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<tr><td valign="top" align=left>© Neuroscience-Net<br> Volume 1, Article #10006
<td width=500 align=right>Received June 5, 1996<br>
Accepted for Publication August 7, 1996<br>
Published August 21, 1996<br>
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<center>
<font size=+1><b>Neuroanatomical Distribution of [<sup>3</sup>H]YM 09151-2 Binding in Monkey Brain: Comparison with Dopamine Transporter and Dopamine D1 Receptor Distribution</b></font><p>
</center>
Running Title: [<sup>3</sup>H]YM 09151-2 Distribution in Primate Brain<P>
Marc J. Kaufman<sup>1</sup> and Bertha K. Madras, Department of Psychiatry, Harvard Medical School, and Division of Behavioral Biology, New
England Regional Primate Research Center, One Pine Hill Drive, Southborough, MA 01772-9102
<P>
Send correspondence to:<br>
Bertha K. Madras, Ph.D.<BR>
Harvard Medical School<BR>
New England Regional Primate Research Center<BR>
One Pine Hill Drive<BR>Box 9102<BR>
Southborough, MA 01772-9102<BR>
(508)-624-8073<BR>
FAX (508)-624-8190<BR>
E-mail: <A HREF="mailto:bmadras@warren.med.harvard.edu">
bmadras@warren.med.harvard.edu</A><P>
<sup>1</sup>Current Address:<br>
Brain Imaging Center<BR>
McLean Hospital<Br>
115 Mill St.<br>
Belmont, MA 02178<br>
E-mail: <a href="mailto:kaufman@mclean.org">
kaufman@mclean.org</a><P>
<ul><ul>
<b>Key Words</b>: [<sup>3</sup>H]YM 09151-2, dopamine receptors, serotonin receptors, dopamine transporter, [<sup>3</sup>H]WIN 35,428, [<sup>3</sup>H]SCH 23390, cocaine, primate, autoradiography<p>
</ul></ul>
<hr>
<ul>
<li><a href="index.html#abstract" TARGET="_parent">Abstract</a><br>
<li><a href="#intro">Introduction</a><br>
<li><a href="#materials">Materials and Methods</a><br>
<li><a href="#results">Results</a><br>
<li><a href="#discuss">Discussion</a><br>
<li><a href="#acknow">Acknowledgments</a><br>
<li><a href="#references">References</a><br>
<li><a href="#legends">Legends</a><br>
</ul>
<hr>
<a name="intro">
<font size=+2><b>INTRODUCTION</b></font><p>
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).<P>
The primary goal of the present study was to examine the distribution and pharmacological
specificity of [<sup>3</sup>H]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 [<sup>3</sup>H]WIN 35,428 ([<sup>3</sup>H]CFT) (Kaufman and Madras, 1993;
Kaufman, et al., 1991; Canfield, et al., 1990; Madras, et al., 1989) was compared to [<sup>3</sup>H]YM
09151-2 and to dopamine D1 receptors labeled with [<sup>3</sup>H]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 [<sup>3</sup>H]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. [<sup>3</sup>H]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 [<sup>3</sup>H]YM 09151-2 also may bind to serotonin 5- HT1A and/or 5-HT4 receptors.<P>
<a name="materials"><hr>
<font size=+2><b>MATERIALS AND METHODS</b></font><P>
<b>Tissue Preparation:</b><BR>
Squirrel monkey (Saimiri sciureus) brains stored at -85<sup>o</sup>C were obtained from the brain bank of
the New England Regional Primate Research Center. Brains were sectioned into 1 cm slabs and
stored at -85<sup>o</sup>C until thin sectioning. Prior to thin sectioning, tissue slabs were placed into a
Reichert-Jung cryostat to equilibrate with the cutting temperature of -18<sup>o</sup>C. Thin sections (20
<sub><img src="../images/micro.gif"></sub>M) were cut and thaw-mounted onto gelatin-coated "subbed" slides, air dried and stored in
sealed containers at -20<sup>o</sup>C until use.<P>
<b>Parametric Studies:</b><BR>
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 MgCl<sub>2</sub>,
