An electron microscope immunocytochemical study of GABA(B) R2 receptors in the monkey basal ganglia: a comparative analysis with GABA(B) R1 receptor distribution.

Abstract

Functional gamma-aminobutyric acid (GABA)(B) receptors are heterodimers made up of GABA(B) R1 and GABA(B) R2 subunits. The subcellular localization of GABA(B) R2 receptors remains poorly known in the central nervous system. Therefore, we performed an ultrastructural analysis of the localization of GABA(B) R2 receptor immunoreactivity in the monkey basal ganglia. Furthermore, to characterize better the neuronal sites at which GABA(B) R1 and GABA(B) R2 may interact to form functional receptors, we compared the relative distribution of immunoreactivity of the two GABA(B) receptors in various basal ganglia nuclei. Light to moderate GABA(B) R2 immunoreactivity was found in cell bodies and neuropil elements in all basal ganglia nuclei. At the electron microscope level, GABA(B) R2 immunoreactivity was commonly expressed postsynaptically, although immunoreactive preterminal axonal segments were also frequently encountered, particularly in the globus pallidus and substantia nigra, where they accounted for the third of the total number of GABA(B) R2-containing elements. A few labeled terminals that displayed the ultrastructural features of glutamatergic boutons were occasionally found in most basal ganglia nuclei, except for the subthalamic nucleus, which was devoid of GABA(B) R2-immunoreactive boutons. The relative distribution of GABA(B) R2 immunoreactivity in the monkey basal ganglia was largely consistent with that of GABA(B) R1, but some exceptions were found, most noticeably in the globus pallidus and substantia nigra, which contained a significantly larger proportion of presynaptic elements labeled for GABA(B) R1 than GABA(B) R2. These findings suggest the possible coexistence and heterodimerization of GABA(B) R1 and GABA(B) R2 at various pre- and postsynaptic sites, but also raise the possibility that the formation of functional GABA(B) receptors in specific compartments of basal ganglia neurons relies on mechanisms other than GABA(B) R1/R2 heterodimerization.

