ABSTRACT
The neuroanatomical distribution of [3H]YM 09151-2 (cis-N-(1-benzyl-2-methylpyrrolidine-3-yl)-5-
chloro-2-methoxy-4-methylaminobenzamide; emonapride) was mapped in squirrel
monkey (Saimiri sciureus) brain using quantitative receptor autoradiography,
and was compared to the neuroanatomical distribution of the dopamine
transporter defined with [3H]WIN 35,428 ([3H]CFT) and of
dopamine D1 receptors defined with [3H]SCH 23390. Preincubation of
caudate-putamen tissue sections increased [3H]YM 09151-2 binding by
nearly 70%, suggesting inhibition of radioligand binding by endogenous
substances. The pharmacological specificity of [3H]YM 09151-2
binding in caudate-putamen tissue sections was suggestive of binding to
dopamine D2-like receptors. Moderate levels of sulpiride-insensitive [3H]YM
09151-2 binding were detected in extrastriatal regions including the medial
prefrontal cortex, the septal area, the claustrum, amygdala and the
hippocampus. Sulpiride-insensitive [3H]YM 09151-2 binding in
hippocampal tissue sections was inhibited in part by the 5-HT1A receptor
antagonist NAN-190 and by the 5-HT4 receptor antagonists GR 113808 and SB
204070 suggesting that a portion of sulpiride-insensitive radioligand binding
may be to serotonin 5-HT1A and/or 5-HT4 receptors. We conclude that [3H]YM
09151-2 labels non-D2-like receptors that are sulpiride- insensitive in a
number of brain regions. This lack of D2-like receptor selectivity combined
with our finding that endogenous substances may inhibit YM 09151-2 binding
suggest that it may not be optimal as a probe for in vivo brain imaging of
dopamine D2-like receptors, and that appropriate masking and baselines must be
instituted in in vitro studies. D1 dopamine receptor distribution in squirrel
monkey brain closely paralleled distributions reported in prior studies in
other primate species. The significance of the differing degrees of
colocalization between [3H]YM 09151-2 labeled dopamine D2
receptors, the dopamine transporter and dopamine D1 receptors is discussed.
INTRODUCTION
The substituted benzamide YM 09151-2 (cis-N-(1-benzyl-2-methylpyrrolidine-3-yl)-5-chloro-2-
methoxy-4-methylaminobenzamide; emonapride) is a D2-like (D2, D3, and D4)
dopamine receptor antagonist (Seeman and Van Tol, 1994; Terai, et al., 1989;
Madras, et al., 1988). Its picomolar affinity and high selectivity for D2-like
receptors has made YM 09151-2 an attractive candidate for use to selectively
label these sites and for development as an imaging probe (Saji, et al., 1996;
Shoup, et al., 1994; Hatano, et al., 1989). However, the selectivity of YM
09151-2 for dopamine receptors has not been fully established. In this regard,
autoradiographic and imaging studies conducted with radiolabeled forms of YM
09151-2 suggest that it may label brain recognition sites other than dopamine
D2-like receptors (Ishiwata, et al., 1994; Yokoyama, et al., 1994; Hatezawa,
et al., 1991; Kazawa, et al., 1990). Moreover, binding studies in
extrastriatal tissues including cortex and hippocampus suggest that YM 09151-2
may interact with serotonin receptors (Kazawa, et al., 1990; Terai, et al.,
1989). Further, striatal YM 09151-2 infusion induced sulpiride-insensitive
dopamine release suggesting that YM 09151-2 evoked striatal dopamine release
by binding to sites exclusive of D2-like receptors (Tomiyama, et al., 1993).
The primary goal of the present study was to examine the distribution and
pharmacological specificity of [3H]YM 09151-2 binding in nonhuman
primate brain using in vitro receptor autoradiography, in order to determine
the selectivity of YM 09151-2 as a probe for brain dopamine D2-like receptors.
This study focused secondarily on mapping the neuroanatomical distribution of
D1 receptors in squirrel monkey brain with quantitative receptor
autoradiography, which, to our knowledge, has yet to be described in the
literature. Finally, the distribution of the dopamine transporter labeled with
[3H]WIN 35,428 ([3H]CFT) (Kaufman and Madras, 1993;
Kaufman, et al., 1991; Canfield, et al., 1990; Madras, et al., 1989) was
compared to [3H]YM 09151-2 and to dopamine D1 receptors labeled
with [3H]SCH 23390 in adjacent tissue sections, to determine the
degree of colocalization of these binding sites at a macroscopic level. This
component of the research was undertaken because the principal target of
cocaine in the brain is the dopamine transporter (Giros, et al., 1996) and in
light of evidence suggesting that both D1 and D2 receptors mediate the
discriminative stimulus effects of cocaine in squirrel monkeys (Spealman, et
al., 1991). The present results indicate that tracer concentrations of [3H]YM
09151-2 bind with high affinity and selectivity to D2-like dopamine receptors
defined as sulpiride- sensitive in many brain regions, but principally the
DA-rich basal ganglia, substantia nigra/ventral tegmentum and nucleus
accumbens/olfactory tubercle. [3H]YM 09151-2 was also found to
label recognition sites that were sulpiride-insensitive in hippocampus and in
other brain regions. Preliminary characterization of these recognition sites
in hippocampal tissue sections suggested that [3H]YM 09151-2 also
may bind to serotonin 5- HT1A and/or 5-HT4 receptors.
MATERIALS AND METHODS
Tissue Preparation:
Squirrel monkey (Saimiri sciureus) brains stored at -85oC were
obtained from the brain bank of the New England Regional Primate Research
Center. Brains were sectioned into 1 cm slabs and stored at -85oC
until thin sectioning. Prior to thin sectioning, tissue slabs were placed into
a Reichert-Jung cryostat to equilibrate with the cutting temperature of -18oC.
Thin sections (20 
M) were
cut and thaw-mounted onto gelatin-coated "subbed" slides, air dried
and stored in sealed containers at -20oC until use.
Parametric Studies:
Tissue sections containing caudate nucleus and putamen were used in parametric
studies to establish appropriate conditions for dopamine D1 and D2 receptor
autoradiography. Incubations were conducted at room temperature in buffer (50
mM Tris HCl/120 mM NaCl, 4 mM MgCl2, pH 7.4 at 23oC) and
postincubation washes were performed at 4oC in wash buffer (50 mM
Tris HCl, pH 7.4 at 4oC). Optimal preincubation, association and
wash times were established in studies in which duplicate slide-mounted tissue
sections from two animals were wiped off the slides with Whatman GF/C filter
discs, placed into scintillation vials along with 4 ml Beckman Ready-Value
counting cocktail, and radioactivity was determined by liquid scintillation
spectrometry with an average counting efficiency of approximately 50%. For D1
receptor parametrics, total binding was determined with 0.5 nM [3H]SCH
23390 and nonspecific binding was determined with 1 >M
(S)-butaclamol. For D2 receptor parametrics, total binding was determined with
0.2 nM [3H]YM 09151-2 and nonspecific binding was determined with 5
M (S)-sulpiride. [3H]WIN
35,428 autoradiography was conducted using a modification of the method of
Canfield et al. (1990). Tissue sections were preincubated in slide mailers
containing ice cold buffer (50 mM Tris HCl/100 mM NaCl, pH 7.4 at 4oC)
for 20 minutes, followed by incubation in buffer containing 5 nM [3H]WIN
35,428 alone or in the presence of 30 M
(-)-cocaine to define nonspecific binding. After a 2 hour incubation, sections
were dipped for 1 second into ice cold fresh buffer, were washed two times (1
minute each) in ice cold fresh buffer, and were dipped for 1 second into ice
cold distilled water. Sections were dried rapidly with a stream of chilled
desiccated air.
Pharmacological Specificity:
The pharmacological specificity of [3H]YM 09151-2 and [3H]SCH
23390 binding was determined in competition studies in caudate-putamen tissue
sections. Tissue sections were preincubated in buffer for 20 minutes and then
with radioligand (and competing drugs) for 150 or 180 minutes ([3H]SCH
23390 or [3H]YM 09151-2, respectively) at 23oC. Sections
then were rinsed in cold buffer for 20 minutes at 4oC, and wiped
off of slides with Whatman GF/C filter paper. Radioactivity was determined by
scintillation spectrometry as described above. Initial studies were conducted
with (S)-butaclamol and (S)-sulpiride to determine the optimal concentrations
of these compounds for determining nonspecific binding. In subsequent studies,
nonspecific binding was defined by 3 M
(S)-butaclamol and by 30 M
(S)-sulpiride in [3H]SCH 23390 (0.5 nM) or [3H]YM
09151-2 (0.2 nM) assays, respectively. In competition experiments with
dopamine, studies were conducted in agonist buffer containing 50 mM Tris HCl,
4 mM MgCl2, 1 mM EDTA, and 0.01% ascorbic acid (pH 7.4 at 23oC).
