araujo et al., 1988; Gurwitz et al., 1985; Kellar et al., 1985; Quirion et al., 1989b; Schwartz, 19861, [3Hloxotremorine-M (Spencer et al., 1986; Gillard et al., 1987), [,HI(-)QNB in the presence of pirenzepine (Mash and Potter, 1986) and [,HIAF-DX Received April 8,1991; accepted in revised form May 24,1991 Dalia M. Araujo is now at Department of Psychobiology, Steinhaus Hall, University of California, Irvine, CA 92717. Address reprint requests there.

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116 (Araujo et al., 1989,1990; Lapchak et al., 1989a, b; Regenold et al., 1989;Wang et al., 1987a).Using in vitro receptor autoradiographic techniques and selective ligands, a clear distinction between the distribution of M, and M, muscarinic receptors in the mammalian brain has been possible (Cortes et al., 1987; Mash and Potter, 1986;Quirion et al., 1989b;Regenold et al., 1989; Spencer et al., 1986). In contrast, differences between the localization of brain M, and M, and between M, and M, muscarinic receptors have yet to be clarified. Pharmacological studies have indicated that 4-diphenylacetoxy-N-methylpiperidinemethiodide (4-DAMP) is selective for M,-muscarinic receptors in the rat brain since it competes for non-M,/non-M, [3HlN-methylscopolamine (NMS) sites (Doods et al., 1987; Fisher and Heacock, 1988). However, recent evidence from membrane binding studies suggests that [,H]4-DAMP may bind to MI, M,, and M, subtypes of muscarinic receptor, depending on the tissue(s) studied (Michel et al., 1989; Michel andwhiting, 1990). In view of these conflicting findings, the main purpose of the present study was to formulate a detailed characterization of [,H]4-DAMP binding in the rat brain. Using full saturation analysis of [,H]4-DAMP binding to homogenates of rat brain, the

Abbreviations ac ACh

anterior commissure acetylcholine

AF-DX 116

11[[2-[(diethylamino~methyll-l-piperidinyllacetyll5, 11-dihydro-6H-pyrido [2,3-b] [ l , 41 benzodiazepin-6-one anterior olfactory nucleus ao anteroventral nucleus of the thalamus av basolateral nucleus of the amygdala bl maximal binding capacity ,,B corpus callosum cc cerebellum ce cingulate cortex ci caudate-putamen dentate gyrus external plexiform layer of the olfactory bulb 4-diphenylacetoxy-N-methylpiperidine methiodide globus pallidus gP hippocampus hi hypothalamus internal capsule interpeduncular nucleus iP apparent affinity constant k.3 laterodorsal nucleus of the thalamus Id lateral septum 1s medial geniculate nucleus mg motor trigeminal nucleus ma5 nucleus accumbens na Hill coefficient of binding N-methylscopolamine %S pyramidal tract Pt ( - )QNB quinuclidinyl benzilate reuniens nucleus of the thalamus re sc superior colliculus substantia inominata si sn substantia nigra nucleus of the solitary tract sol olfactory tubercle tu vertical limb of the diagonal band vldb layers of the frontoparietal cortex I-VI facial nucleus 7 hypoglossal nucleus 12