pH 7.4 at 23<sup>o</sup>C) and postincubation washes were performed at 4<sup>o</sup>C in wash buffer (50 mM Tris
HCl, pH 7.4 at 4<sup>o</sup>C). 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 [<sup>3</sup>H]SCH 23390 and nonspecific binding
was determined with 1 <sub><img src="../images/micro.gif"></sub>M (S)-butaclamol. For D2 receptor parametrics, total binding was
determined with 0.2 nM [<sup>3</sup>H]YM 09151-2 and nonspecific binding was determined with 5 <sub><img src="../images/micro.gif"></sub>M
(S)-sulpiride. [<sup>3</sup>H]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 4<sup>o</sup>C) for 20 minutes, followed by
incubation in buffer containing 5 nM [<sup>3</sup>H]WIN 35,428 alone or in the presence of 30 <sub><img src="../images/micro.gif"></sub>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.<p>
<b>Pharmacological Specificity:</b><BR>
The pharmacological specificity of [<sup>3</sup>H]YM 09151-2 and [<sup>3</sup>H]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 ([<sup>3</sup>H]SCH 23390 or [<sup>3</sup>H]YM 09151-2, respectively) at 23<sup>o</sup>C. Sections then were
rinsed in cold buffer for 20 minutes at 4<sup>o</sup>C, 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 <sub><img src="../images/micro.gif"></sub>M (S)-butaclamol and by 30 <sub><img src="../images/micro.gif"></sub>M (S)-sulpiride in [<sup>3</sup>H]SCH
23390 (0.5 nM) or [<sup>3</sup>H]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
MgCl<sub>2</sub>, 1 mM EDTA, and 0.01% ascorbic acid (pH 7.4 at 23<sup>o</sup>C). IC<sub>50</sub> values, pseudo-Hill
coefficients (nH) and apparent K<sub>d</sub> or K<sub>i</sub> 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 [<sup>3</sup>H]YM 09151-2 binding sites. In those studies, tissue sections were
preincubated in buffer for 20 minutes at room temperature, incubated with [<sup>3</sup>H]YM 09151-2 (0.2
nM, final concentration) in the presence of 30 <sub><img src="../images/micro.gif"></sub>M (S)-sulpiride (to mask D2 receptor binding)
and competing drugs. The pharmacological specificity of [<sup>3</sup>H]WIN 35,428 binding to tissue
sections has been reported previously (Kaufman, et al., 1991; Canfield, et al., 1990).<P>
<b>Autoradiography:</b><BR>
Slides-mounted tissue sections in duplicate and calibrated standards ([<sup>3</sup>H]Microscales,
Amersham) were placed in X-ray cassettes, covered with [<sup>3</sup>H]Ultrofilm (Cambridge Instruments)
and the cassettes were sealed along with desiccant capsules for 5-week exposures at 4<sup>o</sup>C.
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 20<sup>o</sup>C.
Quantitative analysis of autoradiograms:<P>
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 [<sup>3</sup>H]-exposed films to tissue equivalent
binding densities of [<sup>3</sup>H]YM 09151-2, [<sup>3</sup>H]SCH 23390 and [<sup>3</sup>H]WIN 35,428.<P>
<b>Drugs:</b><BR>
[<sup>3</sup>H]YM 09151-2, (84-86 Ci/mmol), [<sup>3</sup>H]SCH 23390 (71.3 Ci/mmol) and [<sup>3</sup>H]CFT ([<sup>3</sup>H]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.<P>
<a name="results"><HR>
<font size=+2><b>RESULTS</b></font><P>
<b>Parametric Studies:</b><BR>
Parametric studies were conducted in caudate-putamen tissue sections to determine optimal
conditions for performing autoradiography with [<sup>3</sup>H]SCH 23390 and [<sup>3</sup>H]YM 09151-2. The
effect of tissue slice preincubation was studied initially to determine whether preincubation
enhanced radioligand binding as was observed with [<sup>3</sup>H]WIN 35,428 (Canfield, et al., 1990).