IC50 values, pseudo-Hill coefficients (nH) and apparent Kd
or Ki values were computed using the EBDA and Ligand programs
(Elsevier-Biosoft, UK). Additional studies were conducted in triplicate
slide-mounted tissue sections containing hippocampus obtained from 2 animals
to determine the identity of sulpiride-insensitive [3H]YM 09151-2
binding sites. In those studies, tissue sections were preincubated in buffer
for 20 minutes at room temperature, incubated with [3H]YM 09151-2
(0.2 nM, final concentration) in the presence of 30 M
(S)-sulpiride (to mask D2 receptor binding) and competing drugs. The
pharmacological specificity of [3H]WIN 35,428 binding to tissue
sections has been reported previously (Kaufman, et al., 1991; Canfield, et
al., 1990).
Autoradiography:
Slides-mounted tissue sections in duplicate and calibrated standards ([3H]Microscales,
Amersham) were placed in X-ray cassettes, covered with [3H]Ultrofilm
(Cambridge Instruments) and the cassettes were sealed along with desiccant
capsules for 5-week exposures at 4oC. Exposed films were developed
with Kodak D-19 developer for 5 minutes, rinsed in water for 30 seconds, fixed
in Kodak Rapid-Fix for 5 minutes, and washed in running water for 20 minutes.
After dipping in Kodak Photoflo (two drops in water) the films were hung to
air-dry. All solutions used in film development were maintained at 20oC.
Quantitative analysis of autoradiograms:
The distribution and levels of radioactivity in brain were determined with
the MCID Image Analysis System (Imaging Research, St. Catharines, Ontario).
Atlases of squirrel monkey brain (Emmers and Akert, 1963; Gergen and MacLean,
1962) were used to establish neuroanatomical landmarks on the autoradiograms.
The developed films were illuminated with a light box and digitized to
quantify relative optical densities corresponding to radioactivity in tissue
sections and in the standards. Relative optical densities of the standard-
exposed films were fitted by linear interpolation in order to convert optical
densities of the [3H]-exposed films to tissue equivalent binding
densities of [3H]YM 09151-2, [3H]SCH 23390 and [3H]WIN
35,428.
Drugs:
[3H]YM 09151-2, (84-86 Ci/mmol), [3H]SCH 23390 (71.3 Ci/mmol)
and [3H]CFT ([3H]WIN 35,428, 81.7 Ci/mmol) were obtained
from DuPont New England Nuclear Corp. (-)-Cocaine hydrochloride was obtained
from the National Institute on Drug Abuse. The following drugs were kindly
donated: SCH 39166 (Schering Corp.), spiperone (Janssen Pharmaceuticals),
mianserin hydrochloride (Organon), (S)- and (R)-butaclamol (Ayerst
Laboratories), (S)-Sulpiride (Ravizza), SB 207710
[(1-butyl-4-piperidinylmethyl)-8-
amino-7-chloro-1,4-benzodioxan-5-carboxylate] (Smith Kline Beecham
Pharmaceuticals), GR113808A [1-[2- methylsulfonyl)amino]ethyl]-4-piperidinyl]methyl
1-methyl-1H-indole-3-carboxylate] (Glaxo Research). NAN-190 HBr was purchased
from Research Biochemicals, Inc.
RESULTS
Parametric Studies:
Parametric studies were conducted in caudate-putamen tissue sections to
determine optimal conditions for performing autoradiography with [3H]SCH
23390 and [3H]YM 09151-2. The effect of tissue slice preincubation
was studied initially to determine whether preincubation enhanced radioligand
binding as was observed with [3H]WIN 35,428 (Canfield, et al.,
1990). Although preincubation of tissue sections was without effect on [3H]SCH
23390 binding, a 20 minute preincubation increased [3H]YM 09151-2
binding by nearly 70% (Figure 1A). Consequently, 20 minute tissue section
preincubation routinely proceeded binding assays for all 3 radioligands in
subsequent studies. [3H]SCH23390 and [3H]YM 09151-2
binding reached steady state by 150 and 180 minutes, respectively (Figure 1B).
Maximal specific binding of the radioligand was achieved following a 2 minute
wash procedure due primarily to the rapid reduction (~50%) in nonspecific
binding (Figures 1C, D).
Pharmacological Specificity of [3H]YM
09151-2 and [3H]SCH 23390:
Radioligand affinities (Kd values) were determined by scatchard
transformation of saturation binding data from studies conducted in caudate-putamen
tissue sections. The Kd and pseudo-Hill (nH) values for [3H]YM
09151-2 binding were 0.21 ± 0.02 nM and 0.86 ± 0.01 (means ± S.D., n=2),
respectively (Figure 2A, Table
1). The Kd and nH values for [3H]SCH 23390 binding
were 0.81 ± 0.20 nM and 0.99 ± 0.01 (means ± S.D., n=2), respectively
(Figure 2B, Table
1).
Table 1. Antagonist and agonist
affinities for dopamine receptors in squirrel monkey caudate-putamen tissue
sections.
| Compound
| IC50 (nM)
| Ki or Kd (nM)
| nH
|
| D1 Receptors
|
| [3H]SCH 23390
|
|
| N.D.
|
|
| 0.81 ± 0.20
|
|
| 0.99 ± 0.01
|
| SCH 39166
|
|
| 1.6 ± 0.21
|
|
| 0.90 ± 0.11
|
|
| 0.94 ± 0.05
|
| (S)-Butaclamol
|
|
| 4.3 ± 0.07
|
|
| 2.4 ± 0.07
|
|
| 0.88 ± 0.01
|
| Dopamine
|
|
| 140 ± 8.5
|
|
| 72 ± 4.2
|
|
| 0.52 ± 0.04
|
| Mianserin
|
|
| 630 ± 0.71
|
|
| 340 ± 0.0
|
|
| 1.01 ± 0.02
|
| (R)-Butaclamol
|
|
| 39000 ± 2900
|
|
| 21000 ± 1600
|
| D2 Receptors
|
| Spiperone
|
|
| 0.55 ± 0.11
|
|
| 0.28 ± 0.06
|
|
| 0.83 ± 0.04
|
| [3H]YM 09151-2
|
|
| N.D.
|
|
| 0.21 ± 0.02
|
|
| 0.86 ± 0.01
|
| (S)-Butaclamol
|
|
| 7.4 ± 0.74
|
|
| 3.7 ± 0.37
|
|
| 0.83 ± 0.01
|
| Dopamine
|
|
| 74 ± 35
|
|
| 21 ± 6.4
|
|
| 0.63 ± 0.01
|
| (S)-Sulpiride
|
|
| 140 ± 16
|
|
| 71 ± 7.8
|
|
| 0.85 ± 0.06
|
| Mianserin
|
|
| 12000 ± 1500
|
|
| 6100 ± 780
|
|
| 1.04 ± 0.01
|
| (R)-Butaclamol
|
|
| 55000 ± 17000
|
|
| 21000 ± 1800
|
[3H]SCH 23390 (0.5 nM), [3H]YM 09151-2 (0.2 nM) and
competing drugs (9 - 13 concentrations) were coincubated with tissue sections
as described under Materials and Methods. The results are the means ± SD of
two experiments in different brains performed in duplicate. The IC50
values are concentrations that inhibited 50% of radioligand binding. The Ki
or Kd and nH values were derived by the EBDA/LIGAND analyses. N.D.
- not determined.
Competition studies were conducted in 2 animals using duplicate caudate-putamen
tissue sections to determine potencies of a number of D1 and D2 receptor
antagonists for inhibiting [3H]SCH 23390 (0.5 nM) and [3H]YM
09151-2 (0.2 nM) binding (Figure 3, Table
1). For [3H]SCH 23390 labeled sites, the rank order of
inhibitory potencies was: SCH 39166 > (S)-butaclamol > dopamine >
mianserin >> (R)-butaclamol (Figure 3A). Stereoselectivity of the [3H]SCH
23390 binding site also was observed with the isomers of butaclamol, the (S)
isomer being approximately 8800-fold more potent than the (R) isomer. Dopamine
inhibition of [3H]SCH 23390 binding was biphasic with an nH of 0.52
± 0.04. A highly significant correlation (p < 0.002) between Kd
(or Ki) values for [3H]SCH 23390 binding to squirrel
monkey caudate-putamen tissue sections and [3H]SCH 23390 binding to
D1 receptors in striatal homogenates was observed (Figure 3C).
