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Quantitative autoradiographic characterisation of the binding of [3H]WAY-100635, a selective 5-HT1A receptor antagonist
BRAIN RESEARCH ELSEVIER
Brain Research 673 (1995) 217-225
Research report
Quantitative autoradiographic characterisation of the binding of [3H]WAY-100635, a selective 5-HTIA receptor antagonist X. Khawaja Department of Molecular Pharmacology, Wyeth Research [UK], Huntercombe Lane South, Taplow, Maidenhead, Berkshire, UK Accepted 22 November 1994
Abstract
The binding characteristics of [3H]WAY-100635 ([O-methyl 3H]-N-(2-(4-(2-methoxyphenyl)-l-piperazinyl)ethyl)-N-(2pyridinyl)cyclohexane carboxamide trihydrochloride), a potent and selective 5-HTIA antagonist radioligand, were examined in the rat brain using in vitro quantitative receptor autoradiography. The regional distribution of specific [3H]WAY-100635 binding sites was heterogeneous and demonstrated a strong correlation with that of [3H]8-OH-DPAT binding. The highest concentrations of [3H]WAY-100635-1abelled sites were found in the lateral septal areas, dorsal raphe n., entorhinal cortex and the hippocampal formation (CA1, CA3 and dentate gyrus). Scatchard transformation of saturation isotherms revealed saturable [3H]WAY-100635 binding sites of high-affinity: in the hippocampal formation, K d was ~ 1 nM and Bmax ranged between 187 and 243 fmol/mg tissue wet weight, in the entorhinal cortex, K d = 0.44 nM and Bmax = 194 fmol/mg tissue wet weight, and in the rostral portion of the dorsal raphe n., K d = 0.52 nM and Bmax = 157 fmol/mg tissue wet weight. The affinity of [3H]WAY-100635 for the 5-HTIA binding site tended to be higher in the dorsal raphe n. and entorhinal cortex compared with that of the hippocampal formation. In contrast, the binding affinity of [3H]8-OH-DPAT in the hippocampal formation was between 1.1 and 2.3 nM and the Bmax was 137 to 183 fmoles/mg tissue wet weight; in the entorhinal cortex, K d = 3.2 nM and Bm~ = 141 fmoles/mg tissue wet weight, and in the rostral portion of the dorsal raphe n., K a = 3.4 nM and B~nax= 163 fmol/mg tissue wet weight. Our findings support the use of [3H]WAY-100635 as a novel antagonist radioligand for studying the binding characteristics of the 5-HTIA receptor by quantitative receptor autoradiography.
Keywords: [3H]WAY-100635; 5-HTIA antagonist; Quantitative autoradiography; Central nervous system; Rat; [3H]8-OH-DPAT
I. Introduction
Recent progress in 5-HT receptor neuropharmacology has grown with the identification of at least seven distinct classes of the receptor in the central nervous system [17]. The 5-HT l receptor has been differentiated into multiple subtypes [1,16,29] with the 5-HTIA receptor being of particular interest because of its potential role as a therapeutic target in the treatment of certain psychopharmacological disorders [5,28]. Whilst numerous potent and selective agonists at the 5-HT~A receptor have been synthesised [17], the paucity of 5-HTIA antagonists has made functional correlates difficult to interpret in animal behavioral studies. Compounds such as S-UH301, SDZ-216525,
* Corresponding author. Fax: (44) (628) 666-587. 0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved
SSDI 0 0 0 6 - 8 9 9 3 ( 9 4 ) 0 1 4 1 6 - 7
BMY 7378 and NAN-190, originally considered as selective 5-HTIA antagonists, were subsequently classified as weak partial agonists a n d / o r as possessing insufficient selectivity for the 5-HT1A receptor [5]. More recently, WAY-100135 and its second generation achiral analogue, WAY-100635, have proved to be highly selective and potent antagonists at the 5-HT~A receptor in animal behavioral [6,7] and in vivo microdialysis studies [12,32]. For the past decade, a variety of 5-HT~A-selective radioligands have been used extensively in in vitro receptor binding assays and autoradiographic studies to characterise the binding properties of the 5-HTIA receptor. The major limitation with most of these radioligands is that they are either full agonists ([3H]8O H - D P A T [4,10,13,31]; [125I]BH-8-MeO-N-PAT [11]) or partial agonists ([3H]ipsapirone [8]), which has constrained the level of information gained from pharma-
218
X. Khawaja./Brain Research 673 (1995) 217-225
cological binding studies on the G-protein coupled 5-HTlA receptor [18]. The tritiated form of WAY-100635 was developed as a selective 5-HTIA receptor antagonist radioligand and we have recently reported on the binding characteristics of [3H]WAY-100635 at the 5-HT1A site in rat brain [19]. The radioligand exhibited high affinity (K d = 0.37 _+ 0.051 nM) for a single population of 5-HTtA binding sites in rat hippocampal membrane preparations, with a maximal binding density (Bmax = 312 _+ 12 fmol/mg protein) which was approximately 36% higher than that of [3H]8-OH-DPAT. The pharmacological profile of [3H]WAY-100635 was consistent with the selective labelling of 5-HT1A sites, and with the ability of the radioligand to recognise with equal affinity multiple conformational states of the 5-HT1A receptor both in the absence and presence of guanyl nucleotides [19]. Moreover, ex vivo and in vivo biodistribution studies with [3H]WAY-100635 in rat and mouse indicated a regional distribution of radioactivity matching the localisation of central 5-HTiA receptor rich regions [14,30]. The present study reports on the use of [3H]WAY100635 as a potent selective antagonist radioligand to study the binding characteristics and regional distribution of the 5-HT1A receptor in the central nervous system by in vitro quantitative receptor autoradiography.
min at 37°C in 50 m M Tris-HCl buffer supplemented with 10 /xM pargyline and 4 m M CaCI 2. Nonspecific binding was determined in adjacent sections exposed to 10 /zM 5-HT. For saturation studies, conditions were essentially identical to those described for the regional studies, except that tissue sections were incubated with increasing concentrations of [3H]WAY-100635 (0.2-14 nM) or [3H]8O H - D P A T (0.7-14 nM). Following incubation, the sections were rinsed in ice-cold 50 m M Tris-HCl buffer ([3H]WAY-100635 2 min and 3 min; [3H]8-OH-DPAT: 2 × 4 rain) and dipped rapidly in ice-cold de-ionised water (5 s).
2.4. Autoradiographic quantitation
2. Materials and methods
Air-dried sections were apposed to [3H]-sensitive film (Hyperfilm-3H; A m e r s h a m plc., UK) together with autoradiographic [3H]microscales (Arrlersham plc., UK) for 6 - 8 weeks at room-temperature. The film was developed for 3 rain in Kodak Industrex manual developer, rinsed in tap water for 1 min, and fixed for 5 min in Kodak fixer. Ligand binding was quantitated by computer-aided densitometry using the Seescan Solitaire processing system (Seescan plc., Cambridge, UK). Specific binding was defined as the difference between total and non-specific values measured on adjacent sections. Bilateral m e a s u r e m e n t s of gray levels were made within various brain regions, identified using the rat brain atlas of Paxinos and Watson [26]. Brain gray matter tissue equivalents ( B q / m g tissue wet weight) were determined by extrapolation from a computer-generated standard curve, which compared film densities produced by the tissue section with those produced by the radio-active standards. The receptor density of a brain area was expressed as femtomoles of radioligand specifically bound per mg tissue wet weight. The regional concentrations from equivalent brain areas per experimental group of animals were averaged. Saturation isotherms were analysed using the non-linear, least-squares, curve-fitting programmes E B D A (Equilibrium Binding Data Analysis) and L I G A N D [24] as modified by McPherson [21], and final K d (nM) and B .... ( f m o l / m g tissue wet weight) parameters were generated.
2.1. Materials
2.5. Rat brain membrane preparation
[3H]WAY-100635 was custom synthesised by A m e r s h a m International plc. (UK) at an initial specific activity of 69 C i / m m o l (98.5% radiochemical purity). [3H]8-OH-DPAT (sp. act. 160-240 C i / m m o l ; 98% radiochemical purity) was supplied by A m e r s h a m International plc (UK). 8 - O H - D P A T (RBI, UK); 5-hydroxytryptamine (Sigma Chemical Co. Poole, UK); WAY-100635 was synthesised by the Chemistry D e p a r t m e n t Wyeth Research (UK).
Male Sprague-Dawley rats (150-170 g) were killed by a blow to the head and cervical dislocation. The brains were rapidly removed and hippocampus, septum, frontal cortex, hypothalamus, striatum and cerebellum were hand-dissected on ice by the method of Glowinski and lversen [9], and stored at - 7 0 ° C . Tissue was thawed at room temperature and initially homogenised using a Brinkman Polytron tissue disrupter (PT 10 probe, setting 5 for 30 s) in 50 vols. (w/v) of ice-cold 50 m M Tris-HCl buffer (pH 7.4). The suspension was centrifuged twice at 27,000× g for 20 min at 4°C. The membrane pellets were resuspended in 50 vols. ( w / v ) Tris-HC1 buffer and incubated at 37°C for 20 min in a shaking water bath, prior to a final centrifugation step (27,000 x g; 20 min; 4°C). The final tissue pellets were stored at - 7 0 ° C until assayed.
2.2. Preparation of slide-mounted sections Male strain Sprague-Dawley rats (150-170 g) were sacrificed by cervical dislocation and decapitation. The brains were carefully removed, frozen in isopentane ( - 4 0 ° C on dry ice) and stored at - 8 0 ° C . Serial coronal sections ( 2 0 / x m thick; 3 - 4 per slide) were cut using a cryomicrotome at - 2 0 ° C , thaw-mounted onto gelatin (5 g/I)-KCr(SO) 4 (0.5 g/I) double-coated glass microscope slides, and stored sealed at - 2 0 ° C until used.
2.3. Quantitative autoradiographic regional distribution and saturation studies Slide-mounted brain sections were warmed to room-temperature and pre-incubated in 50 m M Tris-HCl buffer (pH 7.4) for 30 min at room-temperature. The sections were then incubated with either 3 nM [3H]WAY-100635 for 60 min at 37°C in 50 m M Tris-HCl buffer containing 10 /xM pargyline or with 4 n M [3H]8-OH-DPAT for 30
2.6. Radioreceptor binding assays Hippocampal, cortical, septal, hypothalamic, striatal and cerebellar m e m b r a n e homogenates ( 2 - 4 mg t i s s u e / t u b e ) were incubated in 50 m M Tris-HCI (pH 7.4) with [3H]WAY-100635 (0.5 nM) for 50 min at 37°C in the absence or presence of 10 ~ M unlabelled 8 - O H - D P A T to determine non-specific binding. Parallel binding assays with [3H]8O H - D P A T (1 nM) ( + 4 m M CaCI 2) were performed at 37°C for 20 min, using the same m e m b r a n e preparations. Statistical analyses of the data were carried out using the statistics package S T A T V I E W (Abacus concepts, Inc., Berkeley, CA, 1992) and Student's two-tailed t-test.
