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Dopamine D3 receptor mRNA and binding sites in human brain
187
Molecular Brain Research, 18 (1993) 187-192 © 1993 Elsevier Science Publishers B.V. All rights reserved 0169-328x/93/$06.00
B R E S M 80163
Dopamine D 3 receptor mRNA and binding sites in human brain B. Landwehrmeyer a G. Mengod b,c and J.M. Palacios b,c a Department of Pathology, Dit,ision ofNeuropathology, UniL,ersity of Basel, Basel (Switzerland), h Preclinical Research, Sandoz Pharma Ltd., Basel (Switzerland) and c Departamento de Neuroquimica CID, CSIC, Barcelona (Spain) (Accepted 22 December 1992)
Key words: In situ hybridization; Receptor autoradiography; Dopamine receptor; Ventral striatum; Islands of Calleja; Schizophrenia
Dopamine D 3 receptors (Sokoloff et al., 1990) have been shown to be related to dopamine D 2 receptors and have been suggested to play a role in mediating the antipsychotic effects of neuroleptics. So far studies on the expression of D 3 m R N A and of binding sites with pharmacological characteristics of D 3 receptors have been restricted to rat brain. Using in situ hybridization histochemistry, we demonstrate that D 3 m R N A s are enriched in h u m a n n. accumbens and in the islands of Calleja. In addition, D 3 m R N A was detected at very low levels in anterior caudate and p u t a m e n with a rostro-candally decreasing gradient and in hypothalamic mammillary nuclei. In receptor autoradiographic binding studies, the islands of Calleja were found to be labeled by [125I]iodosulpride and [3H]CV 205 502 but not by [3H]raclopride and [3H]YM 09151-2. Pharmacological analysis of binding of the D 2 / D 3 ligand [3H]CV 205 502 in n. accumbens and caudate-putamen is consistent with the presence of D 3 receptor sites in ventral striatum. Overall distribution and pharmacology of D 3 sites in h u m a n and rat brain appear to be similar. Presence and distribution of D 3 receptors in h u m a n brain are compatible with the notion that D 3 receptors might be involved in mediating the clinical effects of antipsychotics.
The recent cloning of several genes coding for dopamine receptor subtypes expanded the family of dopamine receptors by a number of new members 3'6'7' 16,17,19.23,24,27.29 O2, 0 3 and 04 dopamine receptors share pharmacological, structural and genetical characteristics and may be referred to as Dz-like receptors. The distribution of D 3 receptor m R N A in rat brain 19'2'15 suggested that D 3 receptors might be preferentially involved in the modulation of functions of ventral striatum. The ventral striatum is thought to receive afferents from amygdala, allocortex (hippocampus, parahippocampal areas) and prefrontal cortex and to project via the ventral pallidum and mediodorsal thalamus back to the prefrontal cortex 1°. Dopaminergic afferents modulating the ventral striatum arise from dopaminergic cells in ventral tegmental area 5 (A10) and have been implicated in the pathophysiology of schizophrenia and in the mechanism of action of atypical neuroleptics 28. In studying D3 receptors as potential targets for antipsychotics it seems important to establish whether their distribution in human brain is similar to or different from their distribution in rat
brain. Therefore we examined the distribution of D 3 m R N A in human brain with in situ hybridization histochemistry (ISHH) using oligonucleotide probes for human D 3 m R N A 8. In addition, by combining ISHH and receptor autoradiography we provide evidence for the presence of D 3 binding sites in human brain. Cryostat sections of brains of 18 donors (7 male, 11 female, mean age 74.4 years (range 26-89), mean interval death-autopsy 10.7 hs (range 2-28.5)) with no known neurological or psychiatric diseases and essentially normal neuropathological examination (Prof. Probst, Institute of Pathology Basel) and a brain of a rhesus monkey were studied. For ISHH four oligonucleotide probes complementary to base sequences encoding the amino or carboxy terminus or regions of the putative third cytoplasmatic loop of the human D 3 receptor cDNA 8 with little sequence homology to related known receptors were used ( h D 3 / l : bases 4-51; h D 3 / 2 : bases 1161-1208; hD3/3a: bases 724-771; hD3/3b: 924-971). Oligonucleotide probes for human D 2 receptor were complementary to bases 19-69 ( h D 2 / 1 ) and bases 1019-1064
Correspondence: J.M. Palacios, D e p a r t a m e n t o de Neuroquimica CID, CSIC, Jordi Girona 18-26, Barcelona 08034, Spain. Fax: (34) 3-213-5972.
188
•
'
'
m
Fig. l. ISHH of dopamine D 3 (C) and D 2 (B) m R N A in human brain, d3 mRNA is enriched in nucleus accumbens; low levels of signal are also present in C and P (C). D 2 mRNA appears to be present throughout anterior striatum. There is no ventrally increasing gradient in hybridization signal intensity (B). For anatomical reference see acetylcholinesterase-stained section (A). Bar = 3 ram.
(hD2/3) of the sequence of Grandy et alfl. Probes were 3'-end-labeled using [I~2p]dATP (>3000 C i / mmol; NEN) and terminal deoxynucleotidyltransferase (Boehringer Mannheim, FRG) to specific activities of 0.9-3 × 104 Ci/mmol and hybridized as described 13'14. To increase signal intensity a mixture of two oligonucleotide probes directed to different parts of the transcript were used. Labeled sections were
apposed to /3-max film (Amersham) for 4-5 weeks or dipped in nuclear emulsion. For autoradiographic studies [~251]iodosulpride (spec. act. 2000 Ci/mmol), [3H]raclopride (spec. act. 85 Ci/mmol) and [3H]YM 09151-2 (spec. act. 87 Ci/mmol) were purchased from NEN, DuPont Ltd. [3H]CV 205 502 (spec. act. 95 Ci/mmol) was synthesized and labeled by Dr. R. Voges, Biopharmaceutical
Fig. 2. D 3 mRNA is expressed in the Islands of Calleja, The major island (loCm) and smaller medial islands (loC) are relatively intensely labeled (B). D 3 mRNA is also present in parts of ventral striatum lateral to the islands. A: Nissl stain (Toluidine blue). Bar = 1 ram.