Although preincubation of tissue sections was without effect on [<sup>3</sup>H]SCH 23390 binding, a 20
minute preincubation increased [<sup>3</sup>H]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. [<sup>3</sup>H]SCH23390 and [<sup>3</sup>H]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).<P>
<a href="ph06f01l.html"><img src="../images/ph06f01s.gif" border=0> Figure 1: Click here to view larger image.</a><P>
<a name="fig2"></a>
<b>Pharmacological Specificity of [<sup>3</sup>H]YM 09151-2 and [<sup>3</sup>H]SCH 23390:</b><BR>
Radioligand affinities (K<sub>d</sub> values) were determined by scatchard transformation of saturation
binding data from studies conducted in caudate-putamen tissue sections. The K<sub>d</sub> and pseudo-Hill
(nH) values for [<sup>3</sup>H]YM 09151-2 binding were 0.21 ± 0.02 nM and 0.86 ± 0.01 (means ± S.D.,
n=2), respectively (Figure 2A, <a href="phrtbl1.html" Target=_top">Table 1</a>). The K<sub>d</sub> and nH values for [<sup>3</sup>H]SCH 23390 binding were
0.81 ± 0.20 nM and 0.99 ± 0.01 (means ± S.D., n=2), respectively (Figure 2B, <a href="phrtbl1.html Target=_top">Table 1</a>).<P>
<a href="ph06f02l.html"><img src="../images/ph06f02s.gif" border=0> Figure 2: Click here to view larger image.</a><P>
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
[<sup>3</sup>H]SCH 23390 (0.5 nM) and [<sup>3</sup>H]YM 09151-2 (0.2 nM) binding (Figure 3, <a href="phrtbl1.html Target=_top">Table 1</a>). For
[<sup>3</sup>H]SCH 23390 labeled sites, the rank order of inhibitory potencies was: SCH 39166 >
(S)-butaclamol > dopamine > mianserin >> (R)-butaclamol (Figure 3A). Stereoselectivity of the
[<sup>3</sup>H]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
[<sup>3</sup>H]SCH 23390 binding was biphasic with an nH of 0.52 ± 0.04. A highly significant correlation
(p < 0.002) between K<sub>d</sub> (or K<sub>i</sub>) values for [<sup>3</sup>H]SCH 23390 binding to squirrel monkey
caudate-putamen tissue sections and [<sup>3</sup>H]SCH 23390 binding to D1 receptors in striatal
homogenates was observed (Figure 3C).<a name="fig3"></a> Mianserin (100 nM) was included in the incubation
buffer in all subsequent autoradiographic experiments with [<sup>3</sup>H]SCH 23390 to block serotonin
receptor binding (Nicklaus, et al., 1988). For [<sup>3</sup>H]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 [<sup>3</sup>H]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 [<sup>3</sup>H]YM 09151-2 binding was biphasic with
an nH of 0.63 ± 0.01. A highly significant correlation (p = 0.002) between K<sub>d</sub> (or K<sub>i</sub>) values for
[<sup>3</sup>H]YM 09151-2 binding to squirrel monkey caudate-putamen tissue sections and [<sup>3</sup>H]YM
09151-2 binding to D2 receptors in striatal homogenates was observed (Figure 3D).
Autoradiography with [<sup>3</sup>H]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).<P>
<a href="ph06f03l.html"><img src="../images/ph06f03s.gif" border=0> Figure 3: Click here to view larger image.</a><P>
<a name="table2"></a>
<b>Neuroanatomical Distribution of [<sup>3</sup>H]YM 09151-2 binding:</b><BR>
The highest binding densities of [<sup>3</sup>H]YM 09151-2 (0.2 nM) were found in the caudate nucleus
and putamen. Specific binding, defined as binding blocked by (S)-sulpiride (30 <sub><img src="../images/micro.gif"></sub>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; [<sup>3</sup>H]YM 09151-2 binding was higher in medial compared with lateral caudate
nucleus and the reverse pattern was observed in putamen. Moderate levels of [<sup>3</sup>H]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 <a href="phrtbl2.html" target="_top">Table 2</a>). Sulpiride inhibited 70 - 90% of [<sup>3</sup>H]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 <a href="phrtbl2.html" target="_top">Table 2</a>).<P>
<a name="fig4"></a>
Additional pharmacological studies were conducted to further characterize the nature of
sulpiride- insensitive [<sup>3</sup>H]YM 09151-2 binding in tissue sections of hippocampus and amygdala.