X. Khawaja /Brain Research 673 (1995) 217-225 A
3. Results
The chemical structure of tritiated WAY-100635 is depicted in Fig. 1. Initial experiments were carried out to establish the optimal binding conditions for quantitative autoradiography using [3H]WAY-100635. After each experiment, brain sections were wiped from the microscope slides with pre-wet Whatman G F / B filters and bound radioactivity was measured by liquid scintillation counting. Sections taken from the hippocampal region were incubated with [3H]WAY-100635 (0.7 nM) at 37°C for 60 min and washed for various times in two changes of Tris-HCl buffer at 4°C (Fig. 2). Total and non-specific binding of [3H]WAY-100635 to the sections decreased with time; non-specific binding represented approximately 15-20% of total bound radioactivity after a 2-min wash with no marked change in the level of specific binding. After a 10-min wash, total binding was further reduced vTith no comparable decrease in non-specific binding; this was interpreted as representing dissociation of [3H]WAY-100635 from specific 5-HT~A binding sites. The optimal wash time adopted was a 2 min followed by a 3 min rinse in ice-cold Tris buffer. The effect of temperature on the time course of [3H]WAY-100635 binding to rat hippocampal slide-mounted sections is shown in Fig. 3. Association of [3H]WAY-100635 to central 5-HTIA binding sites was temperature sensitive. Maximal binding occurred at 37°C and at room-temperature, but was 50% lower at 4°C; steady-state was reached after 20 min at 37°C, 100 min at room-temperature, and required over 3 h at 4°C (Fig. 3). An optimal incubation time of 60 min at 37°C was selected for subsequent studies. The regional distribution of specific [3H]WAY100635-sensitive binding sites in rat brain is shown in Fig. 4 (Plates A - F ) and was identical to that of [3H]8O H - D P A T (not shown); the darker areas in the autoradiograms indicated regions of higher receptor density. Table 1 summarises the quantitative regional variations in specific [3H]WAY-100635 and [3H]8-OHD P A T binding throughout the rat central nervous system. [3H]WAY-100635-sensitive 5-HTIA binding sites were heterogeneously distributed, being particularly abundant in the taenia tecta, lateral septum (LSD =
219
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Wash t i m e (rain.) Fig. 2. W a s h curve for [3H]WAY-100635 bound to slide-mounted rat hippocampal sections. Sections were incubated with 1 nM [3H] WAY-100635 at 37°C for 60 min and then rinsed in Tris-HCl buffer at 4°C for various times (1-15 min). Non-specific binding was defined in the presence of 10 /zM 5-HT. Tissue sections were wiped onto G F / B filters and the radio-activity counted. Data shown are the m e a n + S.E.M. of a representative of two experiments performed in quadruplicate.
LSI > LSV), hippocampus (OB1 = CA3 > IB1 = D G > CA1 > > CA2), dorsal raphe nucleus and entorhinal cortex. Lower levels were detected in most cortical regions (frontal, orbital, cingulate, dorsal p e d u n c u l a r / infralimbic, parietal, occipital), amygdalohippocampal area, median raphe, as well as certain discrete nuclei such as the anterior olfactory nucleus (medial/dorsal), and certain septal nuclei (septohippocampal, medial septal, vertical limb diagonal band). Low to intermediate binding densities were detected in the agranular A
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Fig. 3. Temperature-time course of association of [3H]WAY-100635 b o u n d to slide-mounted rat hippocampal sections. Sections were incubated with 1 nM [3H]WAY-100635 for various time intervals at 4°C, room-temperature and 37°C. Non-specific binding was determined in the presence of 10 ~ M 5-HT. Sections were washed in two changes of Tris-HCl buffer (2 m i n + 3 rain) at 4°C. Tissue sections were wiped onto G F / B filters and the radio-activity counted. Data shown are the mean_+ S.E.M. of a representative of two experiments performed in quadruplicate.
X. Khawaja / Brain Research 673 (1995) 217-225
220
and [3H]WAY-100635 binding throughout the central nervous system (Correlation coefficient = 0.978; P < 0.001, d f = 46). In most brain areas examined, the number of 5-HT1A binding sites labelled by [3H]WAY100635 was on average 2.3 + 0.10 times higher than that for high-affinity [3H]8-OH-DPAT binding (Table 1). In certain regions of the cortex (frontal, cingulate, parietal, temporal, retrosplenial), septum (medial, VDB), central gray and caudal portion of the dorsal raphe n., the differences between [3H]antagonist and
insular and lateral/ventrolateral orbital cortices, retrosplenial and temporal cortices, anterior olfactory nucleus (lateral/ventral), claustrum, horizontal limb diagonal band, CA2 hippocampal area, central gray and raphe pontis. Sparse levels of [3H]WAY-100635 binding sites were found in the nucleus accumbens (Acb), corpus callosum (fmi), globus pallidus, caudate-putamen (CPu), thalamic areas and substantia nigra. A significant positive correlation was observed between the regional distributions of [3H]8-OH-DPAT
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Fig. 4. Autoradiographic localisation of specific [~H]WAY-100635 binding to coronal sections of rat brain. The images are dark-field transforms of specific radioligand binding. Representative brain sections are shown in a rostral-caudal (A F) sequence. Sections (20/xm) were incubated with 3 nM [3H]WAY-100635 at 37°C for 60 min. Non-specific binding was defined with 10/xM 5-HT. Sections were washed, dried, and exposed to [3H]-sensitive film for 6-8 weeks. Anatomical nomenclature used in describing the distribution of 5-HT1A binding sites and structures were identified by reference to the atlas of Paxinos and Watson [26]; Fr. Cx, frontal cortex; LO, lateral orbital cortex; A O M / A O D , anterior olfactory nucleus (medial/dorsal); A O L / A O V , anterior olfactory nucleus (lateral/ventral); Par, parietal cortex; DP/IL, dorsal peduncular/infralimbic cortex; TT, taenia tecta; fmi, forceps minor corpus callosum; Cg, Cingulate cortex; Lat. sept., Lateral septum; CPu, caudate-putamen; DG, dentate gyrus; Amygd., amygdala; Ent, entorhinal cortex; DR, dorsal raphe n.; MnR, median raphe n.; RPn, raphe pontis n.
X. Khawaja / Brain Research 673 (1995) 217-225
221
Table 1 Regional distribution of 5-HT1A receptors labelled by [3H]WAY-100635 and [3H]8-OH-DPAT Plate [bregma]
Region
Abbrev.
A
Frontal Cx (area 2) M e d i a l / V e n t r a l orbital Cx Lateral orbital Cx Agranular insular Cx Ant. olf. n. (lateral/ventral) Ant. olf. n. ( m e d i a l / d o r s a l )
Fr2 MO/VO LO AI AOL/AOV AOM/AOD
Frontal Cx (areas 1, 2, 3) Cingulate Cx (areas 1, 3) P a r i e t a l / A g r . insular Cx Dors. ped./infralimbic Cx Taenia tecta L a t . / V e n t r o l a t . orbital Cx Claustrum A c c u m b e n s n. Forceps minor corp. call. Frontal Cx (areas 1, 2) Cingulate Cx (areas 1, 2) Parietal Cx (area 1) Caudate p u t a m e n A c c u m b e n s n. Lateral septal n. (dorsal) Septohippocampal n. Lat. sept. n. (intermediate) Lateral septal n. (ventral) Medial septal n. n, vertical limb diag. band n, horizon limb diag. band
[5.20 mm]
B [2.70 mm]
C [0.70 mm]
D [ - 4 . 3 0 mm]
E [ - 7.64 mm]
F [-8.72mm]
[3H]WAY-100635 ( f m o l / m g tissue wet weight)
n
High affinity [ 3H]8-OH-DPAT ( f m o l / m g tissue wet weight)
n
Ratio
64.2 56.4 43.7 34.7 28.5 51.9
+ 3.5 + 2.0 + 2.9 + 0.9 + 1.6 + 1.2
10 10 10 10 10 10
28.2 33.5 23.6 14.9 15.5 26.7
+ _+ + _+ _+ _+
2.2 2.4 1.9 1.3 0.7 1.0
12 12 12 12 12 12
2.3 1.7 1.8 2.3 1.8 1.9
Frl,2,3 Cgl,3 Parl/AI DP/IL TT LO/VLO C1 Acb fmi Frl,2 Cgl,2 Parl CPu Acb LSD SHi LSI LSV MS VDB HDB
58.5 72.4 43.7 67.3 112.2 43.9 45.1 2.7 5.3 64.4 79.6 62.8 3.5 18.6 165.8 65.6 170.2 119.0 61.9 73.3 45.4
+ 0.9 + 1.4 + 0.8 + 1.7 + 3.6 + 1.6 + 1.1 + 0.4 _+ 1.5 + 1.7 _+ 2.1 _+ 1.6 + 1.1 + 1.5 -+ 5.7 + 10.0 -+ 3.5 + 4.3 + 2.8 -+ 3.7 + 3.2
6 6 6 6 6 6 6 6 6 18 18 18 18 18 18 9 18 18 9 9 18
17.9 25.1 14.3 28.9 51.6 17.1 17.1 4.3 2.7 21.9 31.9 21.3 3.5 7.3 89.0 29.4 80.6 55.6 21.1 24.8 11.7
+ _+ + + _+ _+ + + + + + + + + + + + + + + _+
0.7 0.7 1.4 1.7 2.5 1.0 1.4 0.2 0.8 1.7 2.1 1.7 0.7 0.5 4.3 4.8 4.8 4.4 2.2 2.2 0.9
6 6 6 6 6 6 6 6 6 18 18 18 18 18 18 18 18 18 9 9 18
3.3 2.9 3.1 2.3 2.2 2.6 2.6 0.6 2.0 2.9 2.5 3.0 1.0 2.5 1.9 2.2 2.1 2.1 2.9 3.0 3.9
Retrosplenial Cx Occipital Cx Temporal Cx (areas 1, 3) Amygdalohippoc. area CA1 CA2 CA3 D e n t a t e gyrus Inner blade dentate gyrus O u t e r blade dentate gyrus Thalamic region Medial mammillary n. Entorhinal Cx Dorsal raphe (rostrl end) Median raphe n. Central gray Pontine n.