189 Department, Sandoz Pharma Ltd., Basel, Switzerland. Incubation protocols used for different radioligands were essentially the same as described previously 1'4'13'18'25'26. For displacement studies domperidone, haloperidol and quinpirole (kindly supplied by the original manufacturers) were used at 7 concentrations ranging from 0.1 nM to 10 /.~M. Non-specific binding was determined by incubation in the presence of 1 ~M (+)-butaclamol (or spiperone for [125I]iodosulpride). Autoradiograms were generated by apposing the labeled tissue together with autoradiographic [3H]microscales (Amersham) to Hyperfilm-[3H] (Amersham) for 4 days ([125I]iodosulpride) to 10 weeks ([3H]raclopride) at 4°C. Films were analyzed using a computerized image analysis system (MCID, Imaging Research Inc., St. Catherines, Ont., Canada). Results of displacement studies were further analyzed using a software package for non-linear regression analysis (grafit, Euthacus Software, Staines, UK). Compared to signals obtained with probes for D 2 mRNA, overall hybridization signal for human D 3 mRNA was low (Fig. 1). In dipped sections it appeared that regional signal intensity was low as a consequence of low levels of D 3 mRNA present in each individual D 3 mRNA-expressing cells rather than expression in a small number of cells per region. D 3 mRNA was enriched in fundus striati of Brockhaus (ventral striatum/n, accumbens; Fig. 1). Lower levels of hybridization signal were present in anterior caudate, very low levels also in anterior putamen (n = 7). A rostrocaudal decreasing gradient was visible, most clearly in the single monkey brain studied (not shown). In addition, D 3 mRNAs were found to be expressed in islands of Calleja (IoC; Fig. 2). D 2 mRNA, in contrast, was not detected in IoC (n = 4). In 4 hypothalami studied low
but significant signals were detected in mammillary nuclei (not shown). We were unable, however, to detect any signal in substantia nigra (SN) of 6 subjects including one case studied in serial sections of midbrain (posterior thalamus to rostral pons). Dopamine D 2 receptor probes, in contrast, gave relatively intense signals in the same cases. We were also unable to detect any reproducible signal in bed nucleus of stria terminalis of 7 subjects. Several cortical areas, hippocampus, thalamus, brainstem, pituitary and cerebellum mapped in 4 subjects appeared not to contain D 3 mRNA or at levels below detection thresholds of our ISHH protocol. The specificity of the in situ hybridization signal was studied by applying several criteria. Three oligonucleotide probes (hD3/1, hD3/2, hD3/3b) directed against different parts of the same receptor gene transcript resulted in identical patterns of hybridization when used as independent hybridization probes in close-by sections. Hybridization of labeled probe in the presence of the corresponding unlabeled oligonucleotide in excess (20- to 40-fold) completely abolished the hybridization signal in tissue and reduced signal to background levels. The observed melting temperatures of the hybrids determined empirically were in agreement with their theoretical melting temperature (Tin). Attempts to detect signals with oligonucleotide probes used for ISHH in Northern blots with Poly(A) ÷ RNA purified from several brain regions were unsuccessful. PCR, however, suggested the presence of transcripts of D 3 receptors in striatal RNA (data not shown). Binding with different ' D 2 selective' radioligands showed a differential labeling of sites enriched in D 3 receptor transcripts: [125i]iodosulpride and [3H]CV 205 502, but neither [3H]raclopride nor [3H]YM 09151-2
Fig. 3. Receptor autoradiograms of sections of striatum at the level the major island of Calleja (arrows). Incubations with [125I]lodosulpride (A) and [3H]CV 205 502 (B) but not with [3H]YM 09151-2 (C) or [3H]raclopride (D) resulted in labeling of the island of Calleja magna. Bar = 3 mm.
190 I
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concentration (-log moVI) Fig. 4. Displacement of [-~H]CV 205 502 by domperidone in human anterior striatum at the level of n. accumbens (n = 4) determined by quantitative autoradiography. Acc, nucleus accumbens; C, caudate; P, putamen.
labeled IoCm (Fig. 3). The intense labeling of IoCm by [125i]iodosulpride and [3H]CV 205 502 suggested that both [125I]iodosulpride (an antagonist at D 2 receptors) and [3H]CV 205 502 (an agonist at D 2 receptors) label D 3 sites in addition to D 2 sites in human as in rat brain ~. To characterize these sites further, autoradiographic ligand binding techniques were used in competition studies using domperidone, haloperidol and quinpirole as displacers. Because of an insufficient number of human brain sections containing loCm, the pharmacological properties of labeling by [3H]CV 205 502 were studied in sections of anterior striatum at the
•A
B
level of n. accumbens. At this level, both D 2 and D 3 m R N A were present: caudate and putamen were enriched in D 2 m R N A , ventral aspects of striatum and n. accumbens were enriched in D 3 m R N A (Fig. 4B). Displacement of the dopamine agonist [3H]CV 205 502 by domperidone (Fig. 5), haloperidol and quinpirole was biphasic in both n. accumbens and striatum; monophasic fits resulted in very high chi-square values and were clearly inferior to biphasic fits (for monophasic fit, P < 0.001). In caudate and putamen the majority of binding sites labeled with [3H]CV 205 502 was displaced by nanomolar concentrations of domperidone and haloperidol and micromolar concentrations of quinpirole (D2, Table I). In n. accumbens, however, a high proportion of binding sites was found to be displaced only by concentrations of domperidone and haloperidoi approximately two orders of magnitude higher than in striatum (D3; Table I). In contrast, concentrations of quinpirole required to displace [3H]CV 205 502 from a subpopulation of binding sites in n. accumbens were two orders of magnitude lower in n. accumbens than in caudate and putamen (D3; Table I). Thus, putative D 3 binding sites can be visualized by mixed D 2 / D 3 radioligands like [3H]CV 205 502 or [~25I]iodosulpride in the presence of appropriate concentrations of compounds presenting higher affinities t o D 2 receptors (Fig. 5B). The distribution of D 3 m R N A in human brain appeared to be comparable to the one reported in rat
C
/
Fig. 5. Receptor autoradiogram of [3H]CV 205 502 binding in the presence of 10 ~ M domperidone (B) demonstrates D 3 binding sites in ventral striatum/n, accumbens. Total binding and binding in the presence of 10-6 M domperidone is shown in A and C, respectively.Bar = 3 mm.