Mianserin (100 nM) was included in the incubation buffer in all subsequent
autoradiographic experiments with [3H]SCH 23390 to block serotonin
receptor binding (Nicklaus, et al., 1988). For [3H]YM 09151-2
labeled sites, the rank order of inhibitory potencies was: spiperone > (S)-butaclamol
> dopamine > (S)-sulpiride > mianserin > (R)-butaclamol (Figure
3B). Stereoselectivity of striatal [3H]YM 09151-2 binding sites was
observed with the isomers of butaclamol, the (S) isomer being approximately
5700-fold more potent than the (R) isomer. Dopamine inhibition of [3H]YM
09151-2 binding was biphasic with an nH of 0.63 ± 0.01. A highly significant
correlation (p = 0.002) between Kd (or Ki) values for [3H]YM
09151-2 binding to squirrel monkey caudate-putamen tissue sections and [3H]YM
09151-2 binding to D2 receptors in striatal homogenates was observed (Figure
3D). Autoradiography with [3H]WIN 35,428 has been shown to result
primarily in labeling of dopamine-rich brain regions and the dopamine
transporter (Kaufman and Madras, 1993; Kaufman, et al., 1991; Canfield, et
al., 1990).
|
|
| Figure 3:
Pharmacological specificity of radioligand binding to slide-mounted
caudate- putamen tissue sections. (A - B) Competition curves for
inhibition of [3H]SCH 23390 (0.5 nM) binding (panel A) and
for inhibition of [3H]YM 09151-2 (0.2 nM) binding (panel B).
Shown are averages of duplicate determinations in two brains. (C)
Correlation between Ki (or Kd) values for drug
inhibition of [3H]SCH 23390 binding in monkey caudate-putamen
tissue sections compared with Ki values at D1 receptors
determined in monkey membrane homogenates. (D) Correlation between Ki
(or Kd) values for drug inhibition of [3H]YM
09151-2 binding in monkey caudate-putamen tissue sections compared with
Ki values at D2 receptors in monkey membrane homogenates. Ki
(or Kd) values in tissue section studies were derived from
competition or saturation study data by the EBDA and LIGAND programs.
The sources of Ki values for drugs in membrane homogenates
were (Chipkin, et al., 1988; Madras, et al., 1988; Nicklaus, et al.,
1988; Terai, et al., 1989). In all panels, numbers correspond to the
following drugs: 1, SCH 23390; 2, SCH 39166; 3, (S)- butaclamol; 4,
dopamine; 5, mianserin; 6, (R)-butaclamol; 7, spiperone; 8, YM 09151-2;
9, (S)-sulpiride. |
Neuroanatomical Distribution of [3H]YM 09151-2 binding:
The highest binding densities of [3H]YM 09151-2 (0.2 nM) were found
in the caudate nucleus and putamen. Specific binding, defined as binding
blocked by (S)-sulpiride (30 M)
constituted 90.2 ± 1.2% and 90.6 ± 1.0% of total binding in caudate nucleus
and putamen, respectively (mean ± S.E., n = 3) Within these two nuclei,
medial - lateral gradients of [3H]YM 09151-2 binding were found; [3H]YM
09151-2 binding was higher in medial compared with lateral caudate nucleus and
the reverse pattern was observed in putamen. Moderate levels of [3H]YM
09151-2 binding also were detected in the nucleus accumbens/olfactory
tubercle, in the bed nucleus of the stria terminalis, in the substantia nigra/ventral
tegmentum (primarily the substantia nigra pars compacta), in the ventral
pallidum and in the external segment of the globus pallidus (Figure 4 and Table
2). Sulpiride inhibited 70 - 90% of [3H]YM 09151-2 binding in
extrastriatal regions that included the nucleus accumbens/olfactory tubercle,
substantia nigra, bed nucleus of the stria terminalis, and both anterior and
external segments of the globus pallidus. Several brain regions contained
moderate to high levels of sulpiride-insensitive binding including the medial
prefrontal cortex, the septal area, the claustrum, portions of the amygdala
and hypothalamus, and lateral hippocampus (Figure 4 and Table
2).
Additional pharmacological studies were conducted to
further characterize the nature of sulpiride- insensitive [3H]YM
09151-2 binding in tissue sections of hippocampus and amygdala. Sulpiride (30 M)
was used to mask [3H]YM 09151-2 binding to D2 receptors. Several
drugs were tested for their abilities to inhibit sulpiride-insensitive binding
in the hippocampal region including the potent serotonin 5-HT4 receptor
antagonists GR 113808 and SB 204070, the serotonin 5-HT1A receptor antagonist
NAN 190, and (S)- butaclamol. GR 113808 (50 nM) and SB 204070 (50 nM) reduced
sulpiride-insensitive [3H]YM 09151-2 binding to 80 ± 10 and 83 ±
9 % of control levels, respectively (mean ± S.D., n = 2), while lower
concentrations of these drugs (10 nM) reduced sulpiride-insensitive [3H]YM
09151-2 binding by less than 5%. NAN 190 reduced sulpiride-insensitive [3H]YM
09151-2 binding to 81 ± 10% of control levels (mean ± S.D., n = 2). Finally,
a high concentration of (S)-butaclamol (10 M)
reduced sulpiride-insensitive [3H]YM 09151-2 binding to 79 ± 8% of
control levels (mean ± S.D., n = 2).
|
|
|
|
|
|
| Figure 4:
Pseudocolor images of the neuroanatomical distribution of [3H]WIN
35,428, [3H]SCH 23390 and [3H]YM 09151-2
determined by in vitro receptor autoradiography. Each series of 4 images
displays a representative autoradiograph showing binding of [3H]WIN
35,428 (upper left), [3H]SCH 23390 (upper right), [3H]YM
09151-2 (lower left) and [3H]YM 09151-2 in the presence of 30
M (S)- sulpiride
(lower right). (A) Sections from A-P level 13.5 showing high levels
(reds, yellows) of binding of all three ligands in the Cd, Put, and NaOT,
and moderate levels of [3H]SCH 23390 and [3H]YM
09151-2 binding in MPFC, Cl and Ctx. Moderate levels of sulpiride-insensitive
[3H]YM 09151-2 binding are present in MPFC, Cl and Ctx. (B)
Sections from A-P level 11.5 showing moderate levels of [3H]SCH
23390 and [3H]YM 09151-2 binding in GP, VP and Amy, and
sulpiride-insensitive [3H]YM 09151-2 binding in Amy. (C)
Sections from A-P level 10.5 showing moderate levels of [3H]SCH
23390 binding in GPi and low - moderate levels of [3H]SCH
23390 and [3H]YM 09151-2 binding in GPe, hypo, Cl, and Amy.
Low to moderate levels of sulpiride-insensitive [3H]YM
09151-2 binding are present in Cl, Amy, and Hypo. (D) Sections from
approximately A-P 6.0 showing high and moderate levels of [3H]SCH
23390 and [3H]YM 09151-2 in Sn (pars reticulata and compacta),
respectively, moderate levels of [3H]SCH 23390 and [3H]YM
09151-2 in medial Hipp, and moderate to high levels of [3H]YM
09151-2 in HippL. The majority of hippocampal [3H]YM 09151-2
binding was sulpiride-insensitive. Brain Region Abbreviations: MPFC,
medial prefrontal cortex; MS, septal area; Cd, caudate nucleus; Put,
putamen; NaOT, nucleus accumbens/ olfactory tubercle; ST, bed nucleus of
the stria terminalis; GPi, internal segment of globus pallidus; GPe,
external segment of globus pallidus; VP, ventral pallidum; Hypo,
hypothalamus; PV, paraventricular nucleus of hypothalamus; Cl, claustrum;
Amy, amygdala; Thal, thalamus; Hipp, hippocampus; HippL, lateral
portions of hippocampus; SnVTA, substantia nigra/ventral tegmentum; Ctx,
cortex. |
Table 2. Radioligand Binding
Densities Determined by Quantitative Receptor Autoradiography in adjacent
tissue sections of Squirrel Monkey (Saimiri sciureus) Brain.
|
|
|
| YM 09151-2
|
| YM 09151-2
(sulpiride insensitive)
|
| WIN 35,428
|
| SCH 23390
|
| Region
|
| MPFC
|
|
| +
|
| ++
|
| +
|
| ++
|
| MS
|
|
| +
|
| ++
|
| +
|
| ++
|
| Cd
|
|
| +++++
|
| -
|
| +++++
|
| ++++++
|
| Put
|
|
| +++++
|
| -
|
| +++++
|
| ++++++
|
| NaOT
|
|
| ++++
|
| -
|
| ++++
|
| ++++++
|
| ST
|
|
| ++
|
| -
|
| +++
|
| +++
|
| GPi
|
|
| +
|
| -
|
| +
|
| +++
|
| GPe
|
|
| ++
|
| -
|
| +
|
| ++
|
| VP
|
|
| ++
|
| -
|
| ++
|
| +++
|
| Hypo
|
|
| +
|
| ++
|
| +
|
| +
|
| PV
|
|
| -
|
| -
|
| -
|
| ++
|
| Cl
|
|
| +
|
| ++
|
| +
|
| ++
|
| Amy
|
|
| +
|
| ++
|
| ++
|
| +++
|
| Thal
|
|
| +
|
| -
|
| +
|
| +
|
| Hipp
|
|
| +
|
| -
|
| +
|
| ++
|
| HippL
|
|
| +
|
| +++
|
| +
|
| ++
|
| Sn/VTA
|
|
| ++
|
| -
|
| ++
|
| ++++
|
| Ctx
|
|
| +
|
| +
|
| +
|
| ++
|
Specific binding was determined in autoradiographs of tissue incubated with
[3H]WIN 35,428 (5 nM), [3H]SCH 23390 (0.5 nM) and [3H]YM
09151-2 (0.2 nM). Brain Region Abbreviations are found in the legend to Figure
4.