2

binding parameters (&, B,,, and n,) were determined. In addition, the discrete localization of I3H14DAMP sites in the rat brain was assessed using in vitro receptor autoradiographic techniques. To determine whether [,H]4-DAMP might bind to muscarinic-MI or M3 sites in the rat brain, the ligand selectivity pattern of these sites was assessed. In addition, the binding parameters and the autoradiographic distribution of [,H]4-DAMP muscarinic sites were compared to [,HIpirenzepine/muscarinic-M, binding sites in the corresponding brain regions. The results show that L3H14DAMP binds to a high- and a lower-affinity class of muscarinic sites in the rat brain; while the former may consist mostly of muscarinic-Mi,sites, the latter appears to comprise a large proportion of muscarinic-M, receptors. MATERIALS AND METHODS Materials Male Sprague-Dawley rats (150-200 g) were obtained from Charles River Breeding Farms (St. Constant, Quebec, Canada). Tritium-sensitive microscales and Hyperfilms were purchased from Amersham (Toronto, Canada). (N-rneth~l)-[~H]4-DAMP (85 Ci/mmol) was a generous gift from Dr. S. Hurt (New England Nuclear, Boston, MA). (Meth~l)-[~HIpirenzepine (72 Ci/mmol) was bought from New England Nuclear (Boston, MA). 4-DAMP and (-)quinuclidinyl benzilate (QNB) were from Research Biochemicals Inc. (Natick, MA). Atropine sulfate, L-nicotine (free base), and d-tubocurarine were obtained from Sigma Chemical Co. (St. Louis, MO). Unlabeled AF-DX 116 was a gift from Dr. Karl Thomae (Biberach an der Riss, FRG). Pirenzepine dihydrochloride was generously donated by Drs. M. Watson (UMDNJ, New Jersey) and B. Wolfe (University of Pennsylvania). All other chemicals were from Fisher Scientific (Montreal, Canada). [3H]4-DAMPbinding to muscarinic sites in homogenates of rat brain Male Sprague-Dawley rats (150-200 g) were decapitated and the brains dissected on ice. Hippocampus, striatum, cerebral cortex, thalamus-hypothalamus, and cerebellum were homogenized with a Brinkmann Polytron (setting 6, 20 sec) in Tris-HC1 (50 mM, pH 7.4) buffer containing the following (in mM): NaC1,120; KCl, 5; CaCl,, 2; MgCl,, 1. The homogenate was centrifuged for 10 min at 49,00Og, after which the supernatant was discarded and the pellet washed twice by resuspending in fresh buffer and re-centrifuging. The final membrane pellet was suspended in buffer. Preliminary experiments demonstrated that L3H14-DAMP binding was optimal using Tris-HC1 buffer, although the maximal binding was not significantly different using Krebs buffer (see below, for composition).Aliquots of the final membrane pellet (200-500 pg protein) were incubated in the same buffer as above, containing various concen-

r3H]4-DAMP MUSCARINIC SITES I N R A T BRAIN

trations of [,H]4-DAMP (0.05-250 nM) for 60 min a t 22°C. Bound L3H14-DAMP was separated from free by rapid filtration under reduced pressure, using a Brandel Cell Harvester apparatus (Gaithersburg, MA), through GF/B filters presoaked for at least 1h in 0.1% polyethyleneimine. Filters were rapidly washed with ice-cold buffer (4 x 4 ml). Specific binding was calculated as the difference between radioactivity bound in the absence and presence of 10 pM atropine; this represented approximately 70% of total binding at concentrations of free ligand close to the K, of the high-affinity site. All binding data were analyzed by computerized analysis (LIGAND) by Munson and Rodbard (19801, as modified by G.A. McPherson (Elsevier Biosoft, 1985). In experiments that tested the ligand selectivity of [3H]4-DAMPbinding to the high- and lower-affinity sites, rat forebrain homogenates were incubated with either 0.5 nM (high-affinity) or 50 nM (lower-affinity) free ligand and a wide range of concentrations (0.05 nM-100 pM) of the indicated cholinergic drugs. [3Hlpirenzepine binding to rat brain muscarinic-MI sites Brain tissues were processed as described above and samples were homogenized in Krebs buffer (pH 7.4) of the following composition (in mM): NaC1, 120; MgS04.7H,0, 1.2; KH,P04, 1.2; glucose, 5.6; NaHCO,, 25; CaCl,, 2.5; KC1, 4.7. [3HlPirenzepine binding was measured as described previously (Araujo et al., 1988, 1990: Lapchak et al., 1989a,b). Aliquots of the final membrane pellet were incubated in Krebs buffer with various concentrations of [3Hlpirenzepine (0.1-50 nM) for 60 min at 22°C. Termination of binding assays was as described for [,H]4-DAMP. Specificbinding was defined in the presence of 1 pM atropine and represented 8590% of the total binding with concentrations of free ligand close to the Kd. Protein determination Protein content was measured by the method of Lowry and co-workers (19511, using bovine serum albumin as the standard. [3H]4-DAMPmuscarinic receptor autoradiography Coronal sections (20 pm thick) of rat brain were prepared as described previously (Quirion, 1985, 1987; Quirion et al., 1989b). Slide-mounted sections of rat brain were stored at - 70°C for at least 48 h prior to use. Preliminary tests demonstrated that binding of L3H]4DAMP to its sites was optimal using the incubation procedure described above for binding to homogenates (e.g., Tris-HC1 buffer, 60 min, 22°C). Sections were incubated with either 0.5 nM (high-affinity sites) or 50 nM (lower-affinity sites) [,H]4-DAMP. Non-specific binding was assessed in the presence of 10 pM atropine. After incubation, sections were rinsed in Tris-HC1 (50 mM) buffer (3 x 4 min), with a final dip in ice-cold water