191 TABLE I
Displacement of [3H]CV 205 502 in human and rat brain ICs0 derived from autoradiograms (nmol/l).
D2
D3
Ratio [CD2/ ICD~
Domperidone h r
2.1 2.5
604 470
0.0035 0.0053
Haloperidol h r
6.4 6.5
1260 389
0.0051 0.0167
Quinpirole h r
4 200 343
87 88
48 3.9
brain 19"2'11A5. In both human and rat brain, D 3 m R N A was enriched in n. accumbens and IoC. However, there were some differences. In human brain (as in the single monkey brain studied) we found a more extended presence of D 3 m R N A in anterior striatum than in rat brain. On the other hand, in a number of regions where the presence of D 3 m R N A has been reported in rat brain 19'2'H we were unable to detect hybridization signals. For instance, no signal was detected in the bed nucleus of stria terminalis and in SN. The levels of D 3 m R N A in these regions (if present at all) are therefore probably very low. The failure to detect signals in Northern blots (both total and mRNA-enriched) is consistent with this assumption. The absence of detectable levels of D 3 m R N A in human SN raises the question of the physiological relevance of putative D 3 autoreceptors on dopaminergic cells of SN demonstrated in rat brain 19'2. D 2 receptor transcripts are relatively abundant in dopaminergic cells of SN. It appears likely, therefore, that the vast majority of presynaptic nigrostriatal dopamine receptors are D 2 receptors. Clearly further studies are needed to establish the presence or absence of presynaptic D 3 receptors and their physiological relevance. The binding properties of human D 3 receptors expressed in transfected CHO cells has been published recently 21. Dissociation constants of rodent and human D 3 receptors were found to be comparable for most dopaminergic drugs 23. Both human and rat D 3 receptors can be labeled with high affinity by iodosulpride. D 2 receptor agonists (e.g. quinpirole) tend to have a high affinity, D 2 receptor antagonists a comparatively lower affinity at D 3 receptors. However, the pharmacology of rat and human D 3 receptors expressed in a mammalian cell line 21 were not entirely identical: domperidone and haloperidol appeared to discriminate better between D 2 and D 3 receptors in rodents than in humans (KiD2/KiD 3 in rat 0.032 (domperidone) and 0.041 (haloperidol) vs KiD2/KiD 3 in human 0.18 and 0.21,
respectively2°'21'22). In contrast to these findings on transfected C H O cell membranes we found similar ratios in tissue sections of human and rat brain (see Table I). This difference might be related to differences in coupling to G-proteins in CHO cells and tissue sections. Interestingly, with human but not with rat D 3 receptors a modest but replicatable effect of guanylnucleotides was observed in transfected cells 2°'21. This modulatory effect might be related to marked differences between rat and human D 3 receptors at the level of the third cytoplasmatic loop 8'19. In good aggreement with observations on human D 3 receptors expressed in CHO cells 21 we found a higher affinity of quinpirole to D 3 than to D e receptors (ratio ICsocDe)/ IC50(D3) 48 (Table I) vs Ki(Dz)/Ki(D3)36 21). Human brain regions enriched for transcripts for D 3 receptors differ in their binding properties from those regions with a predominance of 0 2 transcripts. KI and K e values for biphasic displacement of [3H]CV 205 502 binding in human striatum and ICs0 values in IoC and CP of rat brain H were comparable (see Table I). Thus it appears that D 3 binding sites are present in human ventral striatum. However, other dopamine receptor subtypes with a De-like pharmacology are known to exist. Therefore, it could be suggested that other O zlike receptors ( D 4) might account for the different binding properties in ventral striatum. Several findings argue against this possibility. In preliminary ISHH studies on the expression of 0 4 receptor m R N A in human brain very low signals throughout basal ganglia with no apparent ventrally increasing gradient have been observed (BL, GM and JMP, unpublished observations). YM-09151-2, a ligand with high affinity to D 4 receptors z7 did not show higher densities in anteroventral than anterodorsal striatum and was not differentially displaced by domperidone in ventral and dorsal striatum. Together with the close aggreement to data in rat brain, where a 'pure' population of D 3 receptors have been studied in IoC, it is likely that a proportion of De-like binding sites in human ventral striatum indeed represents D 3 receptors. In conclusion, the main result of the studies presented here is the demonstration that in human brain (as in rat brain), D 3 mRNA, as well as D 3 binding sites are enriched in ventral striatum. The presence of D 3 binding sites in human brain, their pharmacology and their peculiar distribution are compatible with the suggestion that D 3 receptors might be involved in mediating the antipsychotic effects of neuroleptics 19. Neuroleptics with preferential D 3 antagonistic activity might have less effects on prolactin secretion and extrapyramidal motor systems than traditional neuroleptics. However, to test this hypothesis the development and
192
