II. Localization and Characterization of Dopamine D4 Binding Sites in Rat and Human Brain by Use of the Novel, D4 Receptor-Selective Ligand [3H]NGD 94–1
- Renee J. Primus,
- Andrew Thurkauf,
- Jing Xu,
- Eileen Yevich,
- Sean Mcinerney,
- Kenneth Shaw,
- John F. Tallman and
- Dorothy W. Gallager
Abstract
The dopamine D4 selective ligand, [3H]NGD 94–1, was used in these studies to characterize binding sites in rat and human brain tissue by membrane binding and autoradiography techniques. Autoradiographic analysis of rat brain showed that specific [3H]NGD 94–1 binding was greatest in entorhinal cortex, lateral septal nucleus, hippocampus and the medial preoptic area of the hypothalamus. This nonstriatal distribution of [3H]NGD 94–1 binding was distinct from the autoradiographic distribution of dopamine D2 and D3 receptor subtypes. In homogenate preparations from rat brain regions, [3H]NGD 94–1 binding sites were low in density (<30.0 fmol/mg protein). The low density of D4binding sites was corroborated by autoradiographic comparisons in which binding density for D4 receptors as measured by [3H]NGD 94–1 was only 1/7 of D2 and 1/5 of D3 receptor densities, despite corrections for differing radioligand binding characteristics. Pharmacological evaluation showed high affinity at rat [3H]NGD 94–1 binding sites for compounds with known D4 receptor affinity and little displacement by compounds with affinity for dopamine D1/D2/D3receptor subtypes. Specific, high-affinity [3H]NGD 94–1 binding was also present in several human brain regions, including hippocampus, hypothalamus, dorsal medial thalamus, entorhinal cortex, prefrontal cortex and lateral septal nucleus. High-affinity [3H]NGD 94–1 binding was not present in any human striatal region examined. The pharmacological profile of [3H]NGD 94–1 binding sites in human brain was consistent with that previously demonstrated for cloned human D4 receptors expressed in mammalian cells. These findings suggest that specific, high-affinity [3H]NGD 94–1 binding exists in rat and human brain and that these sites reflect populations of dopamine D4 receptors with a distribution unique among dopamine receptor subtypes.
Several lines of evidence implicate the dopamine D4receptor as a therapeutic target for the treatment of schizophrenia. Clozapine, a clinically efficacious atypical antipsychotic agent, has approximately a 10-fold greater affinity for the dopamine D4 receptor than for the dopamine D2 receptor (Van Tol et al., 1991). In addition, the therapeutic dose of clozapine correlates well with the dissociation constant of clozapine at the D4receptor (Seeman, 1995). Further evidence for a D4 involvement in schizophrenia are reports of selective increases in D4 receptor binding density in brain tissue from schizophrenics, which have been documented by several laboratories (Seeman et al., 1993; Sumiyoshiet al., 1994; Murray et al., 1995); however, this latter finding remains controversial (Lahti et al., 1996;Reynolds, 1996; Reynolds and Mason, 1995).
The dopamine D4 receptor is a G-protein-coupled receptor that shares sequence homology with D2and D3 receptors (for review, see Civelli, 1995;Van Tol et al., 1991). Although the dopamine D4 receptor is classified as a member of the D2-like receptor family, the pharmacological profile of the D4 receptor is distinct from other known dopamine receptors (Van Tol et al., 1991; Lahtiet al., 1993). Furthermore, the regional distribution of mRNA for the dopamine D4 receptor is also quite different from that of other dopamine receptors. In rats and humans, D4 receptor mRNA expression has been measured in limbic, cortical and other extrastriatal areas with very little expression in striatal regions (O’Malley et al., 1992;Meador-Woodruff et al., 1994). This contrasts with D1, D2, D3 and D5 receptor mRNA localization, which is most abundant in striatal areas (Mansour and Watson, 1995; Meador-Woodruff et al., 1994). The unique extrastriatal distribution of D4 receptor mRNA combined with selectivity of the atypical antipsychotic clozapine at D4 receptors make the D4receptor an attractive target for antipsychotic drug therapy.