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to remove remaining salts. Sections were then dried and apposed to tritium-sensitive Hyperfilm for 1to 8 weeks. Films were subsequently developed and I3H14-DAMP binding was quantified using tritium-labeled standards and computerized image analysis (Microcomputer Imaging Device, Imaging Res. Inc., Ontario, Canada). L3H1pirenzepine muscarinic-M, receptor autoradiography Autoradiography of L3H1pirenzepinebinding sites in the rat brain was carried out as described previously (Quirion and Boksa, 1986; Quirion et al., 1989b; Regenold et al., 1989). Briefly, slide-mounted sections were first pre-incubated in Krebs buffer (see above) for 15 min at 22°C. Subsequently, sections were incubated in fresh Krebs buffer containing 20 nM [3H]pirenzepine. Sections were then sequentially rinsed (3 x 4 min) in 50 mM Tris-HC1 buffer (pH 7.4) and rapidly dipped in ice-cold water. Specifically bound ligand was determined as the amount bound in the absence compared to that bound in the presence of 1uM atropine. Slides were dried and apposed to film and after 3-5 weeks of exposure, films were processed as described above for C3H14DAMP.

RESULTS Characterization of i3H14-DAMPbinding to rat brain homogenates The first series of experiments characterized the binding of [,H]4-DAMP to membrane-enriched homogenates of rat brain. The results (Fig. 1;Table I) show that, under our incubation conditions, [,H]4-DAMP binds saturably, specifically and with high affinity to two classes of muscarinic sites in all tissues studied (hippocampus, striatum, cerebral cortex, thalamus-hypothalamus, and cerebellum). The biphasic Scatchard plot of the [,H]4-DAMP binding data obtained for homogenates of rat forebrain illustrates this point (Fig. 1). [,H]4-DAMP binding to homogenates of brain tissue was inhibited by addition of 10 pM atropine to the incubation medium; specific binding in all brain areas represented approximately 60-70% of total, at concentrations of free ligand close to the & value of the high-affinity sites. A shown in Table I, [3H]4-DAMPbound with similar high affinity (range: 0.14-0.53 nM) to the high-affinity sites, irrespective of the brain region tested. With the exception of the cerebral cortex, little variation in the I(d for the lower-affinity sites (range: 50-59 nM) was also observed for the different brain regions (Table I). Moreover, for all tissues, the & for the lower-affinity sites was at least 100-fold greater than that of the highfor the lower-affinity sites affinity sites, while the B, was a t least five times that for the high-affinity sites (Table I). In addition, the highest densities of both highand lower-affinity [,H]4-DAMP sites were found in homogenates of cerebral cortex, while the lowest densi-

D.M. ARAUJO ET AL.

TABLE I. Parameters of PH14-DAMP binding to rat brain homoeenates'

B,, Kd

Brain region Hippocampus Striatum Cerebral cortex Thalamushypothalamus Cerebellum

(nM)

(fmol/mg protein)

Kdi

Kdz

Bi

B2

0.31 f 0 . 0 5 0.33 f 0.02 0.14 f 0.07 0.48 f 0.04

50f4 51 zk 4 99 f 14 59 f 11

68f8 106 f 9 122 f 30 51 f 6

361 f 2 2 514 f 63 860 f 69 295 f 32

0.53 f0.18

58f4

20 f 2

141 f 21

'Membrane-enriched homogenates of rat hrain were prepared a s described in the text and incubated with various concentrations of['H]4-DAMP(0.05-250 nM). with non-specific binding determined in the presenceof 10pM atropine. Kdi andKdaare the Kd values(nMj for13H]4.UAMPbinding toa high- andlower-affinitymuscarinic site, respectively. Similarly, R I and Bn arp the respective maximal binding capacities (Rmax)of the high- and the lower-affinity site (fmol/mg protein). Specific binding represented approximately 60-70ff of total binding at concentrations of ligand rlosr to the Kd for the high-affinity site. Values are the mean T SEM of 5-6 experiments. with each concentration of [3H14-l)AMPtested in triplicate.