Binding densities (pmol/g) in squirrel monkey brain:
| ++++++
| > 120
|
| +++++
| 85 - 119
|
| ++++
| 50 - 84
|
| +++
| 25 - 49
|
| ++
| 10 - 24
|
| +
| 1 - 9
|
| -
| Background
|
Distribution of [3H]SCH 23390 binding:
The highest levels of D1 receptors labeled by [3H]SCH 23390 (0.5 nM,
in the presence of 100 nM mianserin) were detected in anterior sections of the
caudate nucleus and putamen. In these regions, specific binding, defined as
binding blocked by (S)-butaclamol (3 M)
averaged 95.8 ± 0.5 % and 95.9 ± 0.6% of total binding, respectively (mean
± S.E., n = 3). In contrast to the medial - lateral gradients detected in
caudate nucleus and putamen with [3H]YM 09151-2 and [3H]WIN
35,428, [3H]SCH 23390 distribution in these structures appeared to
be more uniform. High levels of [3H]SCH 23390 binding were detected
in the nucleus accumbens/olfactory tubercle, in the bed nucleus of the stria
terminalis, and in the substantia nigra/ventral tegmentum (primarily in the
substantia nigra pars reticulata). Moderate levels of [3H]SCH 23390
binding were found in the ventral pallidum, the amygdala, the internal segment
of the globus pallidus, the medial prefrontal cortex and the claustrum. [3H]SCH
23390 binding above background (white matter) levels was found in the
paraventricular nucleus of the hypothalamus, the medial septal area, medial
hippocampus, the external segment of the globus pallidus, and in outer layers
of cortex (Figure 4 and Table
2). We did not detect any brain regions that contained (S)-butaclamol-insensitive
[3H]SCH 23390 binding that exceeded white matter binding levels.
Distribution of [3H]WIN 35,428 binding:
The neuroanatomical distribution of [3H]WIN 35,428 binding (5 nM)
to the dopamine transporter paralleled the distribution reported previously
(Kaufman and Madras, 1993; Kaufman, et al., 1991; Canfield, et al., 1990). The
highest levels of binding were found in anterior sections of caudate nucleus
and putamen. Specific binding, defined as cocaine-sensitive (30 M)
binding, averaged 90.0 ± 0.7% and 91.6 ± 0.9% in the caudate nucleus and
putamen, respectively (mean ± S.E., n = 3). Medial - lateral gradients
similar to those reported previously (Kaufman, et al., 1991) and to those
observed presently with [3H]YM 09151-2 were detected with [3H]WIN
35,428. High levels of [3H]WIN 35,428 binding also were detected in
the nucleus accumbens/olfactory tubercle and in the bed nucleus of the stria
terminalis. Moderate levels of [3H]WIN 35,428 binding were found in
the substantia nigra/ventral tegmentum (primarily the substantia nigra pars
compacta) and the ventral pallidum. Low levels were detected in the amygdala,
hypothalamus, globus pallidus, and medial thalamus. [3H]WIN 35,428
binding levels slightly higher than background (white matter) levels were
found in the medial septal area, the hippocampus, portions of thalamus, medial
prefrontal cortex, and other cortical regions (Figure 4 and Table
2).
DISCUSSION
[3H]YM 09151-2 pharmacological specificity and distribution:
In tissue sections of squirrel monkey caudate-putamen, the pharmacological
specificity of [3H]YM 09151-2 binding sites in was consistent with
the dopamine D2-like receptor. The Kd for [3H]YM 09151-2
was 0.21 nM, slightly higher than values typically reported for striatal
homogenates (< 100 pM), (Assié, et al., 1993; Terai, et al., 1989; Niznik,
et al., 1985), but well within the range of Kd values reported in
autoradiographic studies (Yokoyama, et al., 1995; Yokoyama, et al., 1994; Cox
and Waszczak, 1991; Unis, et al., 1990). Scatchard transformation of
saturation binding data yielded a curvilinear distribution of the data points
and an nH of 0.86, suggesting that the radioligand bound to more than one
class of sites. However, a two-site model did not result in a significantly
better fit than a single site model.
The apparent densities of [3H]YM 09151-2 (0.2 nM) binding in
anterior sections of caudate nucleus and putamen averaged approximately 90
pmol/g tissue equivalent. This value is several times higher than D2 receptor
densities measured in homogenate studies with [3H]spiperone which
averaged about 24 pmol/g tissue for mammalian striatum (Seeman, et al., 1993;
Madras, et al., 1988; Niznik, et al., 1985). The higher binding densities
found in the present study are likely to result from the use of the benzamide
[3H]YM 09151-2, which typically detects higher striatal D2 receptor
densities than [3H]spiperone (Seeman, et al., 1993; Seeman, et al.,
1992; Terai, et al., 1989; Niznik, et al., 1985). This feature of [3H]YM
09151-2 has been attributed to its labeling of D2 receptor monomers while [3H]spiperone
labels dimers (Seeman and Van Tol, 1994; Seeman, et al., 1992). In addition, a
20 minute preincubation was found to increase [3H]YM 09151-2
binding densities by nearly 70% in caudate-putamen tissue sections.
Preincubation of tissue slices has been shown to significantly enhance [3H](+)PHNO
binding to caudate-putamen D2 receptors and [125I]iodosulpiride
binding to D3 receptors in the Islands of Calleja, apparently through removal
of endogenous dopamine or other substances that otherwise occupy these
receptor binding sites (Nobrega and Seeman, 1994; Schotte, et al., 1992).
Consequently, endogenous substances, in addition to inhibiting striatal [3H]WIN
35,428 binding (Canfield, et al., 1990), also may inhibit in vitro (and in
vivo) YM 09151-2 binding.
The overall brain distribution of sulpiride-sensitive [3H]YM
09151-2 binding was typical of the reported distribution of dopamine D2
receptors. The regional relative densities of [3H]YM 09151-2
binding in squirrel monkey brain were strongly correlated (r = .96, p <
0.003, Figure 5) with D2 receptor regional relative densities mapped with [3H]
spiperone or [3H]CV 205 502 in brains of primates (Camps, et al.,
1989; DeKeyser, et al., 1988; Richfield, et al., 1987), and with D2 receptor
regional relative densities mapped with [3H](+)PHNO or [3H]YM
09151-2 in rodent brain (Nobrega and Seeman, 1994; Yokoyama, et al., 1994).
These correlations suggest that [3H]YM
09151-2 should be a suitable probe for sulpiride-sensitive D2-like receptors.
However, we also detected substantial levels of sulpiride-insensitive [3H]YM
09151-2 binding in several brain regions including medial prefrontal cortex,
the septal area, the claustrum, and portions of the amygdala, hypothalamus,
and hippocampus. Interestingly, the distribution of in vitro hippocampal [3H]YM
09151-2 binding detected presently parallels cocaine accumulation in the
hippocampus, as detected by ex vivo autoradiographic studies in squirrel
monkeys (Madras and Kaufman, 1994).
Although [3H]YM 09151-2 binds with high affinity to dopamine D4
receptors and sulpiride is a weak antagonist at the human dopamine D4 receptor
(Ki 
M)
(Chabert, et al., 1994; Seeman and Van Tol, 1994), it is unlikely that
sulpiride-insensitive sites are D4-related because the regions in which these
residual sites were found do not fully correspond to mRNA distribution of the
D4 receptor. Furthermore, the concentration of sulpiride used to define
nonspecific binding in the present study (30 M)
should have masked > 90% of brain D4 receptors. It therefore seems unlikely
that sulpiride-insensitive [3H]YM 09151-2 binding is D4
receptor-related.