RSG/RSA Oc2 Tel,3 AHi CA1 CA2 CA3 DG IB1 OB1 MM Ent DR MnR CG Pn
22.5 _+ 1.9 58.9_+ 1.6 43.3 _+ 1.1 73.4-+ 4.6 132.3 _+ 8.5 45.4 _+ 2.5 191.9 _+ 9.0 154.7_+ 7.2 168.9 _+ 7.8 217.1 _+ 8.1 3.7_+ 1.4 8.2 ± 2.6 214.4 _+ 9.1 180.0 _+ 14.5 56.2 _+ 7.3 30.6_+ 1.5 6.2-+ 1.6
12 12 12 12 12 12 12 22 12 12 12 6 4 5 5 10 10
7.0_+ 0.8 23.5 _+ 2.1 14.9 _+ 1.3 31.9_+ 2.4 59.5 + 6.7 19.2_+ 1.6 97.5 ± 9.5 73.3 _+ 4.8 82.4 -+ 7.9 105.6 +_ 9.8 3.2 _+ 0.7 10.8 _+ 2.7 134.9 _+ 17.5 88.1 _+ 9.8 21.9 _+ 1.3 15.5 _+ 1.5 6.0_+ 1.1
12 12 12 12 12 12 12 22 12 12 12 6 4 5 5 10 10
3.2 2.5 2.9 2.3 2.2 2.4 2.0 2.1 2.0 2.1 1.2 0.8 1.6 2.0 2.6 2.0 1.0
Entorhinal Cx Dorsal raphe (Caudal end) Raphepontisn. Central gray
Ent DR RPn CG
232.9_+ 115.6 _+ 41.1_+ 41.9_+
9 10 10 6
107.6_+ 40.2 -+ 10.8_+ 13.4_+
9 10 9 8
2.2 2.9 3.8 3.1
7.4 9.5 2.5 2.5
9.5 2.9 1.0 3.1
The data were derived from quantification of autoradiograms produced from coronal sections (4 per slide) from three animals incubated at 37°C with 4 nM [3H]8-OH-DPAT or 3 n M [3H]WAY-100635 for 30 rain and 60 min, respectively. In the particular case of [3H]8-OH-DPAT, it was a s s u m e d that only high-affinity 5-HT1A sites were measured. Nonspecific binding was d e f n e d with 10 /zM 5-HT. The unit of receptor density ( f m o l / m g wet weight tissue equivalent) was that supplied by the manufacturer of the autoradiographic standards (Amersham). Values represent the m e a n s of radioligand binding densities _+ S.E.M. of separate m e a s u r e m e n t s (n) per region made on sections from a total of 3 rats and have been corrected for receptor occupancy to give 'estimated' Bmax values, Approximately 79% of the [ 3H]WAY_100635 binding sites and 63% of the high-affinity [3H]8-OH-DPAT binding sites were occupied, where% receptor occupancy = 100/(L + Kd) , L = concentration of radioligand and K a = equilibrium dissociation constant [average [3H]WAY-100635 K d = 0.79 _+ 0.12 n M derived from Table 2 and average [3H]8-OH-DPAT K,t = 2.30 + 0.39 nM derived from Table 3]. Ratio represents the bound values for [3H]WAY-100635 divided by [3H]8-OH-DPAT for each brain region.
222
X. Khawaja /Brain Research 673 (1995) 217-225
100- Corr. Coeff. = 0.99746
Table 3 Scatchard plot parameters of specific [3H]8-OH-DPAT binding to slide-mounted rat brain sections
P<0.00Z (DF=4)
Hippocampus I
80-
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60-
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[3H]8-OH-DPAT SPECIFICALLY BOUND (frnol/mg protein)
Fig. 5. Correlation between the brain regional distributions of [3H]8O H - D P A T and [3H]WAY-100635 specific binding. Radioligand binding studies on various brain regions were performed as described in Materials and methods. Absolute levels of [3H]8-OH-DPAT specifically bound are indicated on the abscissa and those of [3H] WAY-100635 are on the ordinate. Data points represent the means of three experiments-+ S.E.M. performed in triplicate.
[3H]agonist binding were about 3-fold greater, and in others, such as the horizontal diagonal band n. (HDB) and raphe pontis n. (RPn), 4 fold greater (Table 1). In membrane homogenate radioreceptor binding studies, the hippocampus exhibited the highest level of specific [3H]WAY-100635 binding, followed by the septurn > frontal cortex > hypothalamus (Fig. 5). Lowest levels of [3H]WAY-100635 binding were measured in the striatum and cerebellum. Parallel studies measur-
Table 2 Scatchard plot parameters of specific [3H]WAY-100635 binding to slide-mounted rat brain sections Brain region
[3H]WAY-100635 K d (nM) _+S.E.M.
Bmax ( f m o l / m g tissue wet weight)-+ S.E.M.
1.02-+0.43 0.87 (0.76; 0.98)
187-+59 236 (182; 290)
3 2
Dentate gyrus (includes i n n e r / outer blades)
" 1.12_+0.21
243_+46
3
Dorsal raphe Rostral [Bregma - 7.3 to - 8 . 0 mm]
0.52 (0.51; 0.53)
157 (149; 165)
2
Entorhinal cortex
0.44 + 0.09
194 _+ 17
3
Hippocampus CAI CA3
g d (nM) -+ S.E.M.
Bmax ( f m o l / m g tissue wet weight)-+ S.E.M.
2.27-+0.59 1.10_+0.38
137-+18 175-+28
3 3
1.59 _+0.08
183 -+ 35
3
Dorsal raphe Rostral [Bregma - 7.3 to - 8 . 0 mm]
3.40 (1.73; 5.08)
163 (136; 189)
2
Entorhinal cortex
3.16-+ 1.01
141 -+ 12
3
Hippocampus CAI CA3 Dentate gyrus (includes i n n e r / outer blades)
Cortex
Serial coronal sections were incubated with increasing concentrations of [3H]WAY-100635 (0.2-14 nM) at 37°C for 60 min Nonspecific binding was defined with 10 ~zM 5-HT. Sections were washed, dried, and exposed to [3H]-sensitive film for 6 - 8 weeks. Structures were identified by reference to the atlas of Paxinos and Watson [26]. Values represent the m e a n s + S E M of separate experiments (n). Data were subjected to Student's t-test: ap < 0.05 vs entorhinal cortex K d.
[ 3H ] 8 - O H - D P A T
Serial coronal sections were incubated with increasing concentrations of [3H]8-OH-DPAT (0.7-14 nM) at 37°C for 30 min Nonspecific binding was defined with 10 /xM 5-HT. Sections were washed, dried, and exposed to [3H]-sensitive film for 6 - 7 weeks. Structures were identified by reference to the atlas of Paxinos and Watson [26]. Values represent the means-+ SEM of separate experiments (n).
ing the specific binding of [3H]8-OH-DPAT to the same membrane suspensions indicated a highly significant regional correlation (Corr. Coeff. = 0.997; P < 0.002, DF = 4) with [3H]WAY-100635 binding (Fig. 5), as well as, the ability of [3H]WAY-100635 to associate with a greater number (2.1 _+ 0.06 fold) of 5-HTIA binding sites. These findings corroborated the autoradiographic regional distribution studies. Saturation isotherms were generated using slidemounted serial sections from hippocampal, dorsal raphe and entorhinal cortical areas of the rat brain. The binding parameters (Kd; B m a x) determined for [3H]WAY-100635 are summarised in Table 2. [3H] WAY-100635 labelled a single population of sites which were saturable and of high affinity in all regions examined. The affinity of [3H]WAY-100635 for the 5-HT]A binding site tended to be higher in the dorsal raphe n. and entorhinal cortex compared with the hippocampal formation (Table 2). Parallel saturation studies with [3H]8-OH-DPAT concentrations ranging between 0.7 and 14 nM produced affinity constants (K d) which were not significantly different in the hippocampal, dorsal raphe and entorhinal cortical areas (Table 3). The B m a x values for [3H]8-OH-DPAT binding (Table 3) were not substantially different from those obtained for [3H]WAY-100635 binding (Table 2).