TABLE II. r3Hluirenzeuine bindine to rat brain homoeenates' Kd

Hippocampus Striatum Cerebral cortex Thalamus-hypothalamus Cerebellum

(nM)

11.1 f 3.2 9.1 f 2.3 8.2 f 1.5 6.3 f 1.9 5.8 f 1.8

B,,

(fmollmg protein)

842 f 46 791 f 39 560 f 27 344 f 31 35 f 6

'Rat brain homogenates were prepared a s describedin the text. For determinationof 500

1000

BOUND (frnolimg protein)

Fig. 1. c3H14-DAMP binding to rat forebrain homogenates. The biphasic Scatchard plot derived from full saturation analysis of L3H14DAMP binding shows that there are two classes of L3H14-DAMP sites, one with low capacity (Bm$ and high-affinity (KJ, and the other with high capacity and lower-affinity. Binding of L3H14-DAMP was tested over a wide range of concentrations (0.05-250 nM). Each point is the mean t SEM of 5-6 experiments, in which each concentration of ligand was tested in triplicate. Specific binding was defined in the presence of atropine (10 KM).

r dioligand binding, homogenates were incubated with various concentrations of ['HH]pirenzepine (0.1-50 nM), to asses8 binding to muscarinic-Mi sites. Specific binding was determined in the presence of 1uM atropine and represented 85-Wfb of total binding a t concentrations of ligand close to the Kd. Values are the mean SEM of four experiments for each brain area, in which each concentration of ligand was tested in triplicate.

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Ligand selectivity pattern of I3H14-DAMPbinding to rat forebrain homogenates To test further whether f3H14-DAMPbinds to specific subtypes of muscarinic sites in rat forebrain, the ligand selectivity of various muscarinic antagonists for f3H]4DAMP sites was studied (Table 111). Atropine, 4-DAMP, and (-)QNB were equipotent in inhibiting [3H]4-DAMP ties for both sites were in the cerebellum (Table I). In binding to the high- and the lower-affinity sites (Table the remaining brain regions, the maximal densities for 111). In contrast, the selective muscarinic-MI antagonist both sites was as follows: striatum > hippocampus > pirenzepine (Watson et al., 1982) was more potent (by thalamus-hypothalamus. approximately 16-fold)at the lower-affinity sites (IC50: With the exception of the thalamus-hypothalamus, 51 k 8 compared to 820 93 nM), although it was still the total densities (BmaX) of lower-affinity f3H14-DAMP less efficacious than 4-DAMP, atropine or (- IQNB. The sites differed somewhat from those measured with the muscarinic-M, antagonist AF-DX 116 (Giachetti et al., selective muscarinic-M, ligand [3Hlpirenzepine (Table 1986; Hammer et al., 1987; Micheletti et al., 1987)was a 11). In the hippocampus and striatum, the densities of poor competitor for both the high- and the lower-affinity [3Hlpirenzepine sites were much greater than those for [3H]4-DAMPsites, with Kd values in the micromolar the lower-affinity L3H14-DAMP sites (Table 11). Con- range (Table 111). The Hill coefficients (n,) for both the versely, in the cerebral cortex and the cerebellum, high- and lower-affinity sites were, with few exceptions I3H14-DAMP lower-affinity sites appeared to be more (e.g., atropine and 4-DAMP, at the lower-affinity sites), abundant than [3Hlpirenzepine sites. Moreover, the & close to unity (Table 111). for f3HI4-DAMPbinding t o the lower-affinity muscaNicotinic drugs such as L-nicotine and d-tuborinic sites was at least fivefold higher than the & for curarine, did not effectively compete for either the highL3Hlpirenzepine binding to muscarinic-M, sites in the or the lower-affinity L3H14-DAMP binding sites (data corresponding brain regions. not shown).