Sulpiride (30 <sub><img src="../images/micro.gif"></sub>M) was used to mask [<sup>3</sup>H]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 [<sup>3</sup>H]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 [<sup>3</sup>H]YM 09151-2 binding by less than 5%. NAN 190
reduced sulpiride-insensitive [<sup>3</sup>H]YM 09151-2 binding to 81 ± 10% of control levels (mean ±
S.D., n = 2). Finally, a high concentration of (S)-butaclamol (10 <sub><img src="../images/micro.gif"></sub>M) reduced
sulpiride-insensitive [<sup>3</sup>H]YM 09151-2 binding to 79 ± 8% of control levels (mean ± S.D., n = 2).<P>
<a href="ph06f04l.html"><img src="../images/ph06f05s.jpg" border=0>
<img src="../images/ph06f06s.jpg" border=0> <BR>
<img src="../images/ph06f07s.jpg" border=0>
<img src="../images/ph06f08s.jpg" border=0> Figure 4: Click here to view larger images.</a><P>
<b>Distribution of [<sup>3</sup>H]SCH 23390 binding:</b><BR>
The highest levels of D1 receptors labeled by [<sup>3</sup>H]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 <sub><img src="../images/micro.gif"></sub>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 [<sup>3</sup>H]YM 09151-2 and
[<sup>3</sup>H]WIN 35,428, [<sup>3</sup>H]SCH 23390 distribution in these structures appeared to be more uniform.
High levels of [<sup>3</sup>H]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 [<sup>3</sup>H]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. [<sup>3</sup>H]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 <a href="phrtbl2.html">Table 2</a>). We did not detect any brain regions that contained
(S)-butaclamol-insensitive [<sup>3</sup>H]SCH 23390 binding that exceeded white matter binding levels.<P>
<b>Distribution of [<sup>3</sup>H]WIN 35,428 binding:</b><br>
The neuroanatomical distribution of [<sup>3</sup>H]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 <sub><img src="../images/micro.gif"></sub>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 [<sup>3</sup>H]YM 09151-2 were detected with [<sup>3</sup>H]WIN
35,428. High levels of [<sup>3</sup>H]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
[<sup>3</sup>H]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. [<sup>3</sup>H]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 <a href="phrtbl2.html">Table 2</a>).<P>
<a name="discuss"><HR>
<font size=+2><b>DISCUSSION</b></font><P>
<b>[<sup>3</sup>H]YM 09151-2 pharmacological specificity and distribution:</b><BR>
In tissue sections of squirrel monkey caudate-putamen, the pharmacological specificity of
[<sup>3</sup>H]YM 09151-2 binding sites in was consistent with the dopamine D2-like receptor. The K<sub>d</sub> for
[<sup>3</sup>H]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 K<sub>d</sub> 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.<P>
The apparent densities of [<sup>3</sup>H]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 [<sup>3</sup>H]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 [<sup>3</sup>H]YM 09151-2, which typically detects higher striatal
D2 receptor densities than [<sup>3</sup>H]spiperone (Seeman, et al., 1993; Seeman, et al., 1992; Terai, et
al., 1989; Niznik, et al., 1985). This feature of [<sup>3</sup>H]YM 09151-2 has been attributed to its
labeling of D2 receptor monomers while [<sup>3</sup>H]spiperone labels dimers (Seeman and Van Tol,
1994; Seeman, et al., 1992). In addition, a 20 minute preincubation was found to increase
[<sup>3</sup>H]YM 09151-2 binding densities by nearly 70% in caudate-putamen tissue sections.