4. D i s c u s s i o n
The findings of the kinetic, saturation and regional distribution studies have shown [3H]WAY-100635 to
X. Khawaja / Brain Research 673 (1995) 217-225
be a practical and sensitive radioligand for identifying and assessing the binding characteristics of 5-HTIA binding sites by quantitative receptor autoradiography. The high affinity of [3H]WAY-100635 for the 5-HTIA binding site in the hippocampal formation (CA1, CA3, dentate gyrus) confirmed previous in vitro saturation binding studies using rat hippocampal membrane preparations [19], and was also demonstrated in the dorsal raphe (DRN) and entorhinal cortex (Table 2); the maximal binding densities were not significantly different in hippocampal, rostral dorsal raphe and entorhinal cortical areas and corresponded closely with their 'estimated' Bmax values from the regional distribution studies (Table 1). [3H]8-OH-DPAT saturation studies (Table 3) demonstrated similar Bmax values to those obtained with [3H]WAY-100635 in the hippocampal formation and rostral DRN; these were higher ( ~ 1.5-2 fold) than 'estimated' high-affinity [3H]8-OH-DPAT binding densities determined in the corresponding regional distribution studies (Table 1). The latter finding, although unexpected, would be consistent with a concentration-dependent agonist recognition of high-affinity versus lower affinity 5-HTIA binding states. In rat hippocampal membrane preparations, both high- and low-affinity agonist states of the 5-HTIA receptor have been identified [23,25] with typical values for the K d of the high-affinity [3H]8-OHDPAT-labelled sites ranging between 0.51 and 0.79 nM, and values for the K o of the low-affinity sites between 8.5 and 21.9 nM. In our regional distribution studies, low concentrations of [3H]8-OH-DPAT (~ 4 nM) would preferentially label high-affinity binding sites, whereas over a broader concentration range of radioligand (as used in the saturation studies), both high-affinity sites and a significantly greater proportion (~ 40-60%) of the lower affinity sites of the 5-HT1A receptor were detected. Scatchard analyses of the [3H]8-OH-DPAT saturation data would thereby extrapolate to give Bmax values (Table 3) comparable with those observed for [3H]WAY-100635 (Table 2). The regional distribution of [3H]WAY-100635-sensitive binding sites correlated closely with that of [3H]8OH-DPAT throughout the central nervous system of the rat, with the highest densities of 5-HTIA binding sites located in the hippocampal formation, septum, dorsal raphe and entorhinal cortex, areas known to be particularly dense in serotonergic nerve terminals [3] and 5-HTIA receptors [11,20,27,35]. Autoradiographic studies using [3H]8-OH-DPAT were in good agreement with the topographical distribution of 5-HTIA binding sites reported previously by other workers, using [3H]8-OH-DPAT, [3H]5-HT (in conjunction with selective blocking agents) and [125I]BH-8-MeO-N-PAT [11,27,35]. It has been proposed that the degree of receptorG-protein coupling will influence the affinity of an
223
agonist for its binding site as well as the proportion of total receptors in any given conformational state [15]. The G-protein-coupled form of the 5-HT~A receptor will exhibit a high affinity for an agonist ligand ([3H]-8OH-DPAT), whereas the G-protein-uncoupled form will display a much lower affinity for that same agonist [4,13,23]. In contrast, an antagonist, such as [3H]WAY100635, should not be influenced by the state of receptor-G-protein coupling and will therefore associate with high affinity to multiple conformational states of the receptor, representing a true estimate of total receptor density [19]. From our regional distribution studies, the ratio of [3H]WAY-100635 binding (total receptor density) to [3H]8-OH-DPAT binding (high-affinity sites only) gave an indication of the level of receptor-G-protein coupling in different areas of the central nervous system. In the majority of brain areas and nuclei examined, [3H]WAY-100635 associated with twice as many binding sites than did [3H]8-OH-DPAT. In some brain regions, the difference between [3H]antagonist and [3H]agonist binding was greater than 3-fold (frontal, cingulate, retrosplenial, temporal and parietal cortices, medial septal n., vertical limb diagonal band, caudal dorsal raphe and central gray), and in others nearly 4 fold (horizontal limb diagonal band and raphe pontis n.). It is evident that the relationship between the high affinity and the low affinity 5-HTIA binding states varied depending on the brain areas and nuclei examined. A ratio of 2 indicated that approximately 50% of the 5-HTIA receptors were in a G-protein pre-coupled high-affinity state; a higher ratio implied that a lower proportion of these sites were pre-coupled to native G-protein and were therefore assumed to be in a predominantly low-affinity state. Whether a particular G-protein subtype favors increased or decreased precoupling to the receptor is unknown, and because the functional relationship between the high- and low-affinity states is also not known, we cannot predict from our findings the level of responsiveness of any given brain area. However, it has been reported that the potency of agonists to inhibit 5-HTiA-adenylyl cyclase activity is functionally related to their affinity for the low-affinity state of [3H]8-OH-DPAT binding (G-protein uncoupled) rather than for the high-affinity (Gprotein coupled) state [2]. It is noteworthy that the density of [3H]WAY-100635 and [3H]8-OH-DPAT binding to somatodendritic 5HTIA autoreceptors was higher in the rostral portion of the DRN compared with the caudal end, with the caudal DRN possessing 5-HTIA receptors in a predominantly receptor-G-protein uncoupled state. Immunocytochemical studies have shown a topographical ordering of the cells in the DRN [34]. The ascending fibers of the rostral DRN project more heavily to the amygdala, cortex, and septum, and those of the caudal DRN project to the lateral septum, hippocampus and
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basal forebrain [34,36]. It can be postulated that local differences in receptor density a n d / o r coupling efficiency may modulate the sensitivity of particular afferent neuronal subregions within the DRN to 5-HTIA ligands, thereby influencing the function of specific target regions within the central nervous system. Radja et al. [31] have reported that the pharmacology of the 5-HT1A recognition sites and the coupling of the receptor to G-proteins is similar between rat hippocampus and DRN. However, electrophysiological studies have shown a region-dependent drug efficacy for 5-HT1A agonists. The signal transduction mechanism in the rat DRN is especially sensitive to 5-HT1A agonists (ECs0 and Ema x) compared with the hippocampus [33]. Certainly, a larger receptor reserve exists for 5-HT1A agonists in the DRN [22] versus hippocampus [37]. To our knowledge, no pharmacological studies detailing the effects of 5-HTIA ligands in the different subregions of the DRN on CNS activity have been reported. It seems that the conformationally active state of the 5-HTIA receptor and the efficacy of an agonist a n d / o r partial agonist would depend on such factors as receptor number a n d / o r efficiency of receptor-effector coupling, which may vary according to the particular subregional zone(s) targetted. In summary, we have demonstrated that [3H]WAY100635 is a potent radioligand that labels the 5-HTIA receptor with nanomolar affinity. [3H]WAY-100635 is suitably adapted for quantitative autoradiographic studies, producing clear, sharp and high quality autoradiograms, that revealed a heterogeneity of binding sites matching recognised central pathways of serotonergic innervation. The availability of [3H]WAY-100635 will be useful as a sensitive tool in in vitro and in vivo autoradiographic studies of the 5-HT1A receptor to further our understanding of receptor-effector coupling mechanisms and the functional regulation of 5HT1A receptors in the central nervous system.
Abbreviations
Central nervous system Dorsal raphe nucleus 5-hydroxytryptamine 8-hydroxy-2-(di-n-propylamino)tetralin N-(2-(4-(2-methoxyphenyl)- 1WAY-100635 piperazinyl)ethyl)-N-(2-pyridinyl)cyclohexane carboxamide trihydrochloride [3H]WAY-100635 [O-methyl3H]-N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2pyridinyl)cyclohexane carboxamide trihydrochloride
CNS DRN 5-HT 8-OH-DPAT
BMY 7378
NAN 190
SDZ-216525
S-UH301
8-[2-[4-(methoxyphenyl)- 1piperazinyl]-ethyl]-8-azaspirol-(4,5)decane-7,9-dione 1-(2-methoxyphenyl)-4-[4-(2-phthalimido)butyl]piperazine hydrobromide methyl-4-[4-[4-[1,1,3-trioxo-2H-1,2benziosothiazol-2-yl]butyl]-1-piperazinyl]- 1H-indole-2-carboxylate 5-fluoro-8-hydroxy-2-(dipropylamino)tetralin
References [1] Adham, N., Kao, H., Schechter, L. E., Bard, J., OIsen, M., Urquhart, D., Durkin, M., Hartig, P. R., Weinshank, R. L. and Branchek, T.A., Cloning of another human serotonin receptor (5-HTw): a fifth 5-HT l receptor subtype coupled to the inhibition of adenylate cyclase, Proc. Natl. Acad. Sci. USA, 90 (1993) 408-412. [2] Chamberlain, J., Offord, S.J., Wolfe, B.B., Tyau, L.S., Wang H.-L. and Frazer, A., Potency of 5-hydroxytryptaminelA agonists to inhibit adenylyl cyclase activity is a function of affinity for the 'low-affinity' state of [3H]8-hydroxy-N,N-dipropylaminotetralin ([3H]8-OH-DPAT) binding, Z Pharmacol. Exp. Ther., 266 (1993) 618-625. [3] Descarries, L., Audet, M.A., Doucet, G., Garcia, S., Oleskevich, S., Seguela, P., Soghomonian, J-J. and Watkins, K.C., Morphology of central serotonin neurons: brief review of quantified aspects of their distribution and ultrastructural relationships. [4] Emerit, M.B., El Mestikawy, S., Gozlan, H., Rouot, B. and Hamon, M., Physical evidence of the coupling of solubilised 5-HT1A binding sites with G regulatory proteins, Biochem. Pharmacol., 39 (1990) 7-18. [5] Fletcher, A., Cliffe, I.A. and Dourish, C.T., Silent 5-HTla receptor antagonists: utility as research tools and therapeutic agents, Trends" Pharmacol. Sci.., 14 (1993) 441-448. [6] Fletcher, A., Bill, D.J., Bill, S.J., Cliffe, I.A., Dover G.M., Forster, E.A., Haskins, J.T., Jones, D., Mansell, H,L. and Reilly, Y., WAY-100135: a novel, selective antagonist at presynaptic and postsynaptic 5-HTIA receptors, Eur. J. Pharmacol., 237 (1993) 283-291. [7] Fletcher, A., Cliffe, i.A., Forster, E.A., Jones, D. and Reilly, Y., A pharmacological profile of WAY-100635, a potent and selective 5-HT1A receptor antagonist, Br. J. Pharmacol., 112 (1994) 91P [8] Glaser, T., Rath, M., Traber, J., Zilles, K. and Schleicher A., Autoradiographic identification and topographical analyses of high affinity serotonin receptor subtypes as a target for the novel putative anxiolytic, TVX Q7821, Brain Res., 358 (1985) 129-136. [9] Glowinski, J. and Iversen, L., Regional studies of catecholamines in the rat brain. I. Disposition of [3H]norepinephfine, [3H]dopamine and [3Hldopa in various regions of the brain, Z Neurochem.., 13 (1966) 655-669. [10] Gozlan, H., El Mestikawy, S., Pichat, A., Glowinski, J. and Hamon, M., Identification of presynaptic serotonin autoreceptors using a new ligand [3H]PAT, Nature, 305 (1983) 140-142. [11] Gozlan, H., Ponchant, M., Daval, G., Verge, D., Menard, F., Vanhove, A., Beaucourt, J.P. and Hamon, M., 1251-BoltonHunter-8-methoxy-2-[N-propyl-N-propylamino]tetralin as a new
