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THE JOURNAL OF COMPARATIVE NEUROLOGY 421:247–255 (2000)
Localization of Dopaminergic Markers in
the Human Subthalamic Nucleus
SARAH J. AUGOOD,1,2* ZANE R. HOLLINGSWORTH,2 DAVID G. STANDAERT,2
PIERS C. EMSON,1 AND JOHN B. PENNEY, JR.2
1
Department of Neurobiology, Babraham Institute, Cambridge CB2 4AT, United Kingdom
2
Neurology Research, Massachusetts General Hospital and Harvard Medical School,
Boston, Massachusetts 02114
ABSTRACT
The potential role for dopamine in the subthalamic nucleus was investigated in human
postmortem tissue sections by examining; (1) immunostaining for tyrosine hydroxylase, the
rate-limiting enzyme in catecholamine synthesis; (2) binding of [3H]-SCH23390 (D1-like),
[3H]-YM-09151-2 (D2-like), and [3H]-mazindol (dopamine uptake); and (3) expression of
dopamine D1 and D2 receptor mRNAs. Immunostaining for tyrosine hydroxylase was visualized in Bouin’s-fixed tissue by using a monoclonal antibody and the avidin-biotin-complex
method. The cellular localization of the dopamine D1 and D2 receptor mRNAs was visualized
by using a cocktail of human specific oligonucleotide probes radiolabeled with 35S-dATP.
Inspection of immunostained tissue revealed a fine network of tyrosine hydroxylaseimmunostained fibers traversing the nucleus; no immunopositive cells were detected. Examination of emulsion-coated tissue sections processed for D1 and D2 receptor mRNA revealed,
as expected, an abundance of D1 and D2 mRNA-positive cells in the caudate nucleus and
putamen. However, no D1 or D2 receptor mRNA-expressing cells were detected in the
subthalamic nucleus. Further, semiquantitative analysis of D1-like, D2-like and dopamine
uptake ligand binding similarly revealed an enrichment of specific binding in the caudate
nucleus and putamen but not within the subthalamic nucleus. However, a weak, albeit
specific, signal for [3H]-SCH23390 and [3H]-mazindol was detected in the subthalamic nucleus, suggesting that the human subthalamic nucleus may receive a weak dopaminergic
input. As weak D1-like binding is detected in the subthalamic nucleus, and subthalamic
neurons do not express dopamine D1 or D2 receptor mRNAs, together these data suggest that
the effects of dopaminergic agents on the activity of human subthalamic neurons may be
indirect and mediated via interaction with dopamine D1-like receptors. J. Comp. Neurol. 421:
247–255, 2000. © 2000 Wiley-Liss, Inc.
Indexing terms: basal ganglia; Parkinson’s disease; brain; dopamine receptor
The role of dopamine (DA) within the subthalamic nucleus (STN) has received considerable interest in recent
years (see Chesselet and Delfs, 1996; Joel and Weiner,
1997, for review), as numerous studies have suggested
that DA and DAergic agents may have a direct effect
within this elliptical structure. One of the early pioneering
studies suggestive of a role for DA in the rat STN was that
of Brown and Wolfson (1978) who reported a marked increase in glucose utilization in the STN of rats treated
with apomorphine, a potent DA agonist (Brown and Wolfson, 1978). Furthermore, the increase in 2-deoxyglucose
signal was blocked by pretreating the animals with haloperidol (Brown and Wolfson, 1978), an antagonist at D2like receptors. Consequently, the nature of the DAergic
input to the rodent STN has been the focus of biochemical
© 2000 WILEY-LISS, INC.
(Rosales et al., 1997), histofluorescence (Meibach and
Katzman, 1979), anatomical tracing (Campbell et al.,
1985; Hassani et al., 1997), and more recently, electrophysiological studies (Mintz et al., 1986; Kreiss et al.,
1996, 1997). Together, these studies have demonstrated
This paper is dedicated to the memory of Dr. Jack Penney for his
friendship and guidance of this work.
Grant sponsor: U.K. Parkinson’s Disease Society; Grant number: 3061;
Grant sponsor: USPHS; Grant number: NS31579.