Preincubation of tissue slices has been shown to significantly enhance [<sup>3</sup>H](+)PHNO binding to
caudate-putamen D2 receptors and [<sup>125</sup>I]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 [<sup>3</sup>H]WIN 35,428 binding
(Canfield, et al., 1990), also may inhibit in vitro (and in vivo) YM 09151-2 binding. <P>
The overall brain distribution of sulpiride-sensitive [<sup>3</sup>H]YM 09151-2 binding was typical of the
reported distribution of dopamine D2 receptors. The regional relative densities of [<sup>3</sup>H]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 [<sup>3</sup>H]spiperone or [<sup>3</sup>H]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 [<sup>3</sup>H](+)PHNO or [<sup>3</sup>H]YM 09151-2 in rodent
brain (Nobrega and Seeman, 1994; Yokoyama, et al., 1994). These correlations suggest that
<a name="fig5"></a>[<sup>3</sup>H]YM 09151-2 should be a suitable probe for sulpiride-sensitive D2-like receptors. However,
we also detected substantial levels of sulpiride-insensitive [<sup>3</sup>H]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
[<sup>3</sup>H]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).<P>
Although [<sup>3</sup>H]YM 09151-2 binds with high affinity to dopamine D4 receptors and sulpiride is a
weak antagonist at the human dopamine D4 receptor (K<sub>i</sub> <IMG SRC="../images/grteql.gif"> 1 <sub><img src="../images/micro.gif"></sub>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 <sub><img src="../images/micro.gif"></sub>M) should have masked > 90% of brain D4 receptors. It therefore seems
unlikely that sulpiride-insensitive [<sup>3</sup>H]YM 09151-2 binding is D4 receptor-related.<P>
<a href="ph06f05l.html"><img src="../images/ph06f04s.gif" border=0> Figure 5: Click here to view larger image.</a><P>
<P>
Two recent reports assessing hippocampal [<sup>3</sup>H]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 [<sup>3</sup>H]YM 09151-2 binding reported in rodent
hippocampus (Yokoyama, et al., 1995), it is also possible that the use of (S)-butaclamol to define
[<sup>3</sup>H]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 [<sup>3</sup>H]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 [<sup>3</sup>H]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 [<sup>3</sup>H]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 (K<sub>i</sub> of 0.6 nM
for the receptor (Glennon, et al., 1988)) was used to determine whether sulpiride-insensitive
binding of [<sup>3</sup>H]YM 09151-2 in hippocampus was associated with the 5-HT1A receptor.
NAN-190 (6 nM) reduced sulpiride-insensitive [<sup>3</sup>H]YM 09151-2 binding by about 20%,
indicating that 5-HT1A receptors may represent a small proportion of [<sup>3</sup>H]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 [<sup>3</sup>H]YM 09151-2.<P>
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 [<sup>3</sup>H]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 [<sup>3</sup>H]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 [<sup>3</sup>H]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 [<sup>3</sup>H]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, (pK<sub>i</sub> > 1 <sub><img src="../images/micro.gif"></sub>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 [<sup>3</sup>H]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 [<sup>3</sup>H]YM 09151-2 binding is to 5-HT4
receptors or that another receptor is a target for both [<sup>3</sup>H]YM 09151-2 and relatively high
concentrations of 5-HT4 antagonists. Further work will be necessary to resolve the nature of
sulpiride-insensitive [<sup>3</sup>H]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.<P>
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 [<sup>3</sup>H]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 [<sup>3</sup>H]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.<P>
<b>[<sup>3</sup>H]SCH 23390 pharmacological specificity and distribution:</b><BR>
The pharmacological specificity of striatal [<sup>3</sup>H]SCH 23390 binding was consistent with labeling
of dopamine D1 receptors. The neuroanatomical distribution of 0.5 nM [<sup>3</sup>H]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.<P>
It is of interest to note that while both [<sup>3</sup>H]YM 09151-2 (present study) and [<sup>3</sup>H]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 [<sup>3</sup>H]SCH 23390 binding. This implies that [<sup>3</sup>H]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).<P>
<b>Colocalization of dopamine receptors and the dopamine transporter:</b><BR>
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 [<sup>3</sup>H]YM 09151-2 and [<sup>3</sup>H]WIN 35,428 binding. In contrast, [<sup>3</sup>H]SCH 23390
density was quite high but more uniform throughout these regions.<P>
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 [<sup>3</sup>H]YM 09151-2 and [<sup>3</sup>H]WIN 35,428 binding were