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selective radioligand of 5-HT1A sites in the rat brain. In vitro binding and autoradiographic studies, J. Pharmacol. Exp. Ther., 244 (1988) 751-759. Gurling, J., Ashworth-Preece, Dourish, C.T. and Routledge C., Effects of acute and chronic treatment with the selective 5-HT1A receptor antagonist WAY-100635 on hippocampal 5-HT release in vivo, Br. J. Pharmacol., 112 (1994) 299P Hall, M.D., El, Mestikawy, S., Emerit, M.B., Pichat, L., Hamon, M., Gozlan, H., [3H]8-Hydroxy-2-(Di-n-Propylamino)Tetralin binding to pre- and postsynaptic 5-Hydroxytryptamine sites in various regions of the rat brain, J. Neurochem., 44 (1985) 16851696. Hamon, M., Lanfumey, L., Lima, L., Laporte, A.M., Jolas, T., Fattaccini, C.M. and Gozlan, H., Electrophysiological and biochemical investigations with central 5-HTIA receptor antagonists. Third IUPHAR Satellite Meeting on Serotonin, Chicago, August, 1994, pp. 24. Harrington, M.A. and Peroutka, S.J., Modulation of 5-HT1A receptor density by non-hydrolysable GTP analogues. J. Neurochem., 54 (1990) 294-299. Hartig, P.R., Adham, N., Zgombick, J., Weinshank, R. and Branchek, T., Molecular biology of the 5-HT 1 receptor subfamily, Drug Dev. Res., 26 (1992) 215-224. Hoyer, D., Clarke, D.E., Fozard, J.R., Hartig, P.R., Martin G.R., Mylecharane, E.J., Saxena, P.R. and Humphrey, P.P.A., International Union of Pharmacology Classification of receptors for 5-Hydroxytryptamine (serotonin), Pharmacol. Rev., 46 (1994) 157-203 Julius, D., Molecular biology of serotonin receptors, Anna. Rev. Neurosci., 14 (1991) 335-360. Khawaja, X., Ifill, A. and Ennis, C., Characterisation of the binding of a novel and selective antagonist radioligand, [3H] WAY-100635, to the 5-HTIA receptor in rat brain, Third IUPHAR Satellite Meeting on Serotonin, Chicago, August, 1994, P74. Marcinkiewicz M., Verge, D., Gozlan, H., Pichat, H. and Hamon, M., Autoradiographic evidence for the heterogeneity of 5-HT~ sites in the rat brain, Brain Res., 291 (1984) 159-163. McPherson, G.A., Kinetic, EBDA, Ligand, Lowry: A Collection of Radioligand Binding Analysis Programs, Elsevier Science Publishers BV, Amsterdam, 1985. Meller, E., Goldstein, M. and Bohmaker, K., Receptor reserve for 5-hydroxytryptaminelA-mediated inhibition of serotonin synthesis: possible relationship to anxiolytic properties of 5-HT1A agonists, Mol. Parmacol., 37 (1990) 231-237. Mongeau, R., Welner, S.A., Quirion, R. and Suranyi-Cadotte, B.E., Further evidence for differential affinity states of the serotoninlA receptor in rat hippocampus, Brain Res., 590 (1992) 229-238. Munson, P.J. and Rodbard, D., LIGAND: a versatile comput-
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erised approach for the characterisation of ligand binding systems, Analyt. Biochem., 107 (1980) 220-239. Nenonene, E.K., Radja, F., Carli, M., Grondin, L. and Reader, T.A., Heterogeneity of cortical and hippocampal 5-HT1A receptors: a reappraisal of homogenate binding with 8-[3H]hydroxydipropylaminotetralin, Z Neurochem.., 62 (1994) 1822-1834. Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, 2nd edn., Academic Press, 1986. Pazos, A. and Palacios, J.M., Quantitative autoradiographic mapping of serotonin receptors in the rat brain. I. Serotonin-1 receptors, Brain Res., 346 (1985) 205-230. Peroutka, S.J., Selective interaction of novel anxiolytics with 5-hydroxytryptaminelA receptors, Biol. Psychiatry, 20 (1985) 971-979. Peroutka, S.J., 5-Hydroxytryptamine receptors, J. Neurochem., 60 (1993) 408-416. Pike, V.W., Hume, S.P., Ashworth, S., Opacka-Juffry, J., McCarron, J.A., Cliffe, I.A and Fletcher, A., EvaLuation of radiolabelled WAY-100635 as a potential ligand for in vivo studies of central 5-HTIA receptors, Third IUPHAR Satellite Meeting on Serotonin, Chicago, August, 1994, P73. Radja, F., Daval, G., Hamon, M. and Verge, D., Pharmacological and physicochemical properties of pre-versus postsynaptic 5-hydroxytryptamine~A receptor binding sites in the rat brain: a quantitative autoradiographic study, J. Neurochem., 58 (1992) 1338-1346. Routledge, C., Gurling, J., Wright, I.K. and Dourish, C.T., Neurochemical profile of the selective and silent 5-HT1A antagonist WAY-100135: an in vivo microdialysis study, Eur. J. Pharmacol., 239 (1993) 195-202. Sprouse, J.S. and Aghajanian, G.K., Responses of hippocampal pyramidal cells to putative serotonin 5-HT1A and 5-HT m agonists: a comparative study with dorsal raphe neurons, Nearopharmacology 27 (1988) 707-715. Van Bockstaele, E.J., Biswas, A. and Pickel, V.M., Topography of serotonin neurons in the dorsal raphe nucleus that send axon collaterals to the rat prefrontal cortex and nucleus accumbens, Brain Res., 624 (1993) 188-198. Verge, D., Daval, G., Marcinkiewicz, M., Patey, A., El Mestikawy, S., Gozlan, H. and Hamon, M., Quantitative autoradiography of multiple 5-HT 1 receptor subtypes in the brain of control or 5,7-dihydroxytryptamine-treated rats, J. Neurosci., 6 (12) (1986) 3474-3482. Vertes, R.P., A PHA-L analysis of ascending projections of the dorsal raphe nucleus in the rat, J. Comp. Neurol., 313 (1991) 643-668. Yocca, F.D., Iben, L. and Meller, E., Lack of receptor reserve (RR) at postsynaptic 5-HTIA receptors linked to adenylyl cyclase (AC) in rat hippocampus (HP), Soc. Neurosci. Abstr., 16 (1990) 1034.
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Research report
Quantitative autoradiographic characterisation of the binding of [3H]WAY-100635, a selective 5-HTIA receptor antagonist X. Khawaja Department of Molecular Pharmacology, Wyeth Research [UK], Huntercombe Lane South, Taplow, Maidenhead, Berkshire, UK Accepted 22 November 1994
Abstract
The binding characteristics of [3H]WAY-100635 ([O-methyl 3H]-N-(2-(4-(2-methoxyphenyl)-l-piperazinyl)ethyl)-N-(2pyridinyl)cyclohexane carboxamide trihydrochloride), a potent and selective 5-HTIA antagonist radioligand, were examined in the rat brain using in vitro quantitative receptor autoradiography. The regional distribution of specific [3H]WAY-100635 binding sites was heterogeneous and demonstrated a strong correlation with that of [3H]8-OH-DPAT binding. The highest concentrations of [3H]WAY-100635-1abelled sites were found in the lateral septal areas, dorsal raphe n., entorhinal cortex and the hippocampal formation (CA1, CA3 and dentate gyrus). Scatchard transformation of saturation isotherms revealed saturable [3H]WAY-100635 binding sites of high-affinity: in the hippocampal formation, K d was ~ 1 nM and Bmax ranged between 187 and 243 fmol/mg tissue wet weight, in the entorhinal cortex, K d = 0.44 nM and Bmax = 194 fmol/mg tissue wet weight, and in the rostral portion of the dorsal raphe n., K d = 0.52 nM and Bmax = 157 fmol/mg tissue wet weight. The affinity of [3H]WAY-100635 for the 5-HTIA binding site tended to be higher in the dorsal raphe n. and entorhinal cortex compared with that of the hippocampal formation. In contrast, the binding affinity of [3H]8-OH-DPAT in the hippocampal formation was between 1.1 and 2.3 nM and the Bmax was 137 to 183 fmoles/mg tissue wet weight; in the entorhinal cortex, K d = 3.2 nM and Bm~ = 141 fmoles/mg tissue wet weight, and in the rostral portion of the dorsal raphe n., K a = 3.4 nM and B~nax= 163 fmol/mg tissue wet weight. Our findings support the use of [3H]WAY-100635 as a novel antagonist radioligand for studying the binding characteristics of the 5-HTIA receptor by quantitative receptor autoradiography.
Keywords: [3H]WAY-100635; 5-HTIA antagonist; Quantitative autoradiography; Central nervous system; Rat; [3H]8-OH-DPAT
I. Introduction
Recent progress in 5-HT receptor neuropharmacology has grown with the identification of at least seven distinct classes of the receptor in the central nervous system [17]. The 5-HT l receptor has been differentiated into multiple subtypes [1,16,29] with the 5-HTIA receptor being of particular interest because of its potential role as a therapeutic target in the treatment of certain psychopharmacological disorders [5,28]. Whilst numerous potent and selective agonists at the 5-HT~A receptor have been synthesised [17], the paucity of 5-HTIA antagonists has made functional correlates difficult to interpret in animal behavioral studies. Compounds such as S-UH301, SDZ-216525,
* Corresponding author. Fax: (44) (628) 666-587. 0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved
SSDI 0 0 0 6 - 8 9 9 3 ( 9 4 ) 0 1 4 1 6 - 7
BMY 7378 and NAN-190, originally considered as selective 5-HTIA antagonists, were subsequently classified as weak partial agonists a n d / o r as possessing insufficient selectivity for the 5-HT1A receptor [5]. More recently, WAY-100135 and its second generation achiral analogue, WAY-100635, have proved to be highly selective and potent antagonists at the 5-HT~A receptor in animal behavioral [6,7] and in vivo microdialysis studies [12,32]. For the past decade, a variety of 5-HT~A-selective radioligands have been used extensively in in vitro receptor binding assays and autoradiographic studies to characterise the binding properties of the 5-HTIA receptor. The major limitation with most of these radioligands is that they are either full agonists ([3H]8O H - D P A T [4,10,13,31]; [125I]BH-8-MeO-N-PAT [11]) or partial agonists ([3H]ipsapirone [8]), which has constrained the level of information gained from pharma-
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cological binding studies on the G-protein coupled 5-HTlA receptor [18]. The tritiated form of WAY-100635 was developed as a selective 5-HTIA receptor antagonist radioligand and we have recently reported on the binding characteristics of [3H]WAY-100635 at the 5-HT1A site in rat brain [19]. The radioligand exhibited high affinity (K d = 0.37 _+ 0.051 nM) for a single population of 5-HTtA binding sites in rat hippocampal membrane preparations, with a maximal binding density (Bmax = 312 _+ 12 fmol/mg protein) which was approximately 36% higher than that of [3H]8-OH-DPAT. The pharmacological profile of [3H]WAY-100635 was consistent with the selective labelling of 5-HT1A sites, and with the ability of the radioligand to recognise with equal affinity multiple conformational states of the 5-HT1A receptor both in the absence and presence of guanyl nucleotides [19]. Moreover, ex vivo and in vivo biodistribution studies with [3H]WAY-100635 in rat and mouse indicated a regional distribution of radioactivity matching the localisation of central 5-HTiA receptor rich regions [14,30]. The present study reports on the use of [3H]WAY100635 as a potent selective antagonist radioligand to study the binding characteristics and regional distribution of the 5-HT1A receptor in the central nervous system by in vitro quantitative receptor autoradiography.
min at 37°C in 50 m M Tris-HCl buffer supplemented with 10 /xM pargyline and 4 m M CaCI 2. Nonspecific binding was determined in adjacent sections exposed to 10 /zM 5-HT. For saturation studies, conditions were essentially identical to those described for the regional studies, except that tissue sections were incubated with increasing concentrations of [3H]WAY-100635 (0.2-14 nM) or [3H]8O H - D P A T (0.7-14 nM). Following incubation, the sections were rinsed in ice-cold 50 m M Tris-HCl buffer ([3H]WAY-100635 2 min and 3 min; [3H]8-OH-DPAT: 2 × 4 rain) and dipped rapidly in ice-cold de-ionised water (5 s).