*Correspondence to: Dr. Sarah Augood, Neurology Research, Warren
408, Massachusetts General Hospital, 32 Fruit Street, Boston, MA
02114. E-mail: [email protected]
Received 7 October 1999; Revised 19 January 2000; Accepted 21 January
2000
248
S.J. AUGOOD ET AL.
unequivocally a direct DAergic input to the rat STN and
that administration of DAergic agents can markedly affect
the firing pattern of these tonically active fast-firing neurons in vivo. The precise nature of this DAergic response is
unclear as binding studies (Savasta et al., 1986; Mansour
et al., 1990, 1992; Johnson et al., 1994; Kreiss et al., 1996)
suggest that the rodent STN is enriched in either D1- or
D2-like receptors, however there is no clear consensus.
The subcellular localization of these DA receptors, that is,
whether they are encoded by STN neurons and/or are
present on STN afferents, has been addressed recently by
using sensitive reverse transcriptase-polymerase chain
reaction (RT-PCR) coupled with receptor autoradiography: the rat STN is enriched in D1-like, D2-like, and
D4-like binding sites, and intrinsic STN neurons express
DA D1, D2, and D3 but not D4 receptor mRNAs (Flores et
al., 1999).
To date, no studies have investigated in detail the
DAergic nature of the human STN. Recent immunochemical studies have reported the presence of fine tyrosine
hydroxylase-immunopositive (TH) fibers traversing this
structure (Cossette et al., 1999; Hedreen, 1999), in line
with rodent (Hassani et al., 1997) and primate (Francois
et al., 1998) data. To our knowledge, no studies have
investigated the nature of the DAergic interaction within
the human STN. Thus, in this study we investigated the
immunostaining for TH coupled with DA D1-like and D2like receptor autoradiography, whilst the DA receptor
phenotype of these STN neurons was addressed by using
in situ hybridization. This molecular approach for localizing the DA receptor subtypes was favored over immunohistochemistry primarily because in situ hybridization
techniques allow the cellular origin of a mRNA to be
identified in addition to the subtype specificity provided by
examining the cellular expression of the individual genes.
Finally, the presence of DA uptake sites, a marker of
functional DA terminals, was assessed by 3H-mazindol
autoradiography.
MATERIALS AND METHODS
Human tissue
Bouin’s (0.9% picric acid, 9% formaldehyde, 5% glacial
acetic acid: Sigma, St. Louis, MO)-fixed tissue blocks (n ⫽
3) containing the medial-lateral extent of the human STN
were provided by the Harvard Brain Tissue Resource Center (Belmont, MA). Frozen human tissue was provided as
fresh-frozen cryostat sections (16 ␮m) thawmounted onto
poly-L-lysine-coated microscope slides by the U.K. Parkinson’s Disease Society Tissue Brain Bank (London, U.K.).
Tissue was stored at ⫺80°C until used. All the Bouin’sfixed and fresh-frozen tissue was from control cases with
no history of neurological disease and no overt signs of
neuropathology. Brain postmortem intervals did not exceed 24 hours for the in situ hybridization studies or 56
hours for the ligand binding studies. All cases used in this
study had brain pH values in the range of 6.1– 6.6 (Kingsbury et al., 1995).
TH immunohistochemistry
Bouin’s-fixed tissue blocks containing the medial-lateral
extent of the STN were cryoprotected in 30% sucrose (w/v),
rinsed briefly with 95% ethanol, and frozen rapidly on
aluminium plates as described previously (Vonsattel et
al., 1995). Fifty-micron-thick sections were cut on a freezing microtome, collected into 0.1 M phosphate-buffered
saline (PBS) pH 7.4/50% glycerol and either processed
immediately for TH immunostaining or stored at ⫺20°C.
For immunostaining, the free-floating sections were first
washed extensively in 0.1 M PBS, treated with 30% methanol ⫹ 1.5% H2O2 to deactivate endogenous peroxidase
activity, rinsed again in 0.1 M PBS, blocked with 10%
normal goat serum (NGS)/0.3% Triton X-100 and then
incubated overnight at 4°C in mouse anti-TH antiserum
(1:1,000; Sigma). Sections were then washed extensively
in PBS, incubated in biotinylated-goat anti-mouse IgG
(1:200; Jackson Labs, Bar Harbor, ME) for 1 hour, rinsed,
then incubated in avidin-biotin-complex (Vectastain Elite
Kit, Vector Laboratories, Burlingame, CA). Finally, THimmunopositive structures were visualized by using 3,3diaminobenzidine (0.5 mg/ml) as chromogen activated
with H2O2 (0.0045%).