[3H]4-DAMP MUSCAKINIC SITES IN RAT BRAIN

TABLE I l l . Comparative potency of various muscarinie antagonists on f3H]4-DAMP sites' Drue Atropine 4-DAMP (-) ,

I

-

0NB

Pirenzepine AF-DX 116

ICMI

nHl

1.9 f 0.2 2.5 f 0.3 2.9 f 0.4 820 5 93 1236 f 105

0.93 f 0.07 1.02 f 0.09 0.93 f 0.04 1.01 0.19 1.15 f 0.22

*

ICMZ 2.3 +_ 0.4 4.3 f 0.2 1.8 f 0.2 51 f 8 926 rt 34

nH7 1.53 f 0.15 1.98 f 0.26 1.02 f 0.11 1.26 f 0.06 1.17 f 0.21

'Homogenates of rat forebrain were incubated with either 5 or 50 nM r3H]4-DAMP and a broad range of concentrations (0.05 nM-100 pM) of the indicated muscarinic drugs; each concentration of drug was tested in triplicate. Non-specificbinding was determined in the presence of 10 pM atropine. IC501 and IC5@ represent the concentration (in nM) of drug required to inhibit 50%of specifically bound L3H]4DAMP at 5 (high-affinitysite) or 50 (lower-affinity site) nM free ligand concentration, respectively.nH1 and nH2 are the respective Hill coefficientsfor the high- and lower-affinity[3H]4-DAMPbinding sites.

Autoradiographic distribution of [3H14-DAMP sites in the rat brain To determine the precise distribution of both highand low-affinity [3H14-DAMPsites, coronal sections (20 km) of rat brain were incubated in buffer containing either 0.5 nM (high-affinity sites) or 50 nM (loweraffinity sites) [,H]4-DAMP. Representative photomicrographs from a broad cross section of the rat brain are depicted in Figures 2-4. For each separate micrograph, different contrasts were selected for optimal visualization. However, quantitative analysis of the autoradiograms obtained from sections incubated with buffer containing 50 nM [,H]4-DAMP, representing the actual densities, are shown in Table IV. Descriptions of the relative binding densities (expressed as fmoVmg tissue, wet weight) are referred to in the text as highest (1451001, very high (99-80), high (79-50), moderate (49-30), low (29-151, very low (14-51, and negligible ( hippocampus 1 thalamus-hypothal-

amus > cerebellum) are in agreement with previous studies that used 4-DAMP displacement of L3H1NMS binding to define muscarinic-M3sites (Ehlert and Tran, 1990; Giraldo et al., 1987). Moreover, the ligand selectivity pattern of [3H]4-DAMPbinding to the high-affinity sites suggests that these sites may consist mainly of muscarinic-M3sites since pirenzepine and AF-DX 116, “selective”antagonists for muscarinic-MI and M, sites,

[3H]4-DAMP MUSCARINIC SITES IN R A T BRAIN

TABLE ZV. Quantitative autoradiographic distribution of 13HJ4-DAMPsites in the rat brain’ Brain region Olfactory bulb External plexifom layer Internal granular layer Olfactory tubercle Corpus callosum Nucleus accumbens Medial forebrain bundle Anterior commissure Caudate-putamen Anterior Posterior Globus pallidus Internal capsule Cortical laminae (fkontoparietal area) Laminae 1-11 Laminae III-IV Laminae V-VI Cingulate cortex Vertical limb of the diagonal bend Septum Medial Lateral Ventral pallidum (substantia inominata) Hippocampus CA1 sub-field of Ammon’s horn CA2 sub-field of Ammon’s horn CA3 sub-field of Ammon’s horn Dentate gyrus Thalamus Anteroventral nuclei Laterodorsal nuclei Reuniens nucleus Hypothalamus (as a whole) Superior colliculus Medial geniculate nucleus Interpeduncular nucleus Substantia nigra, pars reticulata Motor trigeminal nucleus Facial nucleus Nucleus of the solitary tract Hypoglossal nucleus Cerebellum (as a whole)

[3H]4-DAMP binding sites (fmol/mg tissue) 123.4 f 3.0 30.2 f 1.2 129.3 f 1.5 2.4 t 0.9 97.8 f 5.1 19.5 f 5.2 3.9 f 1.3 96.6 f 3.9 80.2 t 4.8 6.5 f 1.7 2.9 f 1.8 117.9 t 4.5 69.3 f 5.6 89.4 f 3.7 65.4 k 2.5 44.3 f 3.7 12.9 t 2.1 27.3 f 1.5 12.3 f 1.1 141.6 f 6.3 81.4 5 3.6 89.1 f 6.6 135.3 f 4.5 77.3 t 5.1 44.7 f 4.6 59.7 f 5.8 36.9 f 7.4 45.0 f 2.5 21.6 t 1.6 47.7 4.9 16.6 f 2.3 12.1 1.9 30.4 f 2.3 39.1 3.2 41.9 2.8 13.5 f 2.7