clinical evaluation of compounds with selectivity for D 3 receptor is needed. B.L. was supported by a grant from DFG, Bonn, FRG. The excellent technical help of M. Rigo in performing the experiments and the support of K.-H. Wiederhold in photographic artwork is gratefully acknowledged. l Bouthenet, M.-L., Martres, M.-P., Sales, N. and Schwartz, J.-C., A detailed mapping of dopamine D2 receptors in rat central nervous system by autoradiography with [12sI]Iodosulpride, Neuroscience, 20 (1987) 117-135. 2 Bouthenet, M.-L., Souil, E., Martres, M.-P., Sokoloff, P., Giros B. and Schwartz, J.-C., Localization of dopamine D 3 receptor mRNA in the rat brain using in situ hybridization histochemistry: comparison with dopamine D~ receptor mRNA, Brain Res., 564 (1991) 203-219. 3 Bunzow, J.R., Van Tol, H.H.M., Grandy, D.K., Albert, P., Salon, J., Christie, M., Machida, C.A., Neve, K.A. and Civelli, O., Cloning and expression of a rat D2 dopamine receptor cDNA, Nature, 336 (1988) 783-787. 4 Charuchinda, C., Supavilai, P., Karobath, M. and Palacios, J.M., Dopamine D2 receptors in the rat brain: autoradiographic visualization using a high-affinity selective agonist ligand, J. Neurosci., 7 11987) 1352-1360. 5 Dahlstr6m, A. and Fuxe, K., Evidence for the existence of monoamine-containing in the central nervous system. I. Demonstration of monoamines in the cell bodies of brain stem neurons, Acta Physiol. Scand., 62 (Suppl. 232) (1964) 1-55. 6 Dearry, A., Gingrich, J.A., Falardeau, P., Fremeau, R.T. Jr. Bates, M.D., and Carom M.G., Molecular cloning and expression of the gene for a human D1 dopamine receptor, Nature, 347 (1990) 72-76. 7 Giros, B., Sokoloff, P., Martres. M.-P., Riou, J.-F., Emorine, L.J. and Schwartz, J.-C., Alternative splicing directs the expression of two D2 dopamine receptor isoforms, Nature, 342 (1989) 923-926. 8 Giros, B., Martres, M.-P., Sokoloff, P. and Schwartz, J.-C., cDNA cloning of the human dopaminergic D3 receptor and chromosome identification, CR Acad. Sci. (III)311 (1990)501-508. 9 Grandy, D.K., Marchionni, M.A., Makam, H., Stofko, R.E., AIfano M., Frothingham, L., Fischer, J.B., Burke-Howie, KJ., Bunzow, J.R., Server, A.C. and Civelli, O., Cloning of the cDNA and gene for a human D2 dopamine receptor, Proc. Natl. Acad. Sci. USA, 86 11989) 9762-9766. l(J Heimer, L., The olfactory cortex and ventral striatum. In K.E. Livingston and O. Hornykiewicz (Eds.), Limbic Mechanism. Plenum, New York, 1978, pp. 95-187. 11 Landwehrmeyer, B., Mengod, G. and Palacios, J.M., Differential visualization of dopamine D2 and D3 receptor sites in rat brain. A comparative study using in situ hybridization histochemistry and ligand binding autoradiography, Eur. J. Neurosci., in press. 12 Martres, M.-P., Bouthenet, M.-L., Sales, N., Sokoloff, P. and Schwartz, J.-C., Widespread distribution of brain dopamine receptors evidenced with [125I]lodosulpride, a highly selective ligand, Science, 228 (1985) 752-755. 13 Mengod, G., Martinez-Mir, M.I., Vilar6 T. and Palacios, J.M., Localization of the mRNA for the dopamine D2 receptor in the rat brain by in situ hybridization histochemistry, Proc. Natl. Acad. Sci. USA, 86 (1989) 8560-8564. 14 Mengod, G., Vilar6 M.T., Niznik, H.B., Sunahara, R.K., Seeman,
P., O'Dowd, B.F. and Palacios, J.M., Visualization of a dopamine D 1 receptor mRNA in human and rat brain, Mol. Brain Res., 10 (1991) 185-191. 15 Mengod, G., Vilard M.T., Landwehrmeyer, G.B., Martinez-Mir, M.I., Niznik, H.B., Sunahara, R.K., Seeman, P., O'Dowd B.F., Probst, A. and Palacios, J.M., Visualization of dopamine D1, D2 and D3 receptor mRNAs in human and rat brain, Neurochem. Int., 20 [Suppl] 11992) 33S-43S. 16 Monsma, F.J. Jr., Mahan, L.C., McVittie, L.D., Gerfen, C.R. and Sibley, D.R., Multiple D2 dopamine receptors produced by alternative RNA splicing, Nature, 342 (1989) 926-929. 17 Monsma, F.J. Jr., Mahan, L.C., McVittie, L.D., Gerfen, C.R. and Sibley, D.R., Molecular cloning and expression of a D1 dopamine receptor linked to adenylyl cyclase activation, Proc. Natl. Acad. Sci. USA, 87 (1990) 6723-6727. 18 Niznik, H.B., Grigoriadis, D.E., Pri-Bar, I., Buchanan, O. and Seeman, P., Dopamine D2 receptors selectively labeled by a benzamide neuroloptic: [3H]YM-09151-2, Naunyn-Schmiedeberg~s" Arch. PharmacoL, 329 (1985) 333-343. 19 Sokoloff, P., Giros, B., Martres, M.P., Bouthenet, M.L. and Schwartz, J.C., Molecular cloning and characterization of a novel dopamine receptor (D3) as a target for neuroleptics, Nature, 347 11990) 146-151. 20 Sokoloff, P., Martres, M.-P., Giros, B., Bouthenet, M.-L. and Schwartz, J.-C., The third dopamine receptor (D3) as a novel target for antipsychotics, Bioehem. Pharmacol., 43 (1992) 659-666. 21 Sokoloff, P., Andrieux, M., Besan~5on, R., Pilon, C., Martres, M.-P., Giros, B. and Schwartz, J.-C., Pharmacology of human dopamine D3 receptor expressed in a mammalian cell line: comparison with D2 receptor, Eur. J. Pharmacol., 225 (1992) 331-337. 22 Sokoloff, P., Giros, B., Martres, M.-P., Andrieux, M., Besan~on, R., Pilon, C., Bouthenet, M.L., Souil, E. and Schwartz, J.-C., Localization and function of the D3 dopamine receptor, Arzneim.-Forsch./Drug Res., 42 (1992) 224-230. 23 Sunahara, R.K., Niznik, H.B., Weiner, D.M., Stormann, T.M., Brann, N.R., Kennedy, J.L., Gelernter, J.E., Rozmahel, R., Yang, Y., Israel, Y., Seeman, P. and O'Dowd, B.F., Human dopamine DI receptor encoded by an intronless gene on chromosome 5, Nature, 347 (1990) 80-83, 24 Sunahara, R.K., Guan, H.-C., O'Dowd, B.F., Seeman, P., Laurier L.G., Gordon, N.G., George, S.R., Torchia J, Van Tol, H.H.M and Niznik H.B., Cloning of the gene for a human dopamine D5 receptor with higher affinity for dopamine than DI, Nature, 350 (1991) 614-619. 