2.4. Autoradiographic quantitation
2. Materials and methods
Air-dried sections were apposed to [3H]-sensitive film (Hyperfilm-3H; A m e r s h a m plc., UK) together with autoradiographic [3H]microscales (Arrlersham plc., UK) for 6 - 8 weeks at room-temperature. The film was developed for 3 rain in Kodak Industrex manual developer, rinsed in tap water for 1 min, and fixed for 5 min in Kodak fixer. Ligand binding was quantitated by computer-aided densitometry using the Seescan Solitaire processing system (Seescan plc., Cambridge, UK). Specific binding was defined as the difference between total and non-specific values measured on adjacent sections. Bilateral m e a s u r e m e n t s of gray levels were made within various brain regions, identified using the rat brain atlas of Paxinos and Watson [26]. Brain gray matter tissue equivalents ( B q / m g tissue wet weight) were determined by extrapolation from a computer-generated standard curve, which compared film densities produced by the tissue section with those produced by the radio-active standards. The receptor density of a brain area was expressed as femtomoles of radioligand specifically bound per mg tissue wet weight. The regional concentrations from equivalent brain areas per experimental group of animals were averaged. Saturation isotherms were analysed using the non-linear, least-squares, curve-fitting programmes E B D A (Equilibrium Binding Data Analysis) and L I G A N D [24] as modified by McPherson [21], and final K d (nM) and B .... ( f m o l / m g tissue wet weight) parameters were generated.
2.1. Materials
2.5. Rat brain membrane preparation
[3H]WAY-100635 was custom synthesised by A m e r s h a m International plc. (UK) at an initial specific activity of 69 C i / m m o l (98.5% radiochemical purity). [3H]8-OH-DPAT (sp. act. 160-240 C i / m m o l ; 98% radiochemical purity) was supplied by A m e r s h a m International plc (UK). 8 - O H - D P A T (RBI, UK); 5-hydroxytryptamine (Sigma Chemical Co. Poole, UK); WAY-100635 was synthesised by the Chemistry D e p a r t m e n t Wyeth Research (UK).
Male Sprague-Dawley rats (150-170 g) were killed by a blow to the head and cervical dislocation. The brains were rapidly removed and hippocampus, septum, frontal cortex, hypothalamus, striatum and cerebellum were hand-dissected on ice by the method of Glowinski and lversen [9], and stored at - 7 0 ° C . Tissue was thawed at room temperature and initially homogenised using a Brinkman Polytron tissue disrupter (PT 10 probe, setting 5 for 30 s) in 50 vols. (w/v) of ice-cold 50 m M Tris-HCl buffer (pH 7.4). The suspension was centrifuged twice at 27,000× g for 20 min at 4°C. The membrane pellets were resuspended in 50 vols. ( w / v ) Tris-HC1 buffer and incubated at 37°C for 20 min in a shaking water bath, prior to a final centrifugation step (27,000 x g; 20 min; 4°C). The final tissue pellets were stored at - 7 0 ° C until assayed.
2.2. Preparation of slide-mounted sections Male strain Sprague-Dawley rats (150-170 g) were sacrificed by cervical dislocation and decapitation. The brains were carefully removed, frozen in isopentane ( - 4 0 ° C on dry ice) and stored at - 8 0 ° C . Serial coronal sections ( 2 0 / x m thick; 3 - 4 per slide) were cut using a cryomicrotome at - 2 0 ° C , thaw-mounted onto gelatin (5 g/I)-KCr(SO) 4 (0.5 g/I) double-coated glass microscope slides, and stored sealed at - 2 0 ° C until used.
2.3. Quantitative autoradiographic regional distribution and saturation studies Slide-mounted brain sections were warmed to room-temperature and pre-incubated in 50 m M Tris-HCl buffer (pH 7.4) for 30 min at room-temperature. The sections were then incubated with either 3 nM [3H]WAY-100635 for 60 min at 37°C in 50 m M Tris-HCl buffer containing 10 /xM pargyline or with 4 n M [3H]8-OH-DPAT for 30
2.6. Radioreceptor binding assays Hippocampal, cortical, septal, hypothalamic, striatal and cerebellar m e m b r a n e homogenates ( 2 - 4 mg t i s s u e / t u b e ) were incubated in 50 m M Tris-HCI (pH 7.4) with [3H]WAY-100635 (0.5 nM) for 50 min at 37°C in the absence or presence of 10 ~ M unlabelled 8 - O H - D P A T to determine non-specific binding. Parallel binding assays with [3H]8O H - D P A T (1 nM) ( + 4 m M CaCI 2) were performed at 37°C for 20 min, using the same m e m b r a n e preparations. Statistical analyses of the data were carried out using the statistics package S T A T V I E W (Abacus concepts, Inc., Berkeley, CA, 1992) and Student's two-tailed t-test.
X. Khawaja /Brain Research 673 (1995) 217-225 A
3. Results
The chemical structure of tritiated WAY-100635 is depicted in Fig. 1. Initial experiments were carried out to establish the optimal binding conditions for quantitative autoradiography using [3H]WAY-100635. After each experiment, brain sections were wiped from the microscope slides with pre-wet Whatman G F / B filters and bound radioactivity was measured by liquid scintillation counting. Sections taken from the hippocampal region were incubated with [3H]WAY-100635 (0.7 nM) at 37°C for 60 min and washed for various times in two changes of Tris-HCl buffer at 4°C (Fig. 2). Total and non-specific binding of [3H]WAY-100635 to the sections decreased with time; non-specific binding represented approximately 15-20% of total bound radioactivity after a 2-min wash with no marked change in the level of specific binding. After a 10-min wash, total binding was further reduced vTith no comparable decrease in non-specific binding; this was interpreted as representing dissociation of [3H]WAY-100635 from specific 5-HT~A binding sites. The optimal wash time adopted was a 2 min followed by a 3 min rinse in ice-cold Tris buffer. The effect of temperature on the time course of [3H]WAY-100635 binding to rat hippocampal slide-mounted sections is shown in Fig. 3. Association of [3H]WAY-100635 to central 5-HTIA binding sites was temperature sensitive. Maximal binding occurred at 37°C and at room-temperature, but was 50% lower at 4°C; steady-state was reached after 20 min at 37°C, 100 min at room-temperature, and required over 3 h at 4°C (Fig. 3). An optimal incubation time of 60 min at 37°C was selected for subsequent studies. The regional distribution of specific [3H]WAY100635-sensitive binding sites in rat brain is shown in Fig. 4 (Plates A - F ) and was identical to that of [3H]8O H - D P A T (not shown); the darker areas in the autoradiograms indicated regions of higher receptor density. Table 1 summarises the quantitative regional variations in specific [3H]WAY-100635 and [3H]8-OHD P A T binding throughout the rat central nervous system. [3H]WAY-100635-sensitive 5-HTIA binding sites were heterogeneously distributed, being particularly abundant in the taenia tecta, lateral septum (LSD =
219
3000
2500
2000 1500
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TO_AL
• " -
-- ~-- NON-SPECIFIC 1000
.....
.
.
• . . + .....
SPECIFIC
,=.,. ,=..
500
a
0
--a
i
i
i
i
i
i
i
i
2
4
6
8
10
12
14
16
Wash t i m e (rain.) Fig. 2. W a s h curve for [3H]WAY-100635 bound to slide-mounted rat hippocampal sections. Sections were incubated with 1 nM [3H] WAY-100635 at 37°C for 60 min and then rinsed in Tris-HCl buffer at 4°C for various times (1-15 min). Non-specific binding was defined in the presence of 10 /zM 5-HT. Tissue sections were wiped onto G F / B filters and the radio-activity counted. Data shown are the m e a n + S.E.M. of a representative of two experiments performed in quadruplicate.
LSI > LSV), hippocampus (OB1 = CA3 > IB1 = D G > CA1 > > CA2), dorsal raphe nucleus and entorhinal cortex. Lower levels were detected in most cortical regions (frontal, orbital, cingulate, dorsal p e d u n c u l a r / infralimbic, parietal, occipital), amygdalohippocampal area, median raphe, as well as certain discrete nuclei such as the anterior olfactory nucleus (medial/dorsal), and certain septal nuclei (septohippocampal, medial septal, vertical limb diagonal band). Low to intermediate binding densities were detected in the agranular A
.°= - ~ooo
u)
~0.
i
T
800-
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. .
/ o
600 !
o
400 ~
~'
/ -
----.oom , . = J.
4°C
200
u
m
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OMe C3H3
"',,,~"
0 Fig. 1. C h e m i c a l s t r u c t u r e o f [ H3 ] W A Y - 1 0 0 6 3 5 .
Fig. 3. Temperature-time course of association of [3H]WAY-100635 b o u n d to slide-mounted rat hippocampal sections. Sections were incubated with 1 nM [3H]WAY-100635 for various time intervals at 4°C, room-temperature and 37°C. Non-specific binding was determined in the presence of 10 ~ M 5-HT. Sections were washed in two changes of Tris-HCl buffer (2 m i n + 3 rain) at 4°C. Tissue sections were wiped onto G F / B filters and the radio-activity counted. Data shown are the mean_+ S.E.M. of a representative of two experiments performed in quadruplicate.
X. Khawaja / Brain Research 673 (1995) 217-225
220
and [3H]WAY-100635 binding throughout the central nervous system (Correlation coefficient = 0.978; P < 0.001, d f = 46). In most brain areas examined, the number of 5-HT1A binding sites labelled by [3H]WAY100635 was on average 2.3 + 0.10 times higher than that for high-affinity [3H]8-OH-DPAT binding (Table 1). In certain regions of the cortex (frontal, cingulate, parietal, temporal, retrosplenial), septum (medial, VDB), central gray and caudal portion of the dorsal raphe n., the differences between [3H]antagonist and
insular and lateral/ventrolateral orbital cortices, retrosplenial and temporal cortices, anterior olfactory nucleus (lateral/ventral), claustrum, horizontal limb diagonal band, CA2 hippocampal area, central gray and raphe pontis. Sparse levels of [3H]WAY-100635 binding sites were found in the nucleus accumbens (Acb), corpus callosum (fmi), globus pallidus, caudate-putamen (CPu), thalamic areas and substantia nigra. A significant positive correlation was observed between the regional distributions of [3H]8-OH-DPAT
/
Fr Cx
LO
~)M/AOD ._vL/AOV
PLATE B
T T
PLATE A
t Sept Pu
DC Amyg
PLATE C
E
l _ J / - ' ~ Jt l . . a
PLATE D
P L A T E F ~*~
Fig. 4. Autoradiographic localisation of specific [~H]WAY-100635 binding to coronal sections of rat brain. The images are dark-field transforms of specific radioligand binding. Representative brain sections are shown in a rostral-caudal (A F) sequence. Sections (20/xm) were incubated with 3 nM [3H]WAY-100635 at 37°C for 60 min. Non-specific binding was defined with 10/xM 5-HT. Sections were washed, dried, and exposed to [3H]-sensitive film for 6-8 weeks. Anatomical nomenclature used in describing the distribution of 5-HT1A binding sites and structures were identified by reference to the atlas of Paxinos and Watson [26]; Fr. Cx, frontal cortex; LO, lateral orbital cortex; A O M / A O D , anterior olfactory nucleus (medial/dorsal); A O L / A O V , anterior olfactory nucleus (lateral/ventral); Par, parietal cortex; DP/IL, dorsal peduncular/infralimbic cortex; TT, taenia tecta; fmi, forceps minor corpus callosum; Cg, Cingulate cortex; Lat. sept., Lateral septum; CPu, caudate-putamen; DG, dentate gyrus; Amygd., amygdala; Ent, entorhinal cortex; DR, dorsal raphe n.; MnR, median raphe n.; RPn, raphe pontis n.