|
|
| Figure 5: Correlation between
neuroanatomical distribution determined by quantitative in vitro
receptor autoradiography of sulpiride-sensitive [3H]YM
09151-2 binding in squirrel monkey brain and D2 receptor binding (Top)
and [3H]SCH 23390 specific binding in squirrel monkey brain
and D1 receptor binding (Bottom). Data are expressed as a percentage of
the binding density determined in anterior caudate nucleus. The
literature sources of D2 and D1 receptor densities determined in
autoradiographic studies are (Camps, et al., 1989; Cortés, et al.,
1989; DeKeyser, et al., 1988; Richfield, et al., 1987). Numbers in both
panels correspond to brain regions as follows: 1, anterior caudate
nucleus; 2, anterior putamen; 3, nucleus accumbens/olfactory tubercle;
4, substantia nigra/ventral tegmentum; 5, amygdala; 6, hypothalamus; 7,
medial thalamus; 8, internal segment of globus pallidus; 9, hippocampus;
10, thalamus; 11, external segment of globus pallidus; 12, claustrum. |
Two recent reports assessing hippocampal [3H]YM 09151-2 binding
indicated that the majority of labeling was of D2 receptors (Lahti, et al.,
1995; Yokoyama, et al., 1995), a finding inconsistent with the present
results. Although species differences could be a basis for the apparently
different pharmacological profile of [3H]YM 09151-2 binding
reported in rodent hippocampus (Yokoyama, et al., 1995), it is also possible
that the use of (S)-butaclamol to define [3H]YM 09151-2 specific
binding in that study (Yokoyama, et al., 1995), and the use of chlorpromazine
in the human study (Lahti, et al., 1995) might account for conflicting
results. In this regard, we found that high concentrations of (S)-butaclamol
inhibited a portion of sulpiride- insensitive binding in hippocampal tissue
sections, suggesting that (S)-butaclamol used to define specific binding may
reveal a different population of [3H]YM 09151-2 binding sites than
(S)-sulpiride. This is particularly important as (S)-butaclamol binds with
high affinity to other receptors whereas (S)-sulpiride is relatively
selective. For example, (S)-butaclamol has nanomolar potency for inhibiting [3H]8-OH-DPAT
binding to 5-HT1A receptors (Assié, et al., 1993; Sundram, et al., 1993)
while sulpiride is a very weak inhibitor (Assié, et al., 1993; Terai, et al.,
1989; El Mestikawy, et al., 1988). We explored whether sulpiride- insensitive
sites in hippocampus represented 5-HT1A receptors. The rationale is based on
the following considerations: YM 09151-2 is a relatively potent inhibitor of
5-HT1A receptor binding (Assié, et al., 1993; Terai, et al., 1989) and the
anatomical distribution of sulpiride-insensitive [3H]YM 09151-2
binding resembles the localization of 5-HT1A-like receptors (Hoyer, et al.,
1994; Pazos, et al., 1987; Hoyer, et al., 1986). Consequently, the 5-HT1A
antagonist NAN-190 (Ki of 0.6 nM for the receptor (Glennon, et al.,
1988)) was used to determine whether sulpiride-insensitive binding of [3H]YM
09151-2 in hippocampus was associated with the 5-HT1A receptor. NAN-190 (6 nM)
reduced sulpiride-insensitive [3H]YM 09151-2 binding by about 20%,
indicating that 5-HT1A receptors may represent a small proportion of [3H]YM
09151- 2 binding sites. Alternatively, as several receptors have nanomolar
affinites for NAN-190 (Glennon, et al., 1988), it is necessary to consider
other targets for [3H]YM 09151-2.
Another and as yet unexplored potential target for YM 09151-2 binding is
the 5-HT4 receptor, which is present in moderate density in striatal and
limbic brain regions (Jakeman, et al., 1994; Waeber, et al., 1993). The
neuroanatomical distribution of 5-HT4 receptors (Patel, et al., 1995;
Reynolds, et al., 1995; Domenech, et al., 1994; Grossman, et al., 1993;
Jakeman, et al., 1994; Waeber, et al., 1993) is consistent with some regions
found presently to contain sulpiride-insensitive [3H]YM 09151-2
binding. From a molecular standpoint, YM 09151-2 is a close congener of
4-amino-5-chloro-2-methoxy-substituted benzamide compounds including cisapride,
zacopride, and renzapride, which are agonists at hippocampal serotonin 5-HT4
receptors (Bockaert, et al., 1990; Chaput, et al., 1990). While structurally
related to this chemical class, sulpiride lacks the 2- methoxy- and 5-chloro-
substitutions present in 5-HT4 receptor agonists of the benzamide class and is
ineffective as a 5-HT4 receptor agonist (Bockaert, et al., 1991). Together,
these observations imply that a proportion of sulpiride-insensitive [3H]YM
09151-2 binding sites may represent 5-HT4 receptors. Consequently, two
selective 5-HT4 receptor antagonists, GR113808 (Grossman, et al., 1993) and SB
204070 (Wardle, et al., 1993) were selected to determine whether sulpiride-insensitive
[3H]YM 09151-2 binding in tissue sections containing hippocampus
and amygdala was to 5-HT4 receptors. Each of these compounds (50 nM) blocked
approximately 20% of the sulpiride-insensitive binding, suggesting that a
small component of [3H]YM 09151-2 binding was to 5-HT4 receptors.
It is important to note that SB 204070 is a very weak inhibitor of 5-HT1A
receptor binding, (pKi > 1 M),
(Wardle, et al., 1993), suggesting that the sulpiride- insensitive sites
blocked by SB 204070 are not 5-HT1A receptor-related. A lower concentration
(10 nM) of the compounds was ineffective at inhibiting sulpiride-insensitive [3H]YM
09151-2 binding. This concentration should have been sufficient to block all
tissue section 5-HT4 receptor binding given the picomolar affinity of GR113808
and SB 204070 for 5-HT4 receptors. Species differences in both affinities and
densities of 5-HT4 receptors (Jakeman, et al., 1994; Waeber, et al., 1993)
also could account for the ineffectiveness of the low dose of these drugs.
Taken together, the results could imply either that a small proportion of the
sulpiride-insensitive [3H]YM 09151-2 binding is to 5-HT4 receptors
or that another receptor is a target for both [3H]YM 09151-2 and
relatively high concentrations of 5-HT4 antagonists. Further work will be
necessary to resolve the nature of sulpiride-insensitive [3H]YM
09151-2 binding. Nevertheless, the results of the present study suggest that
appropriate masking of non-dopamine receptors will be necessary for the
accurate detection of D2-like receptors with YM 09151-2, and may also be
necessary for use of close congeners such as YM-43611 (Hidaka, et al., 1996)
to selectively label D3 and D4 receptors.
If 5-HT4 receptors are targets for YM 09151-2 binding, then it is
conceivable that striatal 5-HT4 receptors may contribute to the apparently
high striatal D2 receptor densities detected with [3H]YM 09151-2 (Seeman,
et al., 1993; Terai, et al., 1989; Niznik, et al., 1985). In this regard,
striatal 5-HT4 receptor densities detected in studies conducted in primate
brain range from approximately 5 - 20 pmol/g tissue (Reynolds, et al., 1995;
Jakeman, et al., 1994; Waeber, et al., 1993) while in primate striatum,
spiperone-defined D2 receptor densities range from approximately 12 - 40 pmol/g
(Madras, et al., 1988; Richfield, et al., 1987; Niznik, et al., 1985).
Therefore, striatal 5-HT4 receptor density is in the range of about half of
striatal spiperone-defined D2 receptor density. Consequently, any 5-HT4
receptor labeling by [3H]YM 09151-2 could substantially alter
apparent D2 receptor density. From a functional standpoint, it is of interest
to note that infusion of YM 09151- 2 into striatum induces sulpiride-insensitive
dopamine release (Tomiyama, et al., 1993) and that 5-HT4 receptors apparently
mediate striatal dopamine release (Steward, et al., 1996; Bonhomme, et al.,
1995). Together, these findings suggest that striatal YM 09151-2 infusion
might induce dopamine release by activating 5-HT4 receptors. Because 5-HT4
receptors are present in high densities in dopamine-rich brain regions, a
modulatory role for the 5-HT4 receptor on dopamine neurotransmission in
striatum and in dopamine-rich limbic regions should be considered.
[3H]SCH 23390 pharmacological specificity and distribution:
The pharmacological specificity of striatal [3H]SCH 23390 binding
was consistent with labeling of dopamine D1 receptors. The neuroanatomical
distribution of 0.5 nM [3H]SCH 23390 in the presence of 100 nM
mianserin closely paralleled D1 receptor distribution reported in primate
brain (Cortés, et al., 1989; DeKeyser, et al., 1988; Richfield, et al., 1987)
(r = .99, p < 0.001, Figure 5). In extrastriatal brain regions, the density
of D1 receptors generally was much higher than D2 receptors. However, as both
SCH 23390 and (S)- butaclamol label serotonin receptors, only some of which
are blocked by mianserin, it is probable that some of the binding sites
designated as D1 receptors are associated with other targets. An appropriate
drug to define D1 receptor sites is clearly needed for D1 density
determinations.
It is of interest to note that while both [3H]YM 09151-2
(present study) and [3H]WIN 35,428 binding (Canfield, et al., 1990)
are sensitive to endogenous substances, as evidenced by increased specific
binding following tissue slice preincubation, tissue slice preincubation was
without significant effect on [3H]SCH 23390 binding. This implies
that [3H]SCH 23390-labeled striatal D1 receptors in situ may exist
in a lower affinity state than D2 receptors. However, this in situ difference
may not be functionally significant as dopamine has an apparently identical
affinity for striatal D1 and D2 receptors (Schoffelmeer, et al., 1994).