DA D1 and D2 receptor in situ hybridization
Hemicoronal tissue sections containing the posterior
putamen, globus pallidus internus and externus, and the
medial-lateral aspect of the STN were processed for in situ
hybridization by using a standardized protocol as reported
previously (Harrington et al., 1995). In brief, sections were
fixed, rinsed in 0.1 M phosphate-buffered saline, dehydrated, and then hybridized at 37°C with human-specific
DA D1 and D2 receptor 35S-oligonucleotide probes as described previously (Harrington et al., 1995; Augood et al.,
1997). Following an overnight hybridization, tissue sections were washed stringently in 1 ⫻ saline sodium citrate
at 55°C and then processed for emulsion autoradiography
(Ilford K5, Polysciences, Warrington, PA) for 8 –12 weeks.
Finally, tissue sections were counterstained with
methylene-blue to visualize all brain nuclei and examined
with light microscopy by using a Leica microscope. Some
tissue sections were also processed with the addition of an
excess of unlabeled oligonucleotide to the hybridization
buffer (pH 7.0) to demonstrate that the binding of the
35
S-probes was displaceable.
Dopamine D1 and D2 receptor binding and
dopamine uptake sites
Hemicoronal tissue sections (n ⫽ 4) containing the posterior putamen, globus pallidus internus and externus,
the medial-lateral aspect of the STN and the rostral substantia nigra pars reticulata were processed for [3H]SCH23390 (DA D1-like), [3H]-YM-08151-2 (DA D2-like)
and [3H]-mazindol (DA uptake) ligand binding by using
standardized protocols as reported previously (Cha et al.,
1998). All [3H]-ligands were obtained from New England
Nuclear (Boston, MA). In brief, assay buffer contained 25
mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM MgCl2, 1 ␮M
pargyline, and 0.001% ascorbate. For D1-like binding,
slides were incubated in the dark with 1.54 nM [3H]-SCH23390 (s.a. 70.3 Ci/mmol) for 2.5 hours at room temperature. Nonspecific binding was defined in the presence of 1
␮M cis-flupentixol. For D2-like binding, slides were incubated in the dark with 187 pM [3H]-YM-09151-2 (s.a. 85.5
Ci/mmol) for 3 hours at room temperature. Nonspecific
binding was defined in the presence of 100 ␮M DA. For DA
uptake, slides were prewashed in ice-cold binding buffer
(50 mM Tris-HCl, 5 mM KCl, and 300 mM NaCl, pH 7.9)
for 5 minutes, then incubated with 5.5 nM [3H]-mazindol
(s.a. 24 Ci/mmol) in the presence of 300 nM desipramine
TH-LI AND DA RECEPTORS IN THE HUMAN STN
for 1 hour at 4°C. Nonspecific binding was defined in the
presence of 10 ␮M nomifensine. After incubation with
[3H]-ligand, slides were rinsed in cold assay buffer for 10
minutes, rinsed quickly in cold distilled water, dried under a stream of cool air, and apposed to tritium-sensitive
film (Hyperfilm 3H, Amersham, Arlington Heights, IL)
with calibrated 14C-standards (ARC, Inc, St. Louis, MO)
for 2– 4 weeks. Films were analyzed by using computerassisted image analysis (M1, Imaging Research, St. Catharine’s, ON, Canada) and the density of the image converted to ␮Ci/g by automated extrapolation from the
calibrated standards. Specific binding was determined by
subtracting the nonspecific value from the total binding
value.