To date, the density of dopamine D4receptors in brain tissue has been measured only indirectly as the difference in maximal binding density between [3H]YM 09151–2 (with affinity for D2, D3 and D4 receptors) and [3H]raclopride (with affinity for only D2 and D3 receptors) (Seeman et al., 1993; Lahti et al., 1995b). The lack of availability of selective dopamine D4receptor ligands so far has precluded the direct characterization of the D4 receptor in brain tissue. NGD 94–1 is a highly selective antagonist at the D4 receptor (Tallman et al., 1997). In cloned human D4.2 receptors expressed in mammalian cells, NGD 94–1 has an affinity of 3 nM at the D4.2receptor subtype, whereas its affinity at D1, D2, D3 and D5 receptor subtypes is ≥2 μM. NGD 94–1 has no significant affinity (≥1 μM) for a wide variety of monoamine or other neurotransmitter receptor or modulatory sites except for 5HT1A sites, with an affinity of 170 nM, and 5HT3 receptors, with an affinity of 750 nM. In the present studies, the regional distribution of dopamine D4 binding sites was evaluated autoradiographically in adult rat brain using [3H]NGD 94–1. The selectivity and pharmacological characterization of [3H]NGD 94–1 was confirmed by regional membrane binding studies. The distribution of sites labeled by [3H]NGD 94–1 was compared with the distribution of dopamine D2and D3 receptor subtypes with use of [3H]raclopride and [3H](+)7-OH-DPAT, respectively. In addition, the localization and characterization of [3H]NGD 94–1 binding sites in regionally dissected human brain homogenates were also examined.
Materials and Methods
Quantitative Autoradiography
Tissue preparation.
Adult male Sprague-Dawley rats (200–300 g; Charles River Laboratories, St. Louis, MO) were deeply anesthetized and then sacrificed by intracardial perfusion with ice-cold buffered saline followed by 0.1% buffered paraformaldehyde solution. Brains were removed, frozen on powdered dry ice and stored at −80°C. Twenty-micron frozen sections were collected and thaw mounted onto gelatin-coated slides. Tissue sections were stored at −80°C for no more than 48 h.
[3H]NGD 94–1 binding (to assess D4 receptors).
Slide-mounted tissue sections were preincubated 2 × 5 min at room temperature in buffer containing 50 mM Tris (pH 7.4), 120 mM NaCl, 1 mM EDTA and 5 mM MgCl2. After preincubation, slides were incubated at room temperature in assay buffer (50 mM Tris, pH 7.4, 120 mM NaCl) containing 2.0 to 3.0 nM [3H]NGD 94–1 (custom labeled by ChemSyn, Lenexa, KS, 44.0 Ci/mmol; fig.1) for 90 min. Nonspecific binding was assessed in the presence of 1 μM eticlopride. Sections were rinsed 2 × 5 min in ice-cold assay buffer followed by a brief dip in ice-cold distilled water. Sections were dried under a stream of cool air, apposed to tritium-sensitive film along with [3H]methylmethacrylate autoradiographic standards (Amersham Microscales, Arlington Heights, IL) and exposed in x-ray cassettes for 3 months at −80°C.
Representative autoradiogram of total (A), nonspecific (B) and (C) specific [3H]NGD 94–1 binding in rat medial preoptic area (MPA). Atlas coordinates for coronal sections of medial preoptic area range from bregma −0.40 mm to −1.40 mm (Paxinos and Watson, 1986).
[3H]Raclopride binding (to assess D2 receptors).
Slide-mounted tissue sections were preincubated 2 × 5 min at room temperature in assay buffer containing 50 mM Tris (pH 7.4 at 25°C), 120 mM NaCl, 5 mM KCl, 2 mM MgCl2 and 2 mM CaCl2. Slides were then incubated at room temperature in assay buffer containing 4 nM [3H]raclopride (NEN, Wilmington, DE; 77.0 Ci/mmol) for 30 min. [3H]Raclopride appears to bind predominantly to D2 receptors under the assay conditions used in this study; however, the binding of [3H]raclopride to D3receptors can not be excluded. Nonspecific binding was defined in the presence of 1 μM haloperidol. The slides were rinsed 2 × 2 min in fresh ice-cold assay buffer followed by a brief dip in ice-cold distilled water. Sections were dried under a stream of cool air, apposed to tritium-sensitive film along with [3H]methylmethacrylate autoradiographic standards and exposed in x-ray cassettes for 1 month at −80°C.