25 Terai, M., Hidaka, K., Nakamura, Y., Comparison of [~H]YM119151-2 with [3H]spiperone and [3H]raclopride for dopamine D2 receptor binding to rat striatum, Eur. J. PharrnacoL, 173 (1989) 177-182. 26 Unis, A.S., Vincent, J.G. and Dillon, B., Brain receptor autoradiography with [3H]YM-09151-2: a ligand for labeling dopamine D2 receptors, Life Sci., 47 11990) 151-155. 27 Van Tol, H.H.M., Bunzow, J.R., Guan, H.-C., Sunahara, R.K.. Seeman, P., Niznik, H.B. and Civelli, O., Cloning of the gene for a human dopamine D4 receptor with high affinity for the antipsychotic clozapine, Nature, 350 (1991) 610-614. 28 White, FJ. and Wang, R.Y., Differential effects of classical and atypical antipsychotic drugs on A9 and A 10 dopamine neurons, Science,, 221 (1984) 11)54-11)57. 29 Zhou, Q.-Y., Grandy, D.K., Tbambi, L., Kushner, J.A., Van Tol H.H.M., Cone, R., Pribnow, D., Salon, J., Bunzow, J.R. and Civelli, O., Cloning and expression of human and rat DI dopamine receptors, Nature, 347 (1990) 76-80.
Molecular Brain Research, 18 (1993) 187-192 © 1993 Elsevier Science Publishers B.V. All rights reserved 0169-328x/93/$06.00
B R E S M 80163
Dopamine D 3 receptor mRNA and binding sites in human brain B. Landwehrmeyer a G. Mengod b,c and J.M. Palacios b,c a Department of Pathology, Dit,ision ofNeuropathology, UniL,ersity of Basel, Basel (Switzerland), h Preclinical Research, Sandoz Pharma Ltd., Basel (Switzerland) and c Departamento de Neuroquimica CID, CSIC, Barcelona (Spain) (Accepted 22 December 1992)
Key words: In situ hybridization; Receptor autoradiography; Dopamine receptor; Ventral striatum; Islands of Calleja; Schizophrenia
Dopamine D 3 receptors (Sokoloff et al., 1990) have been shown to be related to dopamine D 2 receptors and have been suggested to play a role in mediating the antipsychotic effects of neuroleptics. So far studies on the expression of D 3 m R N A and of binding sites with pharmacological characteristics of D 3 receptors have been restricted to rat brain. Using in situ hybridization histochemistry, we demonstrate that D 3 m R N A s are enriched in h u m a n n. accumbens and in the islands of Calleja. In addition, D 3 m R N A was detected at very low levels in anterior caudate and p u t a m e n with a rostro-candally decreasing gradient and in hypothalamic mammillary nuclei. In receptor autoradiographic binding studies, the islands of Calleja were found to be labeled by [125I]iodosulpride and [3H]CV 205 502 but not by [3H]raclopride and [3H]YM 09151-2. Pharmacological analysis of binding of the D 2 / D 3 ligand [3H]CV 205 502 in n. accumbens and caudate-putamen is consistent with the presence of D 3 receptor sites in ventral striatum. Overall distribution and pharmacology of D 3 sites in h u m a n and rat brain appear to be similar. Presence and distribution of D 3 receptors in h u m a n brain are compatible with the notion that D 3 receptors might be involved in mediating the clinical effects of antipsychotics.
The recent cloning of several genes coding for dopamine receptor subtypes expanded the family of dopamine receptors by a number of new members 3'6'7' 16,17,19.23,24,27.29 O2, 0 3 and 04 dopamine receptors share pharmacological, structural and genetical characteristics and may be referred to as Dz-like receptors. The distribution of D 3 receptor m R N A in rat brain 19'2'15 suggested that D 3 receptors might be preferentially involved in the modulation of functions of ventral striatum. The ventral striatum is thought to receive afferents from amygdala, allocortex (hippocampus, parahippocampal areas) and prefrontal cortex and to project via the ventral pallidum and mediodorsal thalamus back to the prefrontal cortex 1°. Dopaminergic afferents modulating the ventral striatum arise from dopaminergic cells in ventral tegmental area 5 (A10) and have been implicated in the pathophysiology of schizophrenia and in the mechanism of action of atypical neuroleptics 28. In studying D3 receptors as potential targets for antipsychotics it seems important to establish whether their distribution in human brain is similar to or different from their distribution in rat
brain. Therefore we examined the distribution of D 3 m R N A in human brain with in situ hybridization histochemistry (ISHH) using oligonucleotide probes for human D 3 m R N A 8. In addition, by combining ISHH and receptor autoradiography we provide evidence for the presence of D 3 binding sites in human brain. Cryostat sections of brains of 18 donors (7 male, 11 female, mean age 74.4 years (range 26-89), mean interval death-autopsy 10.7 hs (range 2-28.5)) with no known neurological or psychiatric diseases and essentially normal neuropathological examination (Prof. Probst, Institute of Pathology Basel) and a brain of a rhesus monkey were studied. For ISHH four oligonucleotide probes complementary to base sequences encoding the amino or carboxy terminus or regions of the putative third cytoplasmatic loop of the human D 3 receptor cDNA 8 with little sequence homology to related known receptors were used ( h D 3 / l : bases 4-51; h D 3 / 2 : bases 1161-1208; hD3/3a: bases 724-771; hD3/3b: 924-971). Oligonucleotide probes for human D 2 receptor were complementary to bases 19-69 ( h D 2 / 1 ) and bases 1019-1064
Correspondence: J.M. Palacios, D e p a r t a m e n t o de Neuroquimica CID, CSIC, Jordi Girona 18-26, Barcelona 08034, Spain. Fax: (34) 3-213-5972.