RESULTS
TH immunohistochemistry
A robust TH-immunopositive signal was detected in all
the Bouin’s-fixed tissue sections processed with the
anti-TH antibody. In a pilot study comparing different
fixatives on the quality of TH-like immunoreactivity, we
found Bouin’s-fixed tissue blocks to give the most robust
staining by using this TH monoclonal antibody. Of the
three Bouin’s-fixed cases processed, b-3932 displayed the
most robust immunostaining. A dense network of intensely labeled TH-immunopositive neurons and fibers
was observed in the lateral tip of the substantia nigra
(Fig. 1A,B). On the same tissue sections, a heterogeneous
pattern of TH immunoreactivity was observed in the STN:
in addition to weak neuropil staining, a fine network of
TH-immunopositive fibers was observed traversing the
nucleus (Fig. 1C,E,F). Many of these fibers have the appearance of long smooth axons with very occasional bifurcated endings. TH-positive fibers were more numerous
medially than laterally. No TH-positive somata were detected. By contrast, on adjacent tissue sections processed
in parallel but with the omission of the anti-TH antibody,
no specific immunostaining was observed (Fig. 1B): The
presence of darkly stained punctate dots in Figure 1D
represents an intensification of the endogenous lipofuscin
pigment contained within the cytoplasm of these human
STN neurons.
DA D1 and D2 receptor mRNA
On film autoradiograms, intense DA D1 and D2 receptor
hybridization signals were observed in the caudate nucleus and putamen of all tissue sections. On sections from
cases containing the lateral tip of the substantia nigra
pars compacta, an intense D2 receptor hybridization signal was observed; no D1 signal was detected in this nucleus. Within the STN, no hybridization signal was detected for either DA receptor transcript. At the cellular
level, DA receptor mRNA-positive cells were identified by
the accumulation of silver grains overlying methylene
blue-counterstained neurons. In the caudate-putamen,
numerous DA receptor mRNA-positive cells were detected
throughout the entire extent of the nucleus. In addition to
the extensive labeling of medium-sized D1 and D2 receptor (Fig. 2A) mRNA-positive cells, D2 mRNA-positive
large cells were also observed. In addition to the striatal
labeling, numerous D2 receptor mRNA-positive neurons
were observed in the lateral tip of the substantia nigra;
these corresponded to the pigmented neurons in the sub-
249
stantia nigra pars compacta (Fig. 2B). No D1 receptor
mRNA-positive cells were observed in the substantia
nigra. Neurons of the human STN were readily identified
by the presence of endogenous yellow pigment (lipofuscin)
contained within the soma of numerous cells, a characteristic of these cells in mature adults. In contrast to the
caudate-putamen, no DA receptor mRNA-positive cells
were detected for either D1 or D2 (Fig. 2C); an occasional
D2 receptor mRNA-positive cell was detected on the ventrolateral edge of the nucleus, although it was not clear if
this was a displaced nigral neuron (Meibach and Katzman, 1979).
DA D1 and D2 receptor binding
Quantitative receptor autoradiography was used to reveal specific sites of DA D1-like, D2-like (Fig. 3) and DA
uptake binding sites (Fig. 4). Sites of specific binding were
determined by subtracting nonspecific binding (Figs.
3B,D; 4B) from total binding (Figs. 3A,C; 4A). Of the four
control brains examined, a robust and reproducible signal
was measured in all brain areas. For D1-like binding sites,
an intense signal was detected within the head of the
caudate and putamen: a less intense signal was observed
in the globus pallidus internus (GPi), substantia nigra
pars reticulata, and insular cortex. A very weak signal
was detected in the STN (Fig. 3A,B). The intensity of
D1-like binding was similar in the head of the caudate
nucleus and putamen. For D2-like binding, an intense
signal was detected within the head of the caudate and
putamen, the intensity of the signal being greater in the
putamen than in the head of the caudate. A less intense
signal was observed within the globus pallidus externus
(GPe). Similarly, for 3H-mazindol binding, a marker of DA
uptake sites, an intense signal was detected within the
head of the caudate and putamen. As for D2-like binding,
the intensity of the signal was greater in the putamen
than in the head of the caudate. A faint specific 3Hmazindol binding signal was detected within the substantia nigra pars reticulata, insular cortex and STN
(see Table 1). The quantitative data are summarized in
Table 1.
DISCUSSION
The present study provides a comprehensive examination of markers of DA transmission in the normal human
STN. We demonstrate that the normal human STN contains a sparse network of fine TH-immunopositive fibers,
a finding reported recently in formalin-fixed tissue (Cossette et al., 1999; Hedreen, 1999). Additionally, we demonstrate that intrinsic STN neurons are not enriched in
DA D1 or D2 receptor mRNA or DA D1-like or D2-like
binding sites. However, a specific, albeit weak, D1-like
binding signal is detected, as is a weak specific DA uptake
signal ([3H]-mazindol). Together these data suggest that
the human STN receives a sparse DAergic innervation.