detected. The significance of medial prefrontal cortex colocalization of [<sup>3</sup>H]YM 09151-2 and
[<sup>3</sup>H]WIN 35,428 binding is unclear. Given the moderate level of sulpiride-insensitive [<sup>3</sup>H]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 [<sup>3</sup>H]SCH 23390
and [<sup>3</sup>H]YM 09151-2 binding appeared to be colocalized. However, a substantial proportion of
[<sup>3</sup>H]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).<P>
D1 and D2 receptors were anatomically segregated in a number of brain regions including the
globus pallidus, in which moderate levels of [<sup>3</sup>H]SCH 23390 binding sites were detected in the
internal segment while low to moderate [<sup>3</sup>H]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, [<sup>3</sup>H]YM 09151- 2 was colocalized with [<sup>3</sup>H]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.<P>
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<sup>+</sup>/K<sup>+</sup>-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.<P>
In summary, [<sup>3</sup>H]YM 09151-2 selectively labels dopamine D2-like receptors in squirrel monkey
caudate nucleus and putamen. However, significant amounts of sulpiride-insensitive [<sup>3</sup>H]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 [<sup>3</sup>H]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 [<sup>3</sup>H]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
[<sup>3</sup>H]YM 09151-2 binding and [<sup>3</sup>H]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 [<sup>3</sup>H]YM 09151-2
labeled D2 receptors, [<sup>3</sup>H]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).<P>
<a name="acknow"><HR>
<font size=+2><b>ACKNOWLEDGMENTS</B></font><P>
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.<P>
<a name="references"><hr>
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<a name="legends"><hr>
<font size=+2><b>FIGURE LEGENDS</b></font><P>
<a href="ph06f01l.html"><img src="../images/ph06f01s.gif" align=left border=0><b>Figure 1</b></a>: Parametric studies for optimizing conditions for receptor
autoradiography. A) Effects of preincubation on radioligand specific binding. Tissue sections
were preincubated in buffer for the indicated times, followed by 2 or 3 hour incubation in buffer
containing [<sup>3</sup>H]SCH 23390 (0.5 nM, 1 <sub><img src="../images/micro.gif"></sub>M (S)- butaclamol for nonspecific binding) or [<sup>3</sup>H]YM
09151-2 (0.2 nM, 10 <sub><img src="../images/micro.gif"></sub>M (S)-sulpiride for nonspecific binding), respectively. Tissue sections then
were washed for 10 minutes in cold buffer. Shown are means ± S.D. from duplicate
determinations in 2 individual brains. B) Association of specific [<sup>3</sup>H]SCH 23390 and [<sup>3</sup>H]YM
09151-2 binding as a function of incubation time. Tissue sections were preincubated in buffer for
20 minutes, incubated with radioligand for the indicated times (nonspecific binding determined as
above), and washed for 10 minutes in cold buffer. Shown are means ± S.D. from duplicate
determinations in 2 individual brains. (C - D) Dissociation of [<sup>3</sup>H]SCH 23390 binding (C) and
[<sup>3</sup>H]YM 09151-2 binding (D) as a function of wash time. Tissue sections were preincubated for
20 minutes, incubated with [<sup>3</sup>H]SCH 23390 or [<sup>3</sup>H]YM 09151-2 for 150 or 180 minutes,
respectively, and washed in cold buffer for the indicated times. Nonspecific binding was
determined as described above. Shown are means ± S.D. for duplicate determinations in 2
individual brains.<P>
<a href="ph06f02l.html"><img src="../images/ph06f02s.gif" align=left border=0> <b>Figure 2</b></a>: Scatchard transformation of saturation data for specific [<sup>3</sup>H]YM
09151-2 (panel A) and [<sup>3</sup>H]SCH 23390 (panel B) binding to slide-mounted tissue sections of
squirrel monkey caudate-putamen. Results from representative experiments conducted in
duplicate tissue sections at each condition are shown. Insets show saturation binding data for
each radioligand. Legend: Total binding, <img src="../images/radio1.gif" ALT="1 - 1">; Specific Binding, <img src="../images/radio2.gif" ALT="l - -l">; Nonspecific binding, <img src="../images/radio3.gif" ALT="o - o">;
A) The concentration of [<sup>3</sup>H]YM 09151-2 ranged from 0.008 to 0.976 nM. Nonspecific
binding was determined with 30 <sub><img src="../images/micro.gif"></sub>M (S)-sulpiride. B) The concentration of [<sup>3</sup>H]SCH 23390
ranged from 0.097 to 1.92 nM. Nonspecific binding was determined with 3 <sub><img src="../images/micro.gif"></sub>M (S)-butaclamol.<P>
<a href="ph06f03l.html"><img src="../images/ph06f03s.gif" align=left border=0>
<b>Figure 3</b></a>: Pharmacological specificity of radioligand binding to slide-mounted
caudate- putamen tissue sections. (A - B) Competition curves for inhibition of [<sup>3</sup>H]SCH 23390
(0.5 nM) binding (panel A) and for inhibition of [<sup>3</sup>H]YM 09151-2 (0.2 nM) binding (panel B).