[3H](+)7-OH-DPAT binding (to assess D3 receptors).
Slide-mounted tissue sections were preincubated 2 × 5 min at room temperature in assay buffer containing 50 mM NaHEPES (pH 7.5), 1 mM EDTA and 0.1% bovine serum albumin. Slides were then incubated in assay buffer containing 2 nM [3H](+)7-OH-DPAT (Amersham; 150 Ci/mmol) for 1 h at room temperature. Nonspecific binding was assessed in the presence of 1 μM dopamine. Slides were rinsed 2 × 2 min in fresh ice-cold assay buffer containing 100 mM NaCl and dipped briefly in ice-cold distilled water. Sections were dried under a stream of cool air and then apposed to tritium-sensitive film. Films were exposed along with [3H]methylmethacrylate autoradiographic standards in x-ray cassettes for 1 month at −80°C.
Quantification of autoradiograms.
Autoradiographic images were digitized and quantified by computer-assisted densitometry (MCID) from Imaging Research (St. Catherines, Ontario). Optical density measurements taken from captured brain images were plotted against best-fit curves of film optical density generated by [3H]methylmethacrylate standards. Data, expressed as femtomoles per milligram tissue equivalent, represent the mean ± S.E.M. from at least three separate experiments.
Homogenate Receptor Binding
Rat and human membrane preparation.
Adult male rats were sacrificed and brains were removed, dissected and immediately frozen at −80°C. For homogenate preparation, dissected brain regions were homogenized (Brinkman Polytron, Westbury, NY) in 50 mM Tris buffer (pH 7.4 at 25°C) containing 120 mM NaCl, 1 mM EDTA and 5 mM MgCl2. The homogenate was centrifuged for 10 min at 48,000 × g, the pellet was resuspended in buffer and centrifugation was repeated. Pellets were resuspended in 20 volumes of ice-cold assay buffer (50 mM Tris, pH 7.4; 120 mM NaCl) and immediately used in homogenate binding assays as described below.
Virus-free human brain tissues obtained from three to five normal, drug-free donors within 6 h of death were regionally dissected and membrane homogenates were prepared by Analytical Biological Services Inc. (ABS; Wilmington, DE) as described above. Pellets were stored at −80°C until shipment. Upon receipt, human membrane pellets were stored at −80°C until use. Before binding assay, pellets were resuspended in 20 volumes of ice-cold assay buffer (50 mM Tris, pH 7.4; 120 mM NaCl).
[3H]NGD 94–1 binding.
To each polypropylene assay tube, 200 μl prepared membranes (200–400 μg), [3H]NGD 94–1 (custom labeled by ChemSyn; 44 Ci/mmol) and assay buffer (50 mM Tris, pH 7.4; 120 mM NaCl) with or without the addition of displacing agents was added to yield a final volume of 1 ml. Incubation was carried out for 120 min at room temperature in the presence of 0.25 to 16.0 nM [3H]NGD 94–1 for saturation analysis or 2.5 nM [3H]NGD 94–1 for displacement studies. Nonspecific binding was assessed in the presence of 600 nM octoclothepin. The reaction was terminated by rapid vacuum filtration through Whatman GF/B filters using ice-cold assay buffer. Scintillation cocktail was added and filters were counted by scintillation spectrometry. Binding characteristics (Kd,B max andKi) were measured using SigmaPlot (Jandel Scientific, San Rafael, CA).
Data Analysis
Binding data were analyzed by SigmaPlot (Jandel). Kinetic data were converted to a Kd value by the following equation: Kd =k −1/k +1, such that k +1 = (k obs −k −1)/[L], where [L] is the radioligand concentration. Calculated IC50 values were converted to Kivalues by use of the Cheng-Prusoff correction with the following equation: Ki = IC50/(1 + [L]/Kd), where [L] is the radioligand concentration andKd is the dissociation constant for the radioligand, as determined by saturation analysis.
Results
Rat autoradiography.