Unlike the rat, intrinsic human STN neurons do not express D1 or D2 receptors. Although low levels of D1-like
(D1/D5) binding are present, this may be associated with
STN afferents and/or may reflect expression of DA D5
receptors.
Whether the TH-immunopositive fibers in the human
STN represent nigrostriatal fibers of passage or a direct
DAergic innervation is unclear and requires further investigation. In the rat (Hassani et al., 1997), tracing studies
250
S.J. AUGOOD ET AL.
Figure 1
TH-LI AND DA RECEPTORS IN THE HUMAN STN
251
Fig. 2. Darkfield images of tissue sections hybridized with the D2
receptor probe and then processed for emulsion autoradiography.
Tissue sections were viewed under darkfield microscopy. Dopamine
D2 receptor mRNA-positive cells (arrows) are visualized by the accu-
mulation of silver grains (white dots). Examples of D2 receptor
mRNA-positive cells are shown in the (A) dorsal striatum and (B)
substantia nigra pars compacta. In marked contrast, no D2 mRNApositive cells are detected in the STN (C). Scale bar ⫽ 50 ␮m.
coupled with immunohistochemistry for TH have provided
convincing evidence for a DAergic innervation of this nucleus, although ultrastructural studies are required to
clarify this point conclusively. In the African green monkey, sparse TH-immunopositive axonal endings (bifurcations and varicosities) are also found traversing the STN
(Francois et al., 1998), suggestive of a direct DAergic innervation. Consistent with these findings, we report here
a specific, albeit weak, 3H-mazindol binding signal in the
human STN, indicating a low level complement of DA
uptake sites and thus supporting the idea of functional DA
terminals within this structure. As the specific [3H]mazindol signal in the STN is weak (approximately 20fold less than the putamen), several important points
must be considered when interpreting this data: (1) the
impact of postmortem interval on DA uptake, (2) the specificity of 3H-mazindol (in the presence of desipramine to
block noradrenaline uptake sites) for DA uptake sites, and
(3) the small number (n ⫽ 4) of human cases studied.
Considering each point in turn, the impact of postmortem
interval on DA uptake is unknown, although our data in
the caudate-putamen (this study) coupled with the recent
studies of Piggott et al., (1999) suggest that the membrane
protein is stable for up to 80 hours (see case 20 in Piggott
et al., 1999). The selectivity of 3H-mazindol for labeling
DA uptake sites (in the presence of desipramine) has been
shown elsewhere and is demonstrated here by the greater
binding in the putamen versus the caudate nucleus, consistent with the respective DA enrichment (Hornykiewicz,
1973). Further, the distribution of other DA uptake ligands such as 125RTI-121 (in the presence of clomipramine to block serotonin uptake sites), 3H-WIN-35,428, and
3
H-CFT suggests that 3H-mazindol does indeed bind to
sites of DA uptake. However, differences in 125I-RTI-55
and 3H-WIN 35,428 binding density have been noted in
the rat STN and have been attributed to variants of the
dopamine transporter and/or posttranslational modifications of the membrane protein (Coulter et al., 1995). To
date, despite the presence of DA uptake binding sites
(Coulter et al., 1995; Flores et al., 1999; Fujita et al.,
1994), no DA transporter-like immunoreactivity has been
detected within this structure (Ciliax et al., 1995), further
suggestive of DA transporter variants.
The weak specific [3H]-mazindol binding measured in
the human STN is comparable to the binding in the insular cortex on the same tissue sections (see Table 1). Within
the insular cortex, scattered TH- and DA transporterimmunopositive axons, both varicose and nonvaricose,
have been reported, which are particularly enriched
within layer 1 (Ciliax et al., 1999; Gaspar et al., 1989).