Colocalization of dopamine receptors and the dopamine transporter:
High densities of D1 and D2 receptors and the dopamine transporter were
detected only in a limited number of brain regions including caudate nucleus,
putamen, and the nucleus accumbens/olfactory tubercle. Within the caudate
nucleus and putamen, a close correspondence between dopamine transporter/D2
receptor density was observed, with parallel medial - lateral gradients of [3H]YM
09151-2 and [3H]WIN 35,428 binding. In contrast, [3H]SCH
23390 density was quite high but more uniform throughout these regions.
D1 and D2 receptors were anatomically colocalized in a number of brain
regions low in dopamine transporter binding. These included the ventral
pallidum and medial prefrontal cortex. In ventral pallidum, similar densities
of [3H]YM 09151-2 and [3H]WIN 35,428 binding were
detected. The significance of medial prefrontal cortex colocalization of [3H]YM
09151-2 and [3H]WIN 35,428 binding is unclear. Given the moderate
level of sulpiride-insensitive [3H]YM 09151-2 binding detected in
this brain region, radioligand labeling of sites other than D2-like receptors
may confound interpretations. In the amygdala, moderate levels of [3H]SCH
23390 and [3H]YM 09151-2 binding appeared to be colocalized.
However, a substantial proportion of [3H]YM 09151-2 binding in this
region also was sulpiride-insensitive. Others studies have reported
segregation of amygdala D1 and D2 receptor distribution (Levey, et al., 1993;
Scibilia, et al., 1992).
D1 and D2 receptors were anatomically segregated in a number of brain
regions including the globus pallidus, in which moderate levels of [3H]SCH
23390 binding sites were detected in the internal segment while low to
moderate [3H]YM 09151-2 labeling was detected primarily in the
external segment. Additionally, D1 and D2 receptor distribution was segregated
in the substantia nigra, the former being enriched in the pars reticulata and
the latter localized in the pars compacta. In the substantia nigra pars
compacta, [3H]YM 09151- 2 was colocalized with [3H]WIN
35,428 binding. These patterns of anatomical overlap and segregation in
general parallel results from prior studies in other primate species and in
rodents.
Determining the degree of anatomical colocalization of dopamine receptors
and the dopamine transporter is important, since both D1 and D2 receptor
subtypes contribute to the discriminative stimulus effects of cocaine (Spealman,
et al., 1991). Additionally, abundant evidence exists demonstrating dopamine
receptor synergism that modulates brain function. For example, D1 and D2
receptor stimulation is required to induce striatal long-term synaptic
depression (Calabresi, et al., 1992) and maximal striatal Fos expression
requires concurrent D1 and D2 receptor agonist stimulation (LaHoste, et al.,
1993). Dopamine depletion generally results in a loss of receptor synergy,
implying that in addition to mediating the discriminative stimulus effects of
cocaine, normal dopamine-related brain function requires simultaneous
stimulation of D1 and D2 receptors (LaHoste, et al., 1993; Calabresi, et al.,
1992; Retaux, et al., 1991; Hu, et al., 1990). Intraneuronal colocalization of
D1 and D2 receptors and the dopamine transporter also may be functionally
significant. For example, activation of D1 and D2 receptor subtypes within
striatal neurons appears to be required for dopamine inhibition of striatal Na+/K+-ATPase
(Bertorello, et al., 1990). From a molecular standpoint, evidence exists
suggesting that when colocalized, presynaptic D2 receptors modulate dopamine
transporter function in striatum (Meiergerd, et al., 1993) and in nucleus
accumbens (Parsons, et al., 1993). Further, functional coupling of striatal D1
and D2 receptors has recently been demonstrated such that dopamine occupancy
of D1 receptors alters antagonist binding to D2 receptors (Seeman, et al.,
1994). Such D1-D2 receptor links appear to be functionally relevant as they
appear to be altered in schizophrenia and Huntington disease (Seeman, et al.,
1989). Recent receptor and mRNA distribution studies suggest that D1 and D2
receptors are intraneuronally localized in only a subpopulation (~25-35%) of
striatal and medial prefrontal neurons (Vincent, et al., 1995; Lester, et al.,
1993; Meador-Woodruff, et al., 1991). However, characterization of single
striatal neurons suggests that D1-D2 receptor co-localization may occur in a
much higher proportion (up to 70 - 80%) in certain neuronal types (Surmier, et
al., 1993). Similarly, messenger RNAs for the dopamine transporter and for D2
receptors are coexpressed in substantia nigra pars compacta cells (Hurd, et
al., 1994). Although the present data do not allow us to determine the degree
of intraneuronal colocalization of dopamine receptors and the dopamine
transporter, they suggest that the caudate nucleus, putamen, and the nucleus
accumbens/olfactory tubercle should be closely examined for such interactions
and their relevance to the effects of cocaine.
In summary, [3H]YM 09151-2 selectively labels dopamine D2-like
receptors in squirrel monkey caudate nucleus and putamen. However, significant
amounts of sulpiride-insensitive [3H]YM 09151-2 recognition sites
were detected in extrastriatal brain regions, suggesting that appropriate
masking drugs are needed to ensure accurate labeling of dopamine D2-like
receptors. The presence of [3H]YM 09151-2 binding to non-dopamine
receptors in brain regions proximate to the caudate nucleus and putamen along
with the apparent inhibition of striatal [3H]YM 09151-2 binding by
endogenous substances may limit the utility of YM 09151-2 as an imaging probe
for striatal D2-like dopamine receptors. The neuroanatomical distribution of
sulpiride-sensitive [3H]YM 09151-2 binding and [3H]SCH
23390 binding in the presence of mianserin in squirrel monkey brain closely
paralleled distribution patterns reported in other primate species and in
rodents. A close anatomical correspondence between relative densities of [3H]YM
09151-2 labeled D2 receptors, [3H]SCH 23390 labeled D1 receptors,
and of dopamine transporters in caudate nucleus and putamen and nucleus
accumbens/olfactory tubercle supports the recent findings that these dopamine
receptors mediate the behavioral effects of cocaine (Spealman, et al., 1991)
following its initial action of blocking the dopamine transporter (Giros, et
al., 1996).
ACKNOWLEDGMENTS
This research was supported by USPHS grants DA 06303 and MH 14275.
Facilities and services were provided by the New England Regional Primate
Research Center (USPHS Division of Research Resources Grant RR00168.
REFERENCES
Assié, M.-B., Sleight, A. J. and Koek, W. (1993) Biphasic displacement of
[3H]YM-09151-2 binding in the rat brain by thioridazine,
risperidone and clozapine, but not by other antipsychotics. Eur. J. Pharmacol.,
237:183-189.
Bertorello, A. M., Hopfield, J. F., Aperia, A. and Greengard, P. (1990)
Inhibition by dopamine of (Na++K+)ATPase activity in
neostriatal neurons through D1 and D2 dopamine receptor synergism. Nature,
347:386-388.
Bockaert, J., Fagni, L., Sebben, M. and Dumuis, A. (1991) Pharmacological
characterization of brain 5-HT4 receptors: Relationship between the effects of
indole, benzamide and azabicycloalkylbenzimidazolone derivatives. In:
Serotonin: Molecular Biology, Receptors and Functional Effects., J. R. Fozard
and P. R. Saxena, eds., Birkhauser Verlag, Basel, pp. 220-231.
Bockaert, J., Sebben, M. and Dumuis, A. (1990) Pharmacological
characterization of 5-hydroxytryptamine4 (5-HT4) receptors positively coupled
to adenylate cyclase in adult guinea pig hippocampal membranes: effects of
substituted benzamide derivatives. Mol. Pharmacol., 37:408-411.
Bonhomme, N., De Deurwaerdere, P., Le Moal, M. and Spaminato, U. (1995)
Evidence for 5-HT4 receptor subtype involvement in the enhancement of striatal
dopamine release induced by serotonin: a microdialysis study in the
halothane-anesthetized rat. Neuropharmacology, 34:269-279.
Calabresi, P., Maj, R., Mercuri, N. B. and Bernardi, G. (1992) Coactivation
of D1 and D2 dopamine receptors is required for long-term synaptic depression
in the striatum. Neuroscience Lett., 142:95-99.
Camps, M., Cortés, R., Gueye, B., Probst, A. and Palacios, J. M. (1989)
Dopamine receptors in human brain: Autoradiographic distribution of D2 sites.
Neuroscience, 28:275-290.
Canfield, D. R., Spealman, R. D., Kaufman, M. J. and Madras, B. K. (1990)
Autoradiographic localization of cocaine binding sites by [3H]CFT
([3H]WIN 35,428) in the monkey brain. Synapse, 6:189-195.