188
•
'
'
m
Fig. l. ISHH of dopamine D 3 (C) and D 2 (B) m R N A in human brain, d3 mRNA is enriched in nucleus accumbens; low levels of signal are also present in C and P (C). D 2 mRNA appears to be present throughout anterior striatum. There is no ventrally increasing gradient in hybridization signal intensity (B). For anatomical reference see acetylcholinesterase-stained section (A). Bar = 3 ram.
(hD2/3) of the sequence of Grandy et alfl. Probes were 3'-end-labeled using [I~2p]dATP (>3000 C i / mmol; NEN) and terminal deoxynucleotidyltransferase (Boehringer Mannheim, FRG) to specific activities of 0.9-3 × 104 Ci/mmol and hybridized as described 13'14. To increase signal intensity a mixture of two oligonucleotide probes directed to different parts of the transcript were used. Labeled sections were
apposed to /3-max film (Amersham) for 4-5 weeks or dipped in nuclear emulsion. For autoradiographic studies [~251]iodosulpride (spec. act. 2000 Ci/mmol), [3H]raclopride (spec. act. 85 Ci/mmol) and [3H]YM 09151-2 (spec. act. 87 Ci/mmol) were purchased from NEN, DuPont Ltd. [3H]CV 205 502 (spec. act. 95 Ci/mmol) was synthesized and labeled by Dr. R. Voges, Biopharmaceutical
Fig. 2. D 3 mRNA is expressed in the Islands of Calleja, The major island (loCm) and smaller medial islands (loC) are relatively intensely labeled (B). D 3 mRNA is also present in parts of ventral striatum lateral to the islands. A: Nissl stain (Toluidine blue). Bar = 1 ram.
189 Department, Sandoz Pharma Ltd., Basel, Switzerland. Incubation protocols used for different radioligands were essentially the same as described previously 1'4'13'18'25'26. For displacement studies domperidone, haloperidol and quinpirole (kindly supplied by the original manufacturers) were used at 7 concentrations ranging from 0.1 nM to 10 /.~M. Non-specific binding was determined by incubation in the presence of 1 ~M (+)-butaclamol (or spiperone for [125I]iodosulpride). Autoradiograms were generated by apposing the labeled tissue together with autoradiographic [3H]microscales (Amersham) to Hyperfilm-[3H] (Amersham) for 4 days ([125I]iodosulpride) to 10 weeks ([3H]raclopride) at 4°C. Films were analyzed using a computerized image analysis system (MCID, Imaging Research Inc., St. Catherines, Ont., Canada). Results of displacement studies were further analyzed using a software package for non-linear regression analysis (grafit, Euthacus Software, Staines, UK). Compared to signals obtained with probes for D 2 mRNA, overall hybridization signal for human D 3 mRNA was low (Fig. 1). In dipped sections it appeared that regional signal intensity was low as a consequence of low levels of D 3 mRNA present in each individual D 3 mRNA-expressing cells rather than expression in a small number of cells per region. D 3 mRNA was enriched in fundus striati of Brockhaus (ventral striatum/n, accumbens; Fig. 1). Lower levels of hybridization signal were present in anterior caudate, very low levels also in anterior putamen (n = 7). A rostrocaudal decreasing gradient was visible, most clearly in the single monkey brain studied (not shown). In addition, D 3 mRNAs were found to be expressed in islands of Calleja (IoC; Fig. 2). D 2 mRNA, in contrast, was not detected in IoC (n = 4). In 4 hypothalami studied low
but significant signals were detected in mammillary nuclei (not shown). We were unable, however, to detect any signal in substantia nigra (SN) of 6 subjects including one case studied in serial sections of midbrain (posterior thalamus to rostral pons). Dopamine D 2 receptor probes, in contrast, gave relatively intense signals in the same cases. We were also unable to detect any reproducible signal in bed nucleus of stria terminalis of 7 subjects. Several cortical areas, hippocampus, thalamus, brainstem, pituitary and cerebellum mapped in 4 subjects appeared not to contain D 3 mRNA or at levels below detection thresholds of our ISHH protocol. The specificity of the in situ hybridization signal was studied by applying several criteria. Three oligonucleotide probes (hD3/1, hD3/2, hD3/3b) directed against different parts of the same receptor gene transcript resulted in identical patterns of hybridization when used as independent hybridization probes in close-by sections. Hybridization of labeled probe in the presence of the corresponding unlabeled oligonucleotide in excess (20- to 40-fold) completely abolished the hybridization signal in tissue and reduced signal to background levels. The observed melting temperatures of the hybrids determined empirically were in agreement with their theoretical melting temperature (Tin). Attempts to detect signals with oligonucleotide probes used for ISHH in Northern blots with Poly(A) ÷ RNA purified from several brain regions were unsuccessful. PCR, however, suggested the presence of transcripts of D 3 receptors in striatal RNA (data not shown). Binding with different ' D 2 selective' radioligands showed a differential labeling of sites enriched in D 3 receptor transcripts: [125i]iodosulpride and [3H]CV 205 502, but neither [3H]raclopride nor [3H]YM 09151-2
Fig. 3. Receptor autoradiograms of sections of striatum at the level the major island of Calleja (arrows). Incubations with [125I]lodosulpride (A) and [3H]CV 205 502 (B) but not with [3H]YM 09151-2 (C) or [3H]raclopride (D) resulted in labeling of the island of Calleja magna. Bar = 3 mm.