These immunohistochemical findings coupled with our DA
uptake autoradiography data suggest that the low level of
[3H]-mazindol binding detected in this structure is indeed
indicative of DA terminals. Similarly, the binding of [3H]SCH23390 in the human insular cortex (this study) is
consistent with the immunohistochemical localization of
D1 and D5 (D1 family) receptors to interneurons (Muly et
al., 1998) and dendritic spines (D1) and dendritic shafts
(D5) of intrinsic pyramidal neurons in the primate prefrontal cortex (Bergson et al., 1995). The lack of D1 receptor mRNA by in situ hybridization analysis (this study) or
Northern analysis (Mrzljak et al., 1996) suggests that
intrinsic STN neurons do not express this DA receptor
subtype. However, as the expression of D5 receptors was
not examined in this study, it is possible that these intrinsic neurons express D5 receptors. Thus, our data showing
a low-level complement of D1-like binding in the human
STN may reflect the local expression of D5 receptors coupled with the presence of D1/D5 receptors on STN affer-
Fig. 1. Montage of tyrosine hydroxylase (TH) immunostaining in
the human subthalamic nucleus (STN) and lateral SN. THimmunopositive neurons and nerve fibers are highly enriched within
the SN (A,B). Within the STN (C), a fine network of THimmunopositive fibers are detected traversing the nucleus. This fine
network of TH-positive fibers (E,F) is more pronounced in the medial
aspect of the nucleus. The specificity of the TH immunostaining is
demonstrated by the absence of specific staining on a control section
processed without any primary antibody. Scale bars ⫽ 200 ␮m in D
(also applies to A,C); 100 ␮m in E (also applies to B); 50 ␮m in F.
252
S.J. AUGOOD ET AL.
Fig. 3. Total (A,C) and nonspecific (B,D) binding of 3H-SCH-23390
(A,B) and 3H-YM-09151-2 (C,D) to human basal ganglia tissue at the
level of the subthalamic nucleus (STN). A: Total binding. Note the
enrichment of D1-like binding sites in the head of the caudate nucleus
(CN), the putamen (Put), the globus pallidus internus (GPi), and the
substantia nigra pars reticulata (SNr) when compared with nonspecific binding in B. C: Total binding. Note the enrichment of D2-like
binding sites in the head of the caudate nucleus (CN), the putamen
(Put), and the globus pallidus externus (GPe) when compared with
nonspecific binding in D. Scale bar ⫽ 1 cm.
ents. Ultrastructural studies using subtype specific antisera will be required to clarify this point.
Functionally, the DA receptor phenotype of STN neurons in mammals is of interest, as this structure has been
identified as a potential therapeutic target in the treatment of Parkinson’s disease. Pharmacological studies
have shown that direct infusion of apomorphine, a DA
receptor agonist, into the STN elicits oral dyskinesias in
rats (Parry et al., 1994), suggestive of functional DA receptors within this structure. Furthermore, systemic administration of ephedrine, an indirect catecholamine agonist (Zarrindast, 1981), results in a massive induction of
Fos protein in the striatum and STN (Blandini et al.,
1997). However, the precise mechanism of this Fos induc-
TH-LI AND DA RECEPTORS IN THE HUMAN STN
253
Fig. 4. Total (A) and nonspecific (B) binding of 3H-mazindol to human basal ganglia tissue at the level
of the subthalamic nucleus (STN). Note the enrichment of dopamine (DA) uptake sites in the head of the
caudate nucleus (CN) and the putamen (Put) when compared with nonspecific binding in (B). SNr,
substantia nigra pars reticulata. Scale bar ⫽ 1 cm.
TABLE 1. Specific Binding of Dopamine (DA) D1-Like, D2-Like and DA Uptake Sites in the Human Basal Ganglia From Four Individual Cases1
3
3
[ H]SCH23390
[3H]YM-09151-24
[3H]mazindol
CN
Put
STN
SNr
Insular ctx
1.56 ⫾ 0.17
1.59 ⫾ 0.12
0.70 ⫾ 0.18
1.43 ⫾ 0.22
1.94 ⫾ 0.16
0.96 ⫾ 0.24
0.06 ⫾ 0.02
0.002
0.05 ⫾ 0.03
0.44 ⫾ 0.00
0.07 ⫾ 0.06
0.02 ⫾ 0.02
0.32 ⫾ 0.06
0.08 ⫾ 0.03
0.06 ⫾ 0.04
1
Values ⫽ mean ⫾ S.E.M. (␮Ci/g ⫻ 10).
N ⫽ 2.
D1-like.
4
D2-like.