Chabert, C., Cavegn, C., Bernard, A. and Mills, A. (1994) Characterization
of the functional activity of dopamine ligands at human recombinant dopamine
D4 receptors. J. Neurochem., 63:62-65.
Chaput, Y., Araneda, R. C. and Andrade, R. (1990) Pharmacological and
functional analysis of a novel serotonin receptor in the rat hippocampus. Eur.
J. Pharmacol., 182:441-456.
Chipkin, R. E., Iorio, L. C., Coffin, V. L., McQuade, R. D., Berger, J. G.
and Barnett, A. (1988) Pharmacological profile of SCH39166: A dopamine D1
selective benzonaphthazepine with potential antipsychotic activity. J.
Pharmacol. Exper. Therap, 247:1093-1102.
Cortés, R., Gueye, B., Pazos, A., Probst, A. and Palacios, J. M. (1989)
Dopamine receptors in human brain: Autoradiographic distribution of D1 sites.
Neuroscience, 28:263-273.
Cox, R. F. and Waszczak, B. L. (1991) Autoradiography of dopamine D2
receptors using [3H]YM-09151-2. Eur. J. Pharmacol., 199:103-106.
DeKeyser, J., Claeys, A., De Backer, J.-P., Ebinger, G., Roels, F. and
Vauquelin, G. (1988) Autoradiographic localization of D1 and D2 dopamine
receptors in the human brain. Neurosci. Lett., 91:142-147.
Domenech, T., Beleta, J., Fernandez, A. G., Gristwood, R. W., Sanchez, F.
C., Tolosa, E. and Palacios, J. M. (1994) Identification and characterization
of serotonin 5-HT4 receptor binding sites in human brain: comparison with
other mammalian species. Mol. Brain Res., 21:176-180.
El Mestikawy, S., Cognard, C., Gozlan, H. and Hamon, M. (1988)
Pharmacological and biochemical characterization of rat hippocampal
5-hydroxytryptamine1A receptors solubilized by 3-[3- (cholamidopropyl)dimethylammonio]-1-propane
sulfonate (CHAPS). J. Neurochem, 51:1031-1040.
Emmers, R. and Akert, K.: A stereotaxic atlas of the brain of the squirrel
monkey (Saimiri sciureus). Univ. of Wisconsin Press, Madison, 1963.
Gergen, J. A. and MacLean, P. D.: A stereotaxic atlas of the squirrel
monkey's brain (Saimiri sciureus). Public Health Service Publication,
Bethesda, MD., 1962.
Giros, B., Jaber, M., Jones, S. R., Wightman, R. M. and Caron, M. G. (1996)
Hyperlocomotion and indifference to cocaine and amphetamine in mice lacking
the dopamine transporter. Nature, 379:606-612.
Glennon, R. A., Naiman, N. A., Pierson, M. E., Titeler, M., Lyon, R. A. and
Weisberg, E. (1988) NAN-190: an arylpiperazine analog that antagonizes the
stimulus effects of the 5-HT1A agonist 8-hydroxy-2(di-n- propylamino)tetralin
(8-OH-DPAT). Eur. J. Pharmacol., 154:339-341.
Grossman, C. J., Kilpatrick, G. J. and Bunce, K. T. (1993) Development of a
radioligand binding assay for 5- HT4 receptors in guinea-pig and rat brain.
Br. J. Pharmacol., 109:618-624.
Hatano, K., Ishiwata, K., Kawashime, K., Hatezawa, J., Itoh, M. and Ido, T.
(1989) D2-dopamine receptor specific brain uptake of carbon-11-labeled
YM-09151-2. J. Nucl. Med., 30:515-522.
Hatezawa, J., Hatano, K., Ishiwata, K., Itoh, M., Ido, T., Kawashima, K.,
Meguro, K., Watanuki, S. and Seo, S. (1991) Measurement of D2 dopamine
receptor-specific carbon-11-YM-09151-2 binding in the canine brain by PET:
Importance of partial volume correction. J. Nucl. Med., 32:713-718.
Hidaka, K., Tada, S., Matsumoto, M., Ohmori, J., Tasaki, Y., Nomura, T.,
Usuda, S. and Yamaguchi, T. (1996) In vitro pharmacological profile of
YM-43611, a novel D2-like receptor antagonist with high affinity and
selectivity for dopamine D3 and D4 receptors. Br. J. Pharmacol.,
117:1625-1632.
Hoyer, D., Clarke, D. E., Fozard, J. R., Hartig, P. R., Martin, G.,
Mylecharane, E. J., Saxena, P. R. and Humphrey, P. A. (1994) VII.
International union of pharmacology classification of receptors for 5-
hydroxytryptamine (serotonin). Pharmacol. Rev., 46:157-203.
Hoyer, D., Pazos, A., Probst, A. and Palacios, J. M. (1986) Serotonin
receptors in the human brain. I. Characterization and autoradiographic
localization of 5-HT1A recognition sites. Apparent absence of 5-HT1B
recognition sites. Brain Res., 376:85-96.
Hu, X.-T., Wachtel, S. R., Galloway, M. P. and White, F. J. (1990) Lesions
of the nigrostriatal dopamine projection increase the inhibitory effects of D1
and D2 dopamine agonists on caudate-putamen neurons and relieve D2 receptors
from the necessity of D1 receptor stimulation. J. Neurosci., 10:2318-2329.
Hurd, Y. L., Pristupa, Z. B., Herman, M. M., Niznik, H. B. and Kleinman, J.
E. (1994) The dopamine transporter and dopamine D2 receptor messenger RNAs are
differentially expressed in limbic- and motor- related subpopulations of human
mesencephalic neurons. Neuroscience, 63:357-362.
Ishiwata, K., Inami, K., Sasaki, T., Nozaki, T. and Senda, M. (1994)
Characerization of [3H]nemonapride binding to mouse brain dopamine
D2 receptors assessed in vivo and ex vivo for metabolic modeling in PET
studies. J. Neural. Transm. Gen. Sect., 97:119-133.
Jakeman, L. B., To, Z. P., Eglen, R. M., Wong, E. H. F. and Bonhaus, D. W.
(1994) Quantitative autoradiography of 5-HT4 receptors in brains of three
species using two structurally distinct radioligands, [3H]GR113808
and [3H]BIMU-1. Neuropharmacology, 33:1027-1038.
Kaufman, M. J. and Madras, B. K. (1993) [3H]CFT ([3H]WIN
35,428) accumulation in dopamine regions of monkey brain: Comparison of a
mature and an aged monkey. Brain Res, 611:322-325.
Kaufman, M. J., Spealman, R. D. and Madras, B. K. (1991) Distribution of
cocaine recognition sites in monkey brain: I. In vitro autoradiography with [3H]CFT.
Synapse, 9:177-187.
Kazawa, T., Mikuni, M., Higuchi, T., Arai, I., Takahashi, K. and Yamauchi,
T. (1990) Characterization of sulpiride-displaceable 3YM-09151-2 binding sites
in rat frontal cortex and the effects of subchronic treatment with haloperidol
on cortical D-2 dopamine receptors. Life Sci., 47:531-537.
LaHoste, G. J., Yu, J. and Marshall, J. F. (1993) Striatal Fos expression
is indicative of dopamine D1/D2 synergism and receptor supersensitivity. Proc.
Natl. Acad. Sci. U.S.A., 90:7451-7455.
Lahti, R. A., Roberts, R. C. and Tamminga, C. A. (1995) D2-family receptor
distribution in human postmortem tissue: an autoradiographic study.
NeuroReport, 6:2505-2512.
Lester, J., Fink, S., Aronin, N. and DiFiglia, M. (1993) Colocalization of
D1 and D2 dopamine receptor mRNAs in striatal neurons. Brain Res.,
621:106-110.
Levey, A. I., Hersch, S. M., Rye, D. B., Sunahara, R. K., Niznik, H. B.,
Kitt, C. A., Price, D. L., Maggio, R., Brann, M. R. and Ciliax, B. J. (1993)
Localization of D1 and D2 dopamine receptors in brain with subtype- specific
antibodies. Proc. Natl. Acad. Sci. U.S.A., 90:8861-8865.
Madras, B. K., Fahey, M. A., Canfield, D. R. and Spealman, R. D. (1988) D1
and D2 dopamine receptors in caudate-putamen of nonhuman primates (Macaca
fascicularis). J. Neurochem., 51:934-943.
Madras, B. K. and Kaufman, M. J. (1994) Cocaine accumulates in
dopamine-rich regions of primate brain after I.V. administration: Comparison
with mazindol distribution. Synapse, 18:261-275.
Madras, B. K., Spealman, R. D., Fahey, M. A., Neumeyer, J. L., Saha, J. K.
and Milius, R. A. (1989) Cocaine receptors labeled by [3H]2B-carbomethoxy-3B-(4-fluorophenyl)tropane.
Mol. Pharmacol., 36:518-524.