190 I
I IIIIIII]
I II11111[
I i liilil I
i I liilil i
I t IHIiI~
I I Illllf I
I I IIIIII i
100
80
-
l
,%.
•
ACC
•
C
•
P
60
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40
20
0
IIIII1[
1 I tlllad
10
I I IIIIIll
9
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5
concentration (-log moVI) Fig. 4. Displacement of [-~H]CV 205 502 by domperidone in human anterior striatum at the level of n. accumbens (n = 4) determined by quantitative autoradiography. Acc, nucleus accumbens; C, caudate; P, putamen.
labeled IoCm (Fig. 3). The intense labeling of IoCm by [125i]iodosulpride and [3H]CV 205 502 suggested that both [125I]iodosulpride (an antagonist at D 2 receptors) and [3H]CV 205 502 (an agonist at D 2 receptors) label D 3 sites in addition to D 2 sites in human as in rat brain ~. To characterize these sites further, autoradiographic ligand binding techniques were used in competition studies using domperidone, haloperidol and quinpirole as displacers. Because of an insufficient number of human brain sections containing loCm, the pharmacological properties of labeling by [3H]CV 205 502 were studied in sections of anterior striatum at the
•A
B
level of n. accumbens. At this level, both D 2 and D 3 m R N A were present: caudate and putamen were enriched in D 2 m R N A , ventral aspects of striatum and n. accumbens were enriched in D 3 m R N A (Fig. 4B). Displacement of the dopamine agonist [3H]CV 205 502 by domperidone (Fig. 5), haloperidol and quinpirole was biphasic in both n. accumbens and striatum; monophasic fits resulted in very high chi-square values and were clearly inferior to biphasic fits (for monophasic fit, P < 0.001). In caudate and putamen the majority of binding sites labeled with [3H]CV 205 502 was displaced by nanomolar concentrations of domperidone and haloperidol and micromolar concentrations of quinpirole (D2, Table I). In n. accumbens, however, a high proportion of binding sites was found to be displaced only by concentrations of domperidone and haloperidoi approximately two orders of magnitude higher than in striatum (D3; Table I). In contrast, concentrations of quinpirole required to displace [3H]CV 205 502 from a subpopulation of binding sites in n. accumbens were two orders of magnitude lower in n. accumbens than in caudate and putamen (D3; Table I). Thus, putative D 3 binding sites can be visualized by mixed D 2 / D 3 radioligands like [3H]CV 205 502 or [~25I]iodosulpride in the presence of appropriate concentrations of compounds presenting higher affinities t o D 2 receptors (Fig. 5B). The distribution of D 3 m R N A in human brain appeared to be comparable to the one reported in rat
C
/
Fig. 5. Receptor autoradiogram of [3H]CV 205 502 binding in the presence of 10 ~ M domperidone (B) demonstrates D 3 binding sites in ventral striatum/n, accumbens. Total binding and binding in the presence of 10-6 M domperidone is shown in A and C, respectively.Bar = 3 mm.
191 TABLE I
Displacement of [3H]CV 205 502 in human and rat brain ICs0 derived from autoradiograms (nmol/l).
D2
D3
Ratio [CD2/ ICD~
Domperidone h r
2.1 2.5
604 470
0.0035 0.0053
Haloperidol h r
6.4 6.5
1260 389
0.0051 0.0167
Quinpirole h r
4 200 343
87 88
48 3.9
brain 19"2'11A5. In both human and rat brain, D 3 m R N A was enriched in n. accumbens and IoC. However, there were some differences. In human brain (as in the single monkey brain studied) we found a more extended presence of D 3 m R N A in anterior striatum than in rat brain. On the other hand, in a number of regions where the presence of D 3 m R N A has been reported in rat brain 19'2'H we were unable to detect hybridization signals. For instance, no signal was detected in the bed nucleus of stria terminalis and in SN. The levels of D 3 m R N A in these regions (if present at all) are therefore probably very low. The failure to detect signals in Northern blots (both total and mRNA-enriched) is consistent with this assumption. The absence of detectable levels of D 3 m R N A in human SN raises the question of the physiological relevance of putative D 3 autoreceptors on dopaminergic cells of SN demonstrated in rat brain 19'2. D 2 receptor transcripts are relatively abundant in dopaminergic cells of SN. It appears likely, therefore, that the vast majority of presynaptic nigrostriatal dopamine receptors are D 2 receptors. Clearly further studies are needed to establish the presence or absence of presynaptic D 3 receptors and their physiological relevance. The binding properties of human D 3 receptors expressed in transfected CHO cells has been published recently 21. Dissociation constants of rodent and human D 3 receptors were found to be comparable for most dopaminergic drugs 23. Both human and rat D 3 receptors can be labeled with high affinity by iodosulpride. D 2 receptor agonists (e.g. quinpirole) tend to have a high affinity, D 2 receptor antagonists a comparatively lower affinity at D 3 receptors. However, the pharmacology of rat and human D 3 receptors expressed in a mammalian cell line 21 were not entirely identical: domperidone and haloperidol appeared to discriminate better between D 2 and D 3 receptors in rodents than in humans (KiD2/KiD 3 in rat 0.032 (domperidone) and 0.041 (haloperidol) vs KiD2/KiD 3 in human 0.18 and 0.21,