Shown are averages of duplicate determinations in two brains. (C) Correlation between K<sub>i</sub> (or
K<sub>d</sub>) values for drug inhibition of [<sup>3</sup>H]SCH 23390 binding in monkey caudate-putamen tissue
sections compared with K<sub>i</sub> values at D1 receptors determined in monkey membrane homogenates.
(D) Correlation between K<sub>i</sub> (or K<sub>d</sub>) values for drug inhibition of [<sup>3</sup>H]YM 09151-2 binding in
monkey caudate-putamen tissue sections compared with K<sub>i</sub> values at D2 receptors in monkey
membrane homogenates. K<sub>i</sub> (or K<sub>d</sub>) values in tissue section studies were derived from
competition or saturation study data by the EBDA and LIGAND programs. The sources of K<sub>i</sub>
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.<P>
<table align=left><tr><td><a href="ph06f04l.html"><img src="../images/ph06f05s.jpg" border=0>
<img src="../images/ph06f06s.jpg" border=0></a><BR></td></tr>
<tr><td> <a href="ph06f04l.html"><img src="../images/ph06f07s.jpg" border=0> <img src="../images/ph06f08s.jpg" border=0></a></td></tr>
</table>
<a href="ph06f04l.html"><b>Figure 4</b></a>: Pseudocolor images of the neuroanatomical distribution of [<sup>3</sup>H]WIN
35,428, [<sup>3</sup>H]SCH 23390 and [<sup>3</sup>H]YM 09151-2 determined by in vitro receptor autoradiography.
Each series of 4 images displays a representative autoradiograph showing binding of [<sup>3</sup>H]WIN
35,428 (upper left), [<sup>3</sup>H]SCH 23390 (upper right), [<sup>3</sup>H]YM 09151-2 (lower left) and [<sup>3</sup>H]YM
09151-2 in the presence of 30 <sub><img src="../images/micro.gif"></sub>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 [<sup>3</sup>H]SCH 23390 and [<sup>3</sup>H]YM 09151-2 binding in MPFC, Cl and Ctx.
Moderate levels of sulpiride-insensitive [<sup>3</sup>H]YM 09151-2 binding are present in MPFC, Cl and
Ctx. (B) Sections from A-P level 11.5 showing moderate levels of [<sup>3</sup>H]SCH 23390 and [<sup>3</sup>H]YM
09151-2 binding in GP, VP and Amy, and sulpiride-insensitive [<sup>3</sup>H]YM 09151-2 binding in Amy.
(C) Sections from A-P level 10.5 showing moderate levels of [<sup>3</sup>H]SCH 23390 binding in GPi and
low - moderate levels of [<sup>3</sup>H]SCH 23390 and [<sup>3</sup>H]YM 09151-2 binding in GPe, hypo, Cl, and
Amy. Low to moderate levels of sulpiride-insensitive [<sup>3</sup>H]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
[<sup>3</sup>H]SCH 23390 and [<sup>3</sup>H]YM 09151-2 in Sn (pars reticulata and compacta), respectively,
moderate levels of [<sup>3</sup>H]SCH 23390 and [<sup>3</sup>H]YM 09151-2 in medial Hipp, and moderate to high
levels of [<sup>3</sup>H]YM 09151-2 in HippL. The majority of hippocampal [<sup>3</sup>H]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.<P>
<a href="ph06f05l.html"><img src="../images/ph06f04s.gif" align=left border=0>
<b>Figure 5</b></a>: Correlation between neuroanatomical distribution determined by
quantitative in vitro receptor autoradiography of sulpiride-sensitive [<sup>3</sup>H]YM 09151-2 binding in
squirrel monkey brain and D2 receptor binding (Top) and [<sup>3</sup>H]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.<P>
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