X. Khawaja / Brain Research 673 (1995) 217-225
221
Table 1 Regional distribution of 5-HT1A receptors labelled by [3H]WAY-100635 and [3H]8-OH-DPAT Plate [bregma]
Region
Abbrev.
A
Frontal Cx (area 2) M e d i a l / V e n t r a l orbital Cx Lateral orbital Cx Agranular insular Cx Ant. olf. n. (lateral/ventral) Ant. olf. n. ( m e d i a l / d o r s a l )
Fr2 MO/VO LO AI AOL/AOV AOM/AOD
Frontal Cx (areas 1, 2, 3) Cingulate Cx (areas 1, 3) P a r i e t a l / A g r . insular Cx Dors. ped./infralimbic Cx Taenia tecta L a t . / V e n t r o l a t . orbital Cx Claustrum A c c u m b e n s n. Forceps minor corp. call. Frontal Cx (areas 1, 2) Cingulate Cx (areas 1, 2) Parietal Cx (area 1) Caudate p u t a m e n A c c u m b e n s n. Lateral septal n. (dorsal) Septohippocampal n. Lat. sept. n. (intermediate) Lateral septal n. (ventral) Medial septal n. n, vertical limb diag. band n, horizon limb diag. band
[5.20 mm]
B [2.70 mm]
C [0.70 mm]
D [ - 4 . 3 0 mm]
E [ - 7.64 mm]
F [-8.72mm]
[3H]WAY-100635 ( f m o l / m g tissue wet weight)
n
High affinity [ 3H]8-OH-DPAT ( f m o l / m g tissue wet weight)
n
Ratio
64.2 56.4 43.7 34.7 28.5 51.9
+ 3.5 + 2.0 + 2.9 + 0.9 + 1.6 + 1.2
10 10 10 10 10 10
28.2 33.5 23.6 14.9 15.5 26.7
+ _+ + _+ _+ _+
2.2 2.4 1.9 1.3 0.7 1.0
12 12 12 12 12 12
2.3 1.7 1.8 2.3 1.8 1.9
Frl,2,3 Cgl,3 Parl/AI DP/IL TT LO/VLO C1 Acb fmi Frl,2 Cgl,2 Parl CPu Acb LSD SHi LSI LSV MS VDB HDB
58.5 72.4 43.7 67.3 112.2 43.9 45.1 2.7 5.3 64.4 79.6 62.8 3.5 18.6 165.8 65.6 170.2 119.0 61.9 73.3 45.4
+ 0.9 + 1.4 + 0.8 + 1.7 + 3.6 + 1.6 + 1.1 + 0.4 _+ 1.5 + 1.7 _+ 2.1 _+ 1.6 + 1.1 + 1.5 -+ 5.7 + 10.0 -+ 3.5 + 4.3 + 2.8 -+ 3.7 + 3.2
6 6 6 6 6 6 6 6 6 18 18 18 18 18 18 9 18 18 9 9 18
17.9 25.1 14.3 28.9 51.6 17.1 17.1 4.3 2.7 21.9 31.9 21.3 3.5 7.3 89.0 29.4 80.6 55.6 21.1 24.8 11.7
+ _+ + + _+ _+ + + + + + + + + + + + + + + _+
0.7 0.7 1.4 1.7 2.5 1.0 1.4 0.2 0.8 1.7 2.1 1.7 0.7 0.5 4.3 4.8 4.8 4.4 2.2 2.2 0.9
6 6 6 6 6 6 6 6 6 18 18 18 18 18 18 18 18 18 9 9 18
3.3 2.9 3.1 2.3 2.2 2.6 2.6 0.6 2.0 2.9 2.5 3.0 1.0 2.5 1.9 2.2 2.1 2.1 2.9 3.0 3.9
Retrosplenial Cx Occipital Cx Temporal Cx (areas 1, 3) Amygdalohippoc. area CA1 CA2 CA3 D e n t a t e gyrus Inner blade dentate gyrus O u t e r blade dentate gyrus Thalamic region Medial mammillary n. Entorhinal Cx Dorsal raphe (rostrl end) Median raphe n. Central gray Pontine n.
RSG/RSA Oc2 Tel,3 AHi CA1 CA2 CA3 DG IB1 OB1 MM Ent DR MnR CG Pn
22.5 _+ 1.9 58.9_+ 1.6 43.3 _+ 1.1 73.4-+ 4.6 132.3 _+ 8.5 45.4 _+ 2.5 191.9 _+ 9.0 154.7_+ 7.2 168.9 _+ 7.8 217.1 _+ 8.1 3.7_+ 1.4 8.2 ± 2.6 214.4 _+ 9.1 180.0 _+ 14.5 56.2 _+ 7.3 30.6_+ 1.5 6.2-+ 1.6
12 12 12 12 12 12 12 22 12 12 12 6 4 5 5 10 10
7.0_+ 0.8 23.5 _+ 2.1 14.9 _+ 1.3 31.9_+ 2.4 59.5 + 6.7 19.2_+ 1.6 97.5 ± 9.5 73.3 _+ 4.8 82.4 -+ 7.9 105.6 +_ 9.8 3.2 _+ 0.7 10.8 _+ 2.7 134.9 _+ 17.5 88.1 _+ 9.8 21.9 _+ 1.3 15.5 _+ 1.5 6.0_+ 1.1
12 12 12 12 12 12 12 22 12 12 12 6 4 5 5 10 10
3.2 2.5 2.9 2.3 2.2 2.4 2.0 2.1 2.0 2.1 1.2 0.8 1.6 2.0 2.6 2.0 1.0
Entorhinal Cx Dorsal raphe (Caudal end) Raphepontisn. Central gray
Ent DR RPn CG
232.9_+ 115.6 _+ 41.1_+ 41.9_+
9 10 10 6
107.6_+ 40.2 -+ 10.8_+ 13.4_+
9 10 9 8
2.2 2.9 3.8 3.1
7.4 9.5 2.5 2.5
9.5 2.9 1.0 3.1
The data were derived from quantification of autoradiograms produced from coronal sections (4 per slide) from three animals incubated at 37°C with 4 nM [3H]8-OH-DPAT or 3 n M [3H]WAY-100635 for 30 rain and 60 min, respectively. In the particular case of [3H]8-OH-DPAT, it was a s s u m e d that only high-affinity 5-HT1A sites were measured. Nonspecific binding was d e f n e d with 10 /zM 5-HT. The unit of receptor density ( f m o l / m g wet weight tissue equivalent) was that supplied by the manufacturer of the autoradiographic standards (Amersham). Values represent the m e a n s of radioligand binding densities _+ S.E.M. of separate m e a s u r e m e n t s (n) per region made on sections from a total of 3 rats and have been corrected for receptor occupancy to give 'estimated' Bmax values, Approximately 79% of the [ 3H]WAY_100635 binding sites and 63% of the high-affinity [3H]8-OH-DPAT binding sites were occupied, where% receptor occupancy = 100/(L + Kd) , L = concentration of radioligand and K a = equilibrium dissociation constant [average [3H]WAY-100635 K d = 0.79 _+ 0.12 n M derived from Table 2 and average [3H]8-OH-DPAT K,t = 2.30 + 0.39 nM derived from Table 3]. Ratio represents the bound values for [3H]WAY-100635 divided by [3H]8-OH-DPAT for each brain region.
222
X. Khawaja /Brain Research 673 (1995) 217-225
100- Corr. Coeff. = 0.99746
Table 3 Scatchard plot parameters of specific [3H]8-OH-DPAT binding to slide-mounted rat brain sections
P<0.00Z (DF=4)
Hippocampus I
80-
Brain region
60-
N
2
Striatum~~
"
"Cerebellum "~ ~'
0
S
~O
(S
Z'o
Z'S
3'0
3S
4'O
[3H]8-OH-DPAT SPECIFICALLY BOUND (frnol/mg protein)
Fig. 5. Correlation between the brain regional distributions of [3H]8O H - D P A T and [3H]WAY-100635 specific binding. Radioligand binding studies on various brain regions were performed as described in Materials and methods. Absolute levels of [3H]8-OH-DPAT specifically bound are indicated on the abscissa and those of [3H] WAY-100635 are on the ordinate. Data points represent the means of three experiments-+ S.E.M. performed in triplicate.
[3H]agonist binding were about 3-fold greater, and in others, such as the horizontal diagonal band n. (HDB) and raphe pontis n. (RPn), 4 fold greater (Table 1). In membrane homogenate radioreceptor binding studies, the hippocampus exhibited the highest level of specific [3H]WAY-100635 binding, followed by the septurn > frontal cortex > hypothalamus (Fig. 5). Lowest levels of [3H]WAY-100635 binding were measured in the striatum and cerebellum. Parallel studies measur-
Table 2 Scatchard plot parameters of specific [3H]WAY-100635 binding to slide-mounted rat brain sections Brain region
[3H]WAY-100635 K d (nM) _+S.E.M.
Bmax ( f m o l / m g tissue wet weight)-+ S.E.M.
1.02-+0.43 0.87 (0.76; 0.98)
187-+59 236 (182; 290)
3 2
Dentate gyrus (includes i n n e r / outer blades)
" 1.12_+0.21
243_+46
3
Dorsal raphe Rostral [Bregma - 7.3 to - 8 . 0 mm]
0.52 (0.51; 0.53)
157 (149; 165)
2
Entorhinal cortex
0.44 + 0.09
194 _+ 17
3
Hippocampus CAI CA3
g d (nM) -+ S.E.M.
Bmax ( f m o l / m g tissue wet weight)-+ S.E.M.