respectively2°'21'22). In contrast to these findings on transfected C H O cell membranes we found similar ratios in tissue sections of human and rat brain (see Table I). This difference might be related to differences in coupling to G-proteins in CHO cells and tissue sections. Interestingly, with human but not with rat D 3 receptors a modest but replicatable effect of guanylnucleotides was observed in transfected cells 2°'21. This modulatory effect might be related to marked differences between rat and human D 3 receptors at the level of the third cytoplasmatic loop 8'19. In good aggreement with observations on human D 3 receptors expressed in CHO cells 21 we found a higher affinity of quinpirole to D 3 than to D e receptors (ratio ICsocDe)/ IC50(D3) 48 (Table I) vs Ki(Dz)/Ki(D3)36 21). Human brain regions enriched for transcripts for D 3 receptors differ in their binding properties from those regions with a predominance of 0 2 transcripts. KI and K e values for biphasic displacement of [3H]CV 205 502 binding in human striatum and ICs0 values in IoC and CP of rat brain H were comparable (see Table I). Thus it appears that D 3 binding sites are present in human ventral striatum. However, other dopamine receptor subtypes with a De-like pharmacology are known to exist. Therefore, it could be suggested that other O zlike receptors ( D 4) might account for the different binding properties in ventral striatum. Several findings argue against this possibility. In preliminary ISHH studies on the expression of 0 4 receptor m R N A in human brain very low signals throughout basal ganglia with no apparent ventrally increasing gradient have been observed (BL, GM and JMP, unpublished observations). YM-09151-2, a ligand with high affinity to D 4 receptors z7 did not show higher densities in anteroventral than anterodorsal striatum and was not differentially displaced by domperidone in ventral and dorsal striatum. Together with the close aggreement to data in rat brain, where a 'pure' population of D 3 receptors have been studied in IoC, it is likely that a proportion of De-like binding sites in human ventral striatum indeed represents D 3 receptors. In conclusion, the main result of the studies presented here is the demonstration that in human brain (as in rat brain), D 3 mRNA, as well as D 3 binding sites are enriched in ventral striatum. The presence of D 3 binding sites in human brain, their pharmacology and their peculiar distribution are compatible with the suggestion that D 3 receptors might be involved in mediating the antipsychotic effects of neuroleptics 19. Neuroleptics with preferential D 3 antagonistic activity might have less effects on prolactin secretion and extrapyramidal motor systems than traditional neuroleptics. However, to test this hypothesis the development and
192
clinical evaluation of compounds with selectivity for D 3 receptor is needed. B.L. was supported by a grant from DFG, Bonn, FRG. The excellent technical help of M. Rigo in performing the experiments and the support of K.-H. Wiederhold in photographic artwork is gratefully acknowledged. l Bouthenet, M.-L., Martres, M.-P., Sales, N. and Schwartz, J.-C., A detailed mapping of dopamine D2 receptors in rat central nervous system by autoradiography with [12sI]Iodosulpride, Neuroscience, 20 (1987) 117-135. 2 Bouthenet, M.-L., Souil, E., Martres, M.-P., Sokoloff, P., Giros B. and Schwartz, J.-C., Localization of dopamine D 3 receptor mRNA in the rat brain using in situ hybridization histochemistry: comparison with dopamine D~ receptor mRNA, Brain Res., 564 (1991) 203-219. 3 Bunzow, J.R., Van Tol, H.H.M., Grandy, D.K., Albert, P., Salon, J., Christie, M., Machida, C.A., Neve, K.A. and Civelli, O., Cloning and expression of a rat D2 dopamine receptor cDNA, Nature, 336 (1988) 783-787. 4 Charuchinda, C., Supavilai, P., Karobath, M. and Palacios, J.M., Dopamine D2 receptors in the rat brain: autoradiographic visualization using a high-affinity selective agonist ligand, J. Neurosci., 7 11987) 1352-1360. 5 Dahlstr6m, A. and Fuxe, K., Evidence for the existence of monoamine-containing in the central nervous system. I. Demonstration of monoamines in the cell bodies of brain stem neurons, Acta Physiol. Scand., 62 (Suppl. 232) (1964) 1-55. 6 Dearry, A., Gingrich, J.A., Falardeau, P., Fremeau, R.T. Jr. Bates, M.D., and Carom M.G., Molecular cloning and expression of the gene for a human D1 dopamine receptor, Nature, 347 (1990) 72-76. 7 Giros, B., Sokoloff, P., Martres. M.-P., Riou, J.-F., Emorine, L.J. and Schwartz, J.-C., Alternative splicing directs the expression of two D2 dopamine receptor isoforms, Nature, 342 (1989) 923-926. 8 Giros, B., Martres, M.-P., Sokoloff, P. and Schwartz, J.-C., cDNA cloning of the human dopaminergic D3 receptor and chromosome identification, CR Acad. Sci. (III)311 (1990)501-508. 9 Grandy, D.K., Marchionni, M.A., Makam, H., Stofko, R.E., AIfano M., Frothingham, L., Fischer, J.B., Burke-Howie, KJ., Bunzow, J.R., Server, A.C. and Civelli, O., Cloning of the cDNA and gene for a human D2 dopamine receptor, Proc. Natl. Acad. Sci. USA, 86 11989) 9762-9766. l(J Heimer, L., The olfactory cortex and ventral striatum. In K.E. Livingston and O. Hornykiewicz (Eds.), Limbic Mechanism. Plenum, New York, 1978, pp. 95-187. 11 Landwehrmeyer, B., Mengod, G. and Palacios, J.M., Differential visualization of dopamine D2 and D3 receptor sites in rat brain. A comparative study using in situ hybridization histochemistry and ligand binding autoradiography, Eur. J. Neurosci., in press. 12 Martres, M.-P., Bouthenet, M.-L., Sales, N., Sokoloff, P. and Schwartz, J.-C., Widespread distribution of brain dopamine receptors evidenced with [125I]lodosulpride, a highly selective ligand, Science, 228 (1985) 752-755. 13 Mengod, G., Martinez-Mir, M.I., Vilar6 T. and Palacios, J.M., Localization of the mRNA for the dopamine D2 receptor in the rat brain by in situ hybridization histochemistry, Proc. Natl. Acad. Sci. USA, 86 (1989) 8560-8564. 14 Mengod, G., Vilar6 M.T., Niznik, H.B., Sunahara, R.K., Seeman,
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