*

* **

‘Sections (20 prn thick) were incubated with 50 nM [3H]4-DAMP, as describedin the text. Autoradiograms were analyzed using computerized densitornetry. Results (fmol/rng of brain tissue, wet weight) are the means 5 SEM of 8-12 determinations, where each individual value was the average of at least 10 readings.

respectively (Doods et al., 1987; Waelbroeck et al., 1986, 1987; Watson et al., 1982), did not effectively compete for these sites. However, the possibility that a t least some of the high-affinity L3H]4-DAMP sites may correspond to muscarinic-M, or non-M,lnon-MrJnon-M, sites cannot be excluded at present. In an earlier report on the binding of [3H14-DAMPto homogenates of rat cerebral cortex, it was not clear whether the second class of sites represented a different subtype of muscarinic receptor or a non-saturable component of [3H]4-DAMPbinding (Michelet al., 1989). Our result demonstrating that pirenzepine is a potent competitor of [3H]4-DAMP binding to the lower-affinity sites, compared to the high-affinity sites, suggests that at least some of these sites may be muscarinic-MI sites.

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Comparison of the autoradiographic distribution of sites obtained using 0.5 nM and 50 nM [3H14-DAMP further supports this: with the higher concentration of ligand additional brain regions that were not labeled with the lower concentration were clearly evident (e.g., the cerebellum, various mesencephalic and hypothalamic nuclei). Moreover, with certain exceptions (e.g., thalamidhypothalmic and mesencephalic nuclei, and cortical layers III-IV and V-VI), the autoradiographic distribution of lower-affinity L3H14-DAMP sites closely parallels that of [3Hlpirenzepine/muscarinic-M,sites in many brain regions. Alternatively, it is possible that in certain brain regions, the higher concentrations of L3H14-DAMPmay label other muscarinic receptor subtypes such as the M, subtype, which appears to be enriched in the basal ganglia and other areas of the rat brain (Vilaro et al., 1991). In situ hybridization of muscarinic receptor mRNA depicts cell bodies that make the respective receptor and in vitro receptor autoradiography localizes the receptor itself, which may be on the cell body or on processes distal to the cell body. Therefore, comparison of the distribution of receptor sites using the two techniques is difficult. Comparison is further hampered by the lack of radioligands that are clearly “selective”for one muscarinic receptor subtype over another. Furthermore, it is not yet apparent whether each muscarinic receptor subtype is the product of a single gene (see Bonner et al., 1987,1988; Buckley et al., 1988; also, review by Hulme et al., 1990). Nevertheless, it appears that the distribution of high-affinity [3H]4-DAMPsites in the rat brain most closely resembles that of m3 mRNA (Buckley et al., 1988). In contrast, lower-affinity L3H]4-DAMP site distribution appears to parallel that of a combination of m l , m3 and m4 mRNA (see Buckley et al., 1988; Weiner et al., 1990). Although [3H14-DAMP appears to label muscarinic-M, sites in some peripheral tissues (Michel and Whiting, 1990), it clearly does not in brain (present results). First, AF-DX 116, a “selective”muscarinic-M, antagonist (Giachetti et al., 1986; Hammer et al., 1986; Micheletti et al., 19871, was a weak (Kd > 1 FM) competitor for both the high- and the lower-affinity L3H14DAMP sites. Second, the parameters of L3H14-DAMP binding to rat brain homogenates are clearly different from those reported for muscarinic-M, sites (Araujo et al., 1989; Lapchak et al., 1989a,b;Quirion et al., 1989b). Third, the autoradiographic distribution of [3H]4DAMP sites is different from that of muscarinic-M, sites, which are enriched in pontine and mesencephalic nuclei, but relatively sparse in forebrain regions (Mash and Potter, 1986; Quirion et al., 1989b; Regenold et al., 1989; Spencer et al., 1986). The physiological importance of I3H14-DAMP sites in the mammalian brain is not entirely understood. Unlike muscarinic-M, receptor sites, which are markedly reduced in neurodegenerative diseases such & Alzhei-

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D.M. ARAUJO ET AL.