2.27-+0.59 1.10_+0.38
137-+18 175-+28
3 3
1.59 _+0.08
183 -+ 35
3
Dorsal raphe Rostral [Bregma - 7.3 to - 8 . 0 mm]
3.40 (1.73; 5.08)
163 (136; 189)
2
Entorhinal cortex
3.16-+ 1.01
141 -+ 12
3
Hippocampus CAI CA3 Dentate gyrus (includes i n n e r / outer blades)
Cortex
Serial coronal sections were incubated with increasing concentrations of [3H]WAY-100635 (0.2-14 nM) at 37°C for 60 min Nonspecific binding was defined with 10 ~zM 5-HT. Sections were washed, dried, and exposed to [3H]-sensitive film for 6 - 8 weeks. Structures were identified by reference to the atlas of Paxinos and Watson [26]. Values represent the m e a n s + S E M of separate experiments (n). Data were subjected to Student's t-test: ap < 0.05 vs entorhinal cortex K d.
[ 3H ] 8 - O H - D P A T
Serial coronal sections were incubated with increasing concentrations of [3H]8-OH-DPAT (0.7-14 nM) at 37°C for 30 min Nonspecific binding was defined with 10 /xM 5-HT. Sections were washed, dried, and exposed to [3H]-sensitive film for 6 - 7 weeks. Structures were identified by reference to the atlas of Paxinos and Watson [26]. Values represent the means-+ SEM of separate experiments (n).
ing the specific binding of [3H]8-OH-DPAT to the same membrane suspensions indicated a highly significant regional correlation (Corr. Coeff. = 0.997; P < 0.002, DF = 4) with [3H]WAY-100635 binding (Fig. 5), as well as, the ability of [3H]WAY-100635 to associate with a greater number (2.1 _+ 0.06 fold) of 5-HTIA binding sites. These findings corroborated the autoradiographic regional distribution studies. Saturation isotherms were generated using slidemounted serial sections from hippocampal, dorsal raphe and entorhinal cortical areas of the rat brain. The binding parameters (Kd; B m a x) determined for [3H]WAY-100635 are summarised in Table 2. [3H] WAY-100635 labelled a single population of sites which were saturable and of high affinity in all regions examined. The affinity of [3H]WAY-100635 for the 5-HT]A binding site tended to be higher in the dorsal raphe n. and entorhinal cortex compared with the hippocampal formation (Table 2). Parallel saturation studies with [3H]8-OH-DPAT concentrations ranging between 0.7 and 14 nM produced affinity constants (K d) which were not significantly different in the hippocampal, dorsal raphe and entorhinal cortical areas (Table 3). The B m a x values for [3H]8-OH-DPAT binding (Table 3) were not substantially different from those obtained for [3H]WAY-100635 binding (Table 2).
4. D i s c u s s i o n
The findings of the kinetic, saturation and regional distribution studies have shown [3H]WAY-100635 to
X. Khawaja / Brain Research 673 (1995) 217-225
be a practical and sensitive radioligand for identifying and assessing the binding characteristics of 5-HTIA binding sites by quantitative receptor autoradiography. The high affinity of [3H]WAY-100635 for the 5-HTIA binding site in the hippocampal formation (CA1, CA3, dentate gyrus) confirmed previous in vitro saturation binding studies using rat hippocampal membrane preparations [19], and was also demonstrated in the dorsal raphe (DRN) and entorhinal cortex (Table 2); the maximal binding densities were not significantly different in hippocampal, rostral dorsal raphe and entorhinal cortical areas and corresponded closely with their 'estimated' Bmax values from the regional distribution studies (Table 1). [3H]8-OH-DPAT saturation studies (Table 3) demonstrated similar Bmax values to those obtained with [3H]WAY-100635 in the hippocampal formation and rostral DRN; these were higher ( ~ 1.5-2 fold) than 'estimated' high-affinity [3H]8-OH-DPAT binding densities determined in the corresponding regional distribution studies (Table 1). The latter finding, although unexpected, would be consistent with a concentration-dependent agonist recognition of high-affinity versus lower affinity 5-HTIA binding states. In rat hippocampal membrane preparations, both high- and low-affinity agonist states of the 5-HTIA receptor have been identified [23,25] with typical values for the K d of the high-affinity [3H]8-OHDPAT-labelled sites ranging between 0.51 and 0.79 nM, and values for the K o of the low-affinity sites between 8.5 and 21.9 nM. In our regional distribution studies, low concentrations of [3H]8-OH-DPAT (~ 4 nM) would preferentially label high-affinity binding sites, whereas over a broader concentration range of radioligand (as used in the saturation studies), both high-affinity sites and a significantly greater proportion (~ 40-60%) of the lower affinity sites of the 5-HT1A receptor were detected. Scatchard analyses of the [3H]8-OH-DPAT saturation data would thereby extrapolate to give Bmax values (Table 3) comparable with those observed for [3H]WAY-100635 (Table 2). The regional distribution of [3H]WAY-100635-sensitive binding sites correlated closely with that of [3H]8OH-DPAT throughout the central nervous system of the rat, with the highest densities of 5-HTIA binding sites located in the hippocampal formation, septum, dorsal raphe and entorhinal cortex, areas known to be particularly dense in serotonergic nerve terminals [3] and 5-HTIA receptors [11,20,27,35]. Autoradiographic studies using [3H]8-OH-DPAT were in good agreement with the topographical distribution of 5-HTIA binding sites reported previously by other workers, using [3H]8-OH-DPAT, [3H]5-HT (in conjunction with selective blocking agents) and [125I]BH-8-MeO-N-PAT [11,27,35]. It has been proposed that the degree of receptorG-protein coupling will influence the affinity of an
223
agonist for its binding site as well as the proportion of total receptors in any given conformational state [15]. The G-protein-coupled form of the 5-HT~A receptor will exhibit a high affinity for an agonist ligand ([3H]-8OH-DPAT), whereas the G-protein-uncoupled form will display a much lower affinity for that same agonist [4,13,23]. In contrast, an antagonist, such as [3H]WAY100635, should not be influenced by the state of receptor-G-protein coupling and will therefore associate with high affinity to multiple conformational states of the receptor, representing a true estimate of total receptor density [19]. From our regional distribution studies, the ratio of [3H]WAY-100635 binding (total receptor density) to [3H]8-OH-DPAT binding (high-affinity sites only) gave an indication of the level of receptor-G-protein coupling in different areas of the central nervous system. In the majority of brain areas and nuclei examined, [3H]WAY-100635 associated with twice as many binding sites than did [3H]8-OH-DPAT. In some brain regions, the difference between [3H]antagonist and [3H]agonist binding was greater than 3-fold (frontal, cingulate, retrosplenial, temporal and parietal cortices, medial septal n., vertical limb diagonal band, caudal dorsal raphe and central gray), and in others nearly 4 fold (horizontal limb diagonal band and raphe pontis n.). It is evident that the relationship between the high affinity and the low affinity 5-HTIA binding states varied depending on the brain areas and nuclei examined. A ratio of 2 indicated that approximately 50% of the 5-HTIA receptors were in a G-protein pre-coupled high-affinity state; a higher ratio implied that a lower proportion of these sites were pre-coupled to native G-protein and were therefore assumed to be in a predominantly low-affinity state. Whether a particular G-protein subtype favors increased or decreased precoupling to the receptor is unknown, and because the functional relationship between the high- and low-affinity states is also not known, we cannot predict from our findings the level of responsiveness of any given brain area. However, it has been reported that the potency of agonists to inhibit 5-HTiA-adenylyl cyclase activity is functionally related to their affinity for the low-affinity state of [3H]8-OH-DPAT binding (G-protein uncoupled) rather than for the high-affinity (Gprotein coupled) state [2]. It is noteworthy that the density of [3H]WAY-100635 and [3H]8-OH-DPAT binding to somatodendritic 5HTIA autoreceptors was higher in the rostral portion of the DRN compared with the caudal end, with the caudal DRN possessing 5-HTIA receptors in a predominantly receptor-G-protein uncoupled state. Immunocytochemical studies have shown a topographical ordering of the cells in the DRN [34]. The ascending fibers of the rostral DRN project more heavily to the amygdala, cortex, and septum, and those of the caudal DRN project to the lateral septum, hippocampus and
224
)d Khawaja /Brain Research 673 (1995) 217-225
basal forebrain [34,36]. It can be postulated that local differences in receptor density a n d / o r coupling efficiency may modulate the sensitivity of particular afferent neuronal subregions within the DRN to 5-HTIA ligands, thereby influencing the function of specific target regions within the central nervous system. Radja et al. [31] have reported that the pharmacology of the 5-HT1A recognition sites and the coupling of the receptor to G-proteins is similar between rat hippocampus and DRN. However, electrophysiological studies have shown a region-dependent drug efficacy for 5-HT1A agonists. The signal transduction mechanism in the rat DRN is especially sensitive to 5-HT1A agonists (ECs0 and Ema x) compared with the hippocampus [33]. Certainly, a larger receptor reserve exists for 5-HT1A agonists in the DRN [22] versus hippocampus [37]. To our knowledge, no pharmacological studies detailing the effects of 5-HTIA ligands in the different subregions of the DRN on CNS activity have been reported. It seems that the conformationally active state of the 5-HTIA receptor and the efficacy of an agonist a n d / o r partial agonist would depend on such factors as receptor number a n d / o r efficiency of receptor-effector coupling, which may vary according to the particular subregional zone(s) targetted. In summary, we have demonstrated that [3H]WAY100635 is a potent radioligand that labels the 5-HTIA receptor with nanomolar affinity. [3H]WAY-100635 is suitably adapted for quantitative autoradiographic studies, producing clear, sharp and high quality autoradiograms, that revealed a heterogeneity of binding sites matching recognised central pathways of serotonergic innervation. The availability of [3H]WAY-100635 will be useful as a sensitive tool in in vitro and in vivo autoradiographic studies of the 5-HT1A receptor to further our understanding of receptor-effector coupling mechanisms and the functional regulation of 5HT1A receptors in the central nervous system.
Abbreviations
Central nervous system Dorsal raphe nucleus 5-hydroxytryptamine 8-hydroxy-2-(di-n-propylamino)tetralin N-(2-(4-(2-methoxyphenyl)- 1WAY-100635 piperazinyl)ethyl)-N-(2-pyridinyl)cyclohexane carboxamide trihydrochloride [3H]WAY-100635 [O-methyl3H]-N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2pyridinyl)cyclohexane carboxamide trihydrochloride
CNS DRN 5-HT 8-OH-DPAT
BMY 7378
NAN 190
SDZ-216525
S-UH301
8-[2-[4-(methoxyphenyl)- 1piperazinyl]-ethyl]-8-azaspirol-(4,5)decane-7,9-dione 1-(2-methoxyphenyl)-4-[4-(2-phthalimido)butyl]piperazine hydrobromide methyl-4-[4-[4-[1,1,3-trioxo-2H-1,2benziosothiazol-2-yl]butyl]-1-piperazinyl]- 1H-indole-2-carboxylate 5-fluoro-8-hydroxy-2-(dipropylamino)tetralin
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