Specific [3H]NGD 94–1 binding was apparent in several nonstriatal areas of the adult rat brain and represented about 50 to 60% of total binding. Autoradiographic images of total, nonspecific and specific [3H]NGD 94–1 binding in the medial preoptic area of the rat hypothalamus are shown in figure 1. The data in table1 show that [3H]NGD 94–1 binding was region-specific and low in density (<20 fmol/mg tissue equivalent). Highest densities were localized in the rat hippocampus, lateral septal nucleus, medial preoptic area and entorhinal cortex. Levels of [3H]NGD 94–1 were not measurable in rat striatal regions. Thus, the autoradiographically measured distribution of [3H]NGD 94–1 binding was distinct from the distribution of binding seen by use of ligands selective for dopamine D2 and D3 receptor subtypes (fig. 2). In agreement with literature reports (Levant and DeSouza, 1993), dopamine D2receptors were concentrated in striatal regions including the caudate/putamen and nucleus accumbens as well as in extrastriatal regions such as the islands of Calleja and olfactory tubercle. Dopamine D3 receptors, labeled by [3H](+)7-OH-DPAT, were localized to the olfactory tubercle and bulb, islands of Calleja, cerebellar lobules 9 and 10, nucleus accumbens, and to a lesser extent, in the striatum (Levant et al., 1993; Levesque et al., 1992). The distinct regional distribution and low number of [3H]NGD 94–1 binding sites as compared with that of D2 and D3 receptor sites are summarized in figure 3. When regions of highest density for the respective ligands were compared, the relative densities of D4/D2 equaled 12.4/90 fmol/mg tissue equivalents and D4/D3 equaled 12.4/57 fmol/mg tissue equivalents (table 2).
Quantitative autoradiographic distribution of [3H]NGD 94-1 binding sites in rat brain1-a
Sagital sections from rat brain showing the distribution of specific [3H]NGD 94–1 (A, D and G), [3H]raclopride (B, E and H) and [3H](+)7-OH-DPAT (C, F and I) binding. Atlas coordinates for A through C, D through F and G through I are lateral 4.20 to 3.90, lateral 2.90 to 1.90 and lateral 1.40 to 0.40, respectively (Paxinos and Watson, 1986). Abbreviations: Acb, accumbens nucleus; CB, cerebellum; CPu, caudate putamen; DG, dentate gyrus; Ent, entorhinal cortex; ICj, islands of Calleja; Tu, olfactory tubercle; LS, lateral septal nucleus; SN, substantia nigra.
Regional distribution and specific binding (fmol/mg tissue equivalence) of [3H]NGD 94–1 labelingversus [3H]raclopride and [3H](+)7-OH-DPAT labeling in rat brain sections (sagittal view). Serial sections from two to three separate animals were analyzed for [3H]NGD 94–1, [3H]raclopride and [3H](+)7-OH-DPAT binding using quantitative autoradiographic techniques.
Autoradiographic comparisons of densities for dopamine D4, D3 and D2 receptors in rat brain
Rat and human homogenate [3H]NGD 94–1 binding.
Figure 4 shows a representative saturation curve for [3H]NGD 94–1 binding in a membrane preparation from rat entorhinal cortex. Under these assay conditions, specific binding ranged between 20 and 30%. Scatchard plot analysis of [3H]NGD 94–1 binding showed high-affinity, saturable binding in rat hippocampus (B max, 24.0 ± 8.4 fmol/mg protein;Kd, 7.2 ± 1.0 nM) and rat entorhinal cortex (B max, 13.1 ± 3.6 fmol/mg protein; Kd, 9.8 ± 1.2 nM). Pharmacological characterization of [3H]NGD 94–1 binding in rat hippocampal membranes showed relatively high affinities for compounds with known dopamine D4receptor affinity (table 3). The D2/D3 selective agents, raclopride and (−)-sulpiride, showed little affinity at the [3H]NGD 94–1 binding site in rat membranes (K i values > 2,000 nM).
Representative saturation curve for [3H]NGD 94–1 binding to membranes prepared from rat entorhinal cortex. Each concentration was tested in triplicate with six to eight concentrations of [3H]NGD 94–1 (0.25–16 nM). The average Kd andB max values, as determined by computer analysis of the saturation isotherm data for three independent experiments, were 9.8 ± 1.2 nM and 13.1 ± 3.6 fmol/mg protein, respectively. The inset shows the corresponding linear Rosenthal plot of the data.