Fig. 5 . Photomicrographs of the autoradiographic distribution of [3Hlpirenzepine/muscarinic-M, sites in the rat brain. Sections were incubated as described in the text with 20 nM L3H1pirenzepine. Non-specific binding is shown in I and was determined in the presence of 1 pM atropine.

mer’s disease (Araujo et al., 1988; Mash et al., 1985; centration and experimental protocol, most notably the Quirion et al., 1989a; Wang et al., 1987b), there is no selection of tissues enriched with one subtype of muscaevidence to date that r3H]4-DAMP sites are compro- rinic receptor over another, may be of critical signifimised in neuropathological states. Moreover, clarifica- cance in the type of effect observed with the drug. For tion of the significance of these sites in brain may be example, in rat hippocampal slices, 4-DAMP has been difficult since it is not clear whether the effects of shown to inhibit the cholinergic slow EPSP (Pitler and 4-DAMP reported in the literature are mediated Alger, 1990). However, effects of 4-DAMP on ACh rethrough muscarinic-M3,M, or non-M,/non-M, receptor lease from nerve terminals in hippocampal, cerebral subtypes (see Discussion above). Thus, both drug con- cortical, striatal and cerebellar slices have not been

[3H]4-DAMP MUSCARINIC SITES I N RAT BRAIN

observed (Lapchak et al., 1989a,b). In contrast, evidence from in vivo studies of ACh release has indicated that muscarinic-M, receptors may function as autoreceptors in the rat striatum (De Boer et al., 1990). The use of transformed cell lines individually expressing the various subtypes of muscarinic receptor to characterize the antagonist binding properties of muscarinic receptor subtypes appears to be a promising tool in deciphering the subtype “selectivity”of various muscarinic drugs. In one such study, 4-DAMP exhibited high affinity for the m3 subtype of receptor, although it was equally potent in competing for L3H1NMSbinding to ml, m4 and m5 receptors (Dorje et al., 1991). However, its affinity for m2 receptor sites was significantly lower (Dorje et al., 1991). These results imply that 4-DAMP may be classified as a “selective”muscarinic-M, ligand only in tissues that contain a predominant population of muscarinic-M, sites relative to muscarinic-M, and M, sites. Nonetheless, 4-DAMP can be a useful drug to distinguish between muscarinic-M, (low affinity for 4-DAMP) and non-M, receptor sites. Thus, in human SK-N-SHneuroblastoma cells, which are enriched with muscarinic-M, sites (Fisher and Heacock, 1988) and in guinea-pig ileal smooth muscle, a tissue that contains both muscarinic-M, and M3 subtypes (see Michel and Whiting, 1990), 4-DAMP is a much more potent inhibitor of [,H]NMS binding than either pirenzepine or AF-DX 116. In summary, it appears that F3H14-DAMP, in low concentrations, may be a useful ligand to label muscarinic-M, subtypes in certain regions of the rat brain that are enriched with this subtype of receptor. In other brain areas, the exact nature of the subtype(s) of muscarinic receptor labeled by L3H]4-DAMPis not unambiguous, although the present results and those of other investigators suggest that L3H14-DAMPmay label both the MI and the M3 subtype, but not the M, subtype of muscarinic receptor. ACKNOWLEDGMENTS This work was supported by a grant from the Medical Research Council of Canada (MRCC).D.M.A. and P.A.L. are post-doctoral fellows and R.Q. is a “Chercheurboursier” of the Fonds de Recherches en Sante du Quebec. REFERENCES Araujo, D.M., Lapchak, P.A., Robitaille, Y., Gauthier, S., and Quirion, R. (1988) Differential alteration of various cholinergic markers in cortical and subcortical regions of human brain in Alzheimer’s disease. J . Neurochem., 50:1914-1923. Araujo, D.M., Lapchak, P.A., Regenold, W., and Quirion, R. (1989) Characterization of 13H]AF-DX 116 binding sites in the rat brain: evidence for heterogeneity of muscarinic-M, receptor sites. Synapse, 4:106-114. Araujo, D.M., Lapchak, P.A., Meaney, M.J., Collier, B., and Quirion, R. (1990) Effects of aging on nicotinic and muscarinic autoreceptor function in the rat brain: relationship to presynaptic cholinergic markers and binding sites. J. Neurosci., 10:3069-3078. Bloom, J.W., Halonen, M., Seaver, N.A., and Yamamura, H.I. (1987) Heterogeneity of the M, muscarinic receptor subtype between pe-

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