Meador-Woodruff, J. H., Mansour, A., Healy, D. J., Kuehn, R., Zhou, Q.-Y.,
Bunzow, J. R., Akil, H., Civelli, O. and Watson, S. (1991) Comparison of the
distribution of D1 and D2 dopamine receptor mRNAs in rat brain.
Neuropsychopharmacology, 5:231-242.
Meiergerd, S. M., Patterson, T. A. and Schenk, J. O. (1993) D2 receptors
may modulate the function of the striatal transporter for dopamine: Kinetic
evidence from studies in vitro and in vivo. J. Neurochem., 61:764- 767.
Nicklaus, K. J., McGonigle, P. and Molinoff, P. B. (1988) [3H]SCH
23390 labels both dopamine-1 and 5- hydroxytryptamine1c receptors in choroid
plexus. J. Pharmacol. Exper. Therap., 247:343-348.
Niznik, H. B., Grigoriadis, D. E., Pri-Bar, I., Buchman, O. and Seeman, P.
(1985) Dopamine D2 receptors selectively labeled by a benzamide neuroleptic: [3H]YM-09151-2.
Naunyn-Schmiedeberg's Arch. Pharmacol., 329:333-343.
Nobrega, J. N. and Seeman, P. (1994) Dopamine D2 receptors mapped in rat
brain with [3H](+)PHNO. Synapse, 17:167-172.
Parsons, L. H., Schad, C. A. and Justice, J. B. J. (1993) Co-administration
of the D2 antagonist pimozide inhibits up-regulation of dopamine release and
uptake induced by repeated cocaine. J. Neurochem., 60:376- 379.
Patel, S., Roberts, J., Moorman, J. and Reavill, C. (1995) Localization of
serotonin-4 receptors in the striatonigral pathway in rat brain. Neuroscience,
69:1159-1167.
Pazos, A., Probst, A. and Palacios, J. M. (1987) Serotonin receptors in the
human brain-III. Autoradiographic mapping of serotonin-1 receptors.
Neuroscience, 21:97-122.
Retaux, S., Besson, M. J. and Penit-Soria, J. (1991) Synergism between D1
and D2 dopamine receptors in the inhibition of the evoked release of [3H]GABA
in the rat prefrontal cortex. Neuroscience, 43:323-329.
Reynolds, G. P., Mason, S. L., Meldrum, A., De Keczer, S., Parnes, H.,
Eglen, R. M. and Wong, E. H. F. (1995) 5-Hydroxytryptamine (5-HT)4 receptors
in post mortem human brain tissue: distribution, pharmacology and effects of
neurodegenerative diseases. Br. J. Pharmacol., 114:993-998.
Richfield, E. K., Young, A. B. and Penney, J. B. (1987) Comparative
distribution of dopamine D-1 and D-2 receptors in the basal ganglia of
turtles, pigeons, rats, cats and monkeys. J. Comp. Neurol., 262:446-463.
Saji, H., Tanahashi, K., Kinoshita, T., Iida, Y., Magata, Y. and Yokoyama,
A. (1996) Synthesis, in vitro binding profile and biodistribution of a
125I-labeled N-benzyl pyrrolidinyl benzamide derivative: A potential
radioligand for mapping dopamine D2 receptors. Nucl. Med. and Biol.,
23:121-127.
Schoffelmeer, A. N. M., Hogenboom, F., Mulder, A. H., Ronken, E., Stoof, J.
C. and Drukarch, B. (1994) Dopamine displays an identical apparent affinity
towards functional dopamine D1 and D2 receptors in rat striatal slices:
Possible implications for the regulatory role of D2 receptors. Synapse,
17:190-195.
Schotte, A., Janssen, P. F. M., Gommeren, W., Luyten, W. H. L. M. and
Leysen, J. E. (1992) Autoradiographic evidence for the occlusion of rat brain
dopamine D3 receptors in vivo . Eur. J. Pharmacol., 218:373-375.
Scibilia, R. J., Lachowicz, J. E. and Kilts, C. D. (1992) Topographic
nonoverlapping distribution of D1 and D2 dopamine receptors in the amygdaloid
nuclear complex of the rat brain. Synapse, 11:146-154.
Seeman, P., Guan, H. C., Civelli, O., Van Tol, H. H. M., Sunahara, R. K.
and Niznik, H. B. (1992) The cloned dopamine D2 receptor reveals different
densities for dopamine receptor antagonist ligands. Implications for human
brain positron emission tomography. Eur. J. Pharmacol.-Mol. Pharmacol. Sect.,
227:139-146.
Seeman, P., Niznik, H. B., Guan, H.-C., Booth, G. and Ulpian, C. (1989)
Link between D1 and D2 dopamine receptors is reduced in schizophrenia and
Huntington diseased brain. Proc. Natl. Acad. Sci. U.S.A., 86:10156-10160.
Seeman, P., Sunahara, R. K. and Niznik, H. B. (1994) Receptor-receptor link
in membranes revealed by ligand competition: Example for dopamine D1 and D2
receptors. Synapse, 17:62-64.
Seeman, P., Ulpian, C., Larsen, R. D. and Anderson, P. S. (1993) Dopamine
receptors labelled by PHNO. Synapse, 14:254-262.
Seeman, P. and Van Tol, H. H. M. (1994) Dopamine receptor pharmacology.
Trends Pharmacol. Sci., 15:264-270.
Shoup, T., M,, Brownell, A.-L., Fischman, A. J., Elmaleh, D. R. and Madras,
B. K. (1994) Selective dopamine D-2 receptor imaging of monkey brain using
[11C]methyl-YM-09151-2 and PET. J. Nucl. Med., 35:121.
Spealman, R. D., Bergman, J., Madras, B. K. and Melia, K. F. (1991)
Discriminative stimulus effects of cocaine in squirrel monkeys: Involvement of
dopamine receptor subtypes. J. Pharmacol. Exper. Therap., 258:945-953.
Steward, L. J., Ge, J., Stowe, R. L., Brown, D. C., Bruton, R. K., Stokes,
P. R. A. and Barnes, N. M. (1996) Ability of 5-HT4 receptor ligands to
modulate rat striatal dopamine release in vitro and in vivo. Br. J. Pharmacol.,
117:55-62.
Sundram, H., Newman-Tancredi, A. and Strange, P. G. (1993) Characterization
of recombinant human serotonin 5-HT1A receptors expressed in chinese hamster
ovary cells. Biochem. Pharmacol., 45:1003-1009.
Surmier, D. J., Reiner, A., Levine, M. S. and Ariano, M. A. (1993) Are
neostriatal dopamine receptors co- localized? Trends Neurosci., 16:299-305.
Terai, M., Hidaka, K. and Nakamura, Y. (1989) Comparison of [3H]YM-09151-2
with [3H]spiperone and [3H]raclopride for dopamine D-2
receptor binding to rat striatum. Eur. J. Pharmacol., 173:177-182.
Tomiyama, K., Noguchi, M., Koshikawa, N. and Kobayashi, M. (1993)
YM-09151-2 but not l-sulpiride induces transient dopamine release in rat
striatum via a tetrodotoxin-insensitive mechanism. J. Neurochem.,
60:1690-1695.
Unis, A. S., Vincent, J. G. and Dillon, B. (1990) Brain receptor
autoradiography with [3H]YM-09151-2: A ligand for labeling dopamine
D-2 receptors. Life Sci., 47:PL151-PL155.
Vincent, S. L., Khan, Y. and Benes, F. M. (1995) Cellular colocalization of
dopamine D1 and D2 receptors in rat medial prefrontal cortex. Synapse,
19:112-120.
Waeber, C., Sebben, M., Grossman, C., Javoy-Agid, F., Bockaert, J. and
Dumuis, A. (1993) [3H]-GR113808 labels 5-HT4 receptors in the human
and guinea-pig brain. NeuroReport, 4:1239-1242.
Wardle, K. A., Ellis, E. S., Gaster, L. M., King, F. D. and Sanger, G. J.
(1993) SB 204070: A highly potent and selective 5-HT4 receptor antagonist. Br.
J. Pharmacol. Proc. Suppl., 110:15P.
Yokoyama, C., Okamura, H. and Ibata, Y. (1995) Dopamine D2-like receptors
labeled by [3H]YM-09151-2 in the rat hippocampus: characterization
and autoradiographic distribution. Brain Res., 681:153-159.
Yokoyama, C., Okamura, H., Nakajima, T., Taguchi, J.-I. and Ibata, Y.
(1994) Autoradiographic distribution of [3H]YM-09151-2, a
high-affinity and selective antagonist ligand for the dopamine D2 receptor
group, in the rat brain and spinal cord. J. Comp. Neurol., 344:121-136.
;
Specific Binding, 
;
Nonspecific binding, 
;
A) The concentration of [3H]YM 09151-2 ranged from 0.008 to
0.976 nM. Nonspecific binding was determined with 30