Pharmacological characterization of [3H]NGD 94-1 binding in hippocampal membranes from rat and human brain tissue3-a
High-affinity, saturable [3H]NGD 94–1 binding was also measured in human homogenate preparations (fig.5). Under these assay conditions, specific binding ranged between 30 and 40%. Scatchard plot analysis of [3H]NGD 94–1 binding showed saturable binding in several brain regions, including hippocampus, hypothalamus, dorsal medial thalamus, entorhinal cortex, prefrontal cortex and lateral septal nucleus (table 4). High-affinity [3H]NGD 94–1 binding was not detected in any striatal regions examined (nucleus accumbens, caudate/putamen) or in cerebellar tissue homogenates. Pharmacological characterization of [3H]NGD 94–1 binding to human membrane preparations showed relatively high affinities for compounds with known dopamine D4 receptor affinity (table 3). Raclopride and (−)-sulpiride, both reported to be selective dopamine D2 receptor ligands, showed very weak affinity (Ki values > 2,000 nM).
Representative saturation curve for [3H]NGD 94–1 binding to membranes prepared from human lateral septal nucleus. Each concentration was tested in triplicate with six to eight concentrations of [3H]NGD 94–1 (0.25–16 nM). The averageKd and B maxvalues, as determined by computer analysis of the saturation isotherm data for two independent experiments, were 5.3 ± 0.8 nM and 28.9 ± 3.9 fmol/mg protein, respectively. The inset shows the corresponding linear Rosenthal plot of the data.
Characteristics of high-affinity [3H]NGD 94-1 binding in membrane preparations from dissected human brain regions4-a
Discussion
The present study was undertaken to characterize pharmacologically the [3H]NGD 94–1 binding sites in rat and human brain tissue and to determine the regional distribution of these binding sites. Specific, high-affinity [3H]NGD 94–1 binding has been demonstrated previously at cloned human D4.2 receptors expressed in mammalian cells (Tallman et al., 1997). In these studies, high-affinity, saturable [3H]NGD 94–1 binding was present in both rat and human brain and was shown to be regionally specific and low in density. However, the affinity of NGD 94–1 appears to be somewhat higher for human (K d= 2–5 nM) than that observed in rat (K d = 7–10 nM) tissue using membrane saturation binding procedures. Such data are consistent with reports of marked differences in sequence homology between human and rat D4 receptors (O’Malley et al., 1992;Van Tol et al., 1991).
In the adult rat brain, the regional distribution of [3H]NGD 94–1 binding sites was compared with the distribution of dopamine D2 and D3 receptor subtypes with the ligands [3H]raclopride and [3H](+)7-OH-DPAT, respectively. Specific [3H]NGD 94–1 binding was greatest in hippocampus, lateral septal nucleus, entorhinal cortex and medial preoptic area of the hypothalamus. The nonstriatal distribution of [3H]NGD 94–1 binding was therefore distinct from the distribution of dopamine D2 and D3 receptor subtypes. Quantitative analysis of autoradiographs showed the density of dopamine D4binding sites to be much lower than the density of binding sites for either D2 or D3 receptor subtypes, consistent with the literature (Lahti et al., 1995b). With use of homogenate preparations from rat hippocampus or entorhinal cortex, saturation analysis of [3H]NGD 94–1 binding also showed a low density of binding sites (<30.0 fmol/mg protein). High-affinity, saturable [3H]NGD 94–1 binding was also shown in homogenate preparations from rat heart tissue (data not shown), consistent with the high level of D4 mRNA expression observed in this organ (O’Malley et al., 1992). These studies, together with the selective pharmacological profile at the [3H]NGD 94–1 binding site, suggest that specific, high-affinity [3H]NGD 94–1 binding in rat reflect populations of the dopamine D4receptor.
The localization of [3H]NGD 94–1 binding in rat brain appears to share considerable overlap with that of the serotonin 1A (5-HT1A) receptor subtype (labeled with [3H]8-OH-DPAT, data not shown). However, although 5-HT1A receptors were shown to be concentrated in the raphe nucleus, [3H]NGD 94–1 showed no specific binding in this brain region. This regional difference in receptor labeling together with the low affinity of NGD 94–1 at 5-HT1A sites (Ki = ∼170 nM) support a distinct binding site for [3H]NGD 94–1, presumably that of the dopamine D4 receptor. Functional studies also fail to support significant interaction of NGD 94–1 at 5HT1A sites in the dorsal raphe (Wilson, personal communication).