2
3
tion is not clear and may relate, in part, to the vasoactive
properties of this compound; the STN is one of the most
densely vascularized structures in the human central nervous system. By contrast, no such induction of Fos immunoreactivity is observed following DA depletion (Cooper
and Mitchell, 1995) or systemic haloperidol (Suzuki et al.,
1998). Together these data invite the question of whether
DA exerts a direct effect on intrinsic STN neurons and, if
so, what is the DA receptor phenotype of STN neurons?
Extracellular recordings in anesthetized rats have revealed a predominantly excitatory effect of DA on the
basal activity of STN neurons (Mintz et al., 1986; Kreiss et
al., 1996), although earlier conflicting data exist (Campbell et al., 1985). Similarly, an increase in STN firing rate
is observed following systemic administration of apomorphine and SKF-82958 (a D1 receptor agonist) and following DA depletion (Hassani et al., 1996; Kreiss et al., 1997)
but not after administration of quinpirole, a D2 receptor
agonist (Kreiss et al., 1997). Further, the increase in tonic
firing rate of these STN neurons in the DA-depleted rat
model of parkinsonism can be “normalized” by systemic
administration of the nonselective DA agonists, apomorphine and I-DOPA (Kreiss et al., 1997), underscoring the
importance of DAergic interactions within this structure.
To dissect the DA receptor phenotype of the rat STN,
ligand binding studies have been carried out by using a
variety of radiolabeled ligands. In general, studies using
tritiated ligands favor a preferential enrichment of D1like over D2-like binding sites (Savasta et al., 1986; Mansour et al., 1990, 1992; Kreiss et al., 1996; Flores et al.,
1999), although conflicting data exist using iodinated ligands (Johnson et al., 1994). Further as D1, D2, and D3
receptor mRNAs are expressed by intrinsic STN neurons
in the rat (Flores et al., 1999), yet no D1-like or D2-like
immunopositive cells have been observed (Yung et al.,
1995), these data suggest that the DA receptor mRNAs
expressed by rat STN neurons are translated and the
receptor proteins exported outside of the STN.
With regard to the cellular localization of DA D1 and/or
D2 receptors within the human STN, our study demonstrates clearly that DA D1 and D2 receptor mRNAs are
differentially expressed within the human basal ganglia,
as might be predicted from in situ mapping studies in
rodents (Mansour et al., 1990, 1992). Of particular note is
the apparent lack of D1 or D2 receptor mRNAs within the
STN despite prominent expression within other basal ganglia nuclei on the same tissue section. These findings
agree with Northern analysis data (Matsumoto et al.,
254
S.J. AUGOOD ET AL.
1996) which similarly report a lack of expression of D1 and
D2 receptor mRNAs within the human STN, although an
enrichment of D4 receptor mRNA has been observed.
These mRNA studies are exactly contrary to rat data
which report an enrichment of D1, D2, and D3 receptor
mRNA but not D4 mRNA (Flores et al., 1999). Collectively
these findings add to the known species differences in
dopaminergic systems between rodents and human
(Berger et al., 1991; Betarbet et al., 1997) and underscore
the importance of human brain studies.
In summary, we have demonstrated that THimmunopositive processes traverse the human STN and
that these processes may constitute functional DA terminals. By using in situ hybridization and DA receptor receptor autoradiography, we were unable to detect a robust
DA D1-like, D2-like, or DA uptake signal within the human STN of any of the normal brains examined. However,
weak and specific 3H-mazindol and D1-like binding was
observed in this structure (see Table 1), suggestive of a
low-level complement of DA terminals and DA D1 receptor
binding sites. It will be important to validate these findings by using specific DA receptor subtype-specific antisera coupled with sensitive ultrastructural techniques.
Understanding the DA receptor complement of the human
STN is of clinical importance, as this structure plays a
central role in motor (Lee and Marsden, 1994; Wichmann
et al., 1994; Joel and Weiner, 1997) and possibly executive
functioning (Baunez et al., 1995; Ardouin et al., 1999).
ACKNOWLEDGMENTS
Bouin’s-fixed human tissue was provided by the Harvard Brain Tissue Resource Center, which is supported, in
part, by USPHS grant number NS 31579. K. Westmore
and A. Kingsbury are thanked for their expert assistance.
Dr. S. Daniel is thanked for her neuropathological evaluation of the frozen tissue. Dr. John Hedreen is thanked for
his comments on this manuscript.
LITERATURE CITED
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