Specific, high-affinity [3H]NGD 94–1 binding sites were also detected in homogenate membrane preparations from several different human brain regions, including the hippocampus, hypothalamus, dorsal medial thalamus, entorhinal cortex, prefrontal cortex and lateral septal nucleus. The densities of [3H]NGD 94–1 binding sites in these brain regions were comparable with dopamine D4 receptor densities determined as the difference in maximal binding density between [3H]YM 09151–2 and [3H]raclopride binding (D4 receptor methodology of Seeman et al., 1993). For example, in human hippocampus, [3H]NGD 94–1 binding showed aB max of 8.9 ± 1.7 fmol/mg protein. In human hippocampus from the same tissue homogenate preparation, dopamine D4 receptor density was 10.7 fmol/mg protein as determined by the difference in maximal binding density between [3H]YM 09151–2 and [3H]raclopride binding (data not shown). In the human hypothalamus, saturation binding analysis of [3H]NGD 94–1 binding showed aB max of 11.8 ± 5.0 fmol/mg protein. The difference in maximal binding density between [3H]YM 09151–2 and [3H]raclopride binding in the hypothalamus from the same tissue homogenate preparation was 8.0 fmol/mg protein (data not shown). No high-affinity [3H]NGD 94–1 binding was detected in any striatal region (caudate/putamen and nucleus accumbens) examined. Preliminary autoradiographic studies in postmortem human brain support specific localization of [3H]NGD 94–1 binding sites to cortical and limbic regions with little or no [3H]NGD 94–1 binding to striatal regions (Lahti et al., 1995a). This localization is consistent with recent autoradiographic studies with use of subtraction of [3H]raclopride binding from [3H]YM-09151–2 binding to define D4 sites in human postmortem tissue (Lahtiet al., 1995b). The distribution of D4sites in human brain is also consistent with the recent findings byMrzljak et al. (1996) in which D4receptor immunoreactivity on GABAergic neurons was present in cerebral cortex, hippocampus and other subcortical regions, but not in striatal regions of the primate brain.
These studies suggest that specific, high-affinity [3H]NGD 94–1 binding in rat and human brain reflect populations of the dopamine D4 receptor with a regional distribution unique among dopamine receptor subtypes. With special regard to schizophrenia, numerous postmortem studies in brains of schizophrenics have shown abnormalities in several different corticolimbic regions, including the prefrontal area, entorhinal cortex, cingulate cortex and hippocampus (for review, see Benes, 1995;Goldsmith and Joyce, 1995; Jakob and Beckmann, 1994). The proposed involvement of these brain regions in the mediation of emotional, motivational and attentional drives (for reviews, see Weinberger and Lipska, 1995; Benes, 1995) supports a role for this circuitry in the pathophysiology of schizophrenia. The selective localization of [3H]NGD 94–1 binding sites to corticolimbic regions, as shown in the present study, may suggest a role for the dopamine D4 receptor in the mediation of affective and attentional processes. The introduction of NGD 94–1 into clinical populations should address the D4receptor hypothesis of schizophrenia.
The unique distribution of [3H]NGD 94–1 binding and the similarity of its pharmacology to that in cloned human D4 receptors support the existence of dopamine D4 receptors in both rat and human brain. The extrastriatal location of [3H]NGD 94–1 binding sites suggests that NGD 94–1, as a therapeutic agent for the treatment of schizophrenia, may be free of extrapyramidal side effects. In fact, behavioral data show that acute NGD 94–1 treatment in the rat does not produce catalepsy nor block apomorphine- or amphetamine-induced stereotypy (Hoffmanet al., 1995). In conclusion, the present studies support the existence of a population of dopamine D4receptors in rat and human brain. The regional distribution of these sites suggests that therapeutic agents directed at these sites may possess antipsychotic efficacy without extrapyramidal side-effect liability.
Footnotes
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Send reprint requests to: Dr. Renee Primus, Neurogen Corporation, 35 N.E. Industrial Road, Branford, CT 06405.
- Abbreviations:
- NGD 94–1
- 2-phenyl-4(5)-[4-(2-pyrimidinyl)-piperazin-1-yl)-methyl]-imidazole dimaleate
- HEPES
- N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid
- DPAT
- dipropylaminotetralin
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- Received October 18, 1996.
- Accepted April 8, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