Full text

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An Electron Microscope
Immunocytochemical Study of GABABR2
Receptors in the Monkey Basal Ganglia:
A Comparative Analysis with GABABR1
Receptor Distribution
ALI CHARARA,1,2ADRIANA GALVAN,1,2MASAAKI KUWAJIMA,1,3RANDY A. HALL,3
AND YOLAND SMITH1,2*
1Yerkes National Primate Research Center, Emory University, Atlanta, Georgia 30322
2Department of Neurology, Emory University, Atlanta, Georgia 30322
3Department of Pharmacology, Emory University, Atlanta, Georgia 30322
ABSTRACT
Functional ?-aminobutyric acid (GABA)Breceptors are heterodimers made up of GABABR1
and GABABR2 subunits. The subcellular localization of GABABR2 receptors remains poorly
known in the central nervous system. Therefore, we performed an ultrastructural analysis of the
localization of GABABR2 receptor immunoreactivity in the monkey basal ganglia. Furthermore,
tocharacterizebettertheneuronalsitesatwhichGABABR1andGABABR2mayinteracttoform
functional receptors, we compared the relative distribution of immunoreactivity of the two
GABABreceptors in various basal ganglia nuclei. Light to moderate GABABR2 immunoreactiv-
ity was found in cell bodies and neuropil elements in all basal ganglia nuclei. At the electron
microscope level, GABABR2 immunoreactivity was commonly expressed postsynaptically, al-
though immunoreactive preterminal axonal segments were also frequently encountered, partic-
ularly in the globus pallidus and substantia nigra, where they accounted for the third of the total
number of GABABR2-containing elements. A few labeled terminals that displayed the ultra-
structural features of glutamatergic boutons were occasionally found in most basal ganglia
nuclei, except for the subthalamic nucleus, which was devoid of GABABR2-immunoreactive
boutons. The relative distribution of GABABR2 immunoreactivity in the monkey basal ganglia
was largely consistent with that of GABABR1, but some exceptions were found, most noticeably
in the globus pallidus and substantia nigra, which contained a significantly larger proportion of
presynapticelementslabeledforGABABR1thanGABABR2.Thesefindingssuggestthepossible
coexistence and heterodimerization of GABABR1 and GABABR2 at various pre- and postsyn-
aptic sites, but also raise the possibility that the formation of functional GABABreceptors in
specific compartments of basal ganglia neurons relies on mechanisms other than GABABR1/R2
heterodimerization. J. Comp. Neurol. 476:65–79, 2004.
© 2004 Wiley-Liss, Inc.
Indexing terms: striatum; globus pallidus; subthalamic nucleus; substantia nigra; GABABR1
receptors; immunocytochemistry
Grant sponsor: National Institutes of Health (NIH); Grant number: R01
NS37423-01 (Y.S.), Grant number: R01 NS42937 (Y.S.); Grant number:
R01-NS045644 (R.A.H.); Grant sponsor: the Yerkes National Primate Re-
search Center NIH base grant; Grant number: RR 00165; Grant sponsor:
the Parkinson’s Disease Foundation; Grant sponsor: the W. M. Keck Foun-
dation (a Distinguished Young Scholar in Medical Research award)
(R.A.H.).
The first two authors contributed equally to this work.
Ali Charara’s current address is College of Arts and Sciences, American
University of Kuwait, Salmiya, Kuwait.
*Correspondence to: Yoland Smith, Yerkes Primate Research Center,
Emory University, 954 Gatewood Road NE, Atlanta, GA 30322.
E-mail: yolands@rmy.emory.edu
Received 26 August 2003; Revised 5 February 2004; Accepted 15 April 2004
DOI 10.1002/cne.20210
Published online in Wiley InterScience (www.interscience.wiley.com).
THE JOURNAL OF COMPARATIVE NEUROLOGY 476:65–79 (2004)
© 2004 WILEY-LISS, INC.

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The inhibitory amino acid ?-aminobutyric acid (GABA)
plays a major role in regulating neuronal activity within
the basal ganglia. Studies in rodents and primates have
shown that the intrinsic synaptic circuits as well as output
pathways of the basal ganglia utilize GABA as a neuro-
transmitter (Parent and Hazrati, 1995; Smith et al.,
1998). Three types of receptors mediate the effects of
GABA in the central nervous system (CNS). The fast
inhibitory responses result from the stimulation of the
postsynaptic chloride-gated GABAAand GABACreceptor
channels (Olsen and Tobin, 1990; Macdonald and Olsen,
1994), whereas the slow GABA-mediated postsynaptic hy-
perpolarization or presynaptic modulatory effects on neu-
rotransmission result from the activation of G protein-
coupled GABABreceptors (Bormann, 1988, 2000; Bowery,
1989; Misgeld et al., 1995; Deisz, 1997; Bettler et al.,
1998).
Expression cloning initially showed that GABABrecep-
tors consist of at least four splice variants, termed GABAB
R1a, -b, -c, and -d, which differ in their N termini (Kaup-
mann et al., 1997; Isomoto et al., 1998; Pfaff et al., 1999).
Moreover, several groups have cloned a second GABAB
receptor, termed GABABR2, which exhibits a similar
molecular weight and 30–40% sequence homology to
GABABR1 (Jones et al., 1998; Kaupmann et al., 1998;
White et al., 1998). Unexpectedly for a G protein-coupled
receptor, heteromeric assembly of GABABR1 and GABAB
R2 is necessary to form functional GABABreceptors (Mar-
shall et al., 1999; Bowery and Enna, 2000).
In situ hybridization and immunohistochemical studies
revealed that GABABR1 and GABABR2 mRNAs expres-
sion show considerable overlap in the rat CNS. However,
in some areas like the striatum, there is very little GABAB
R2 relative to GABABR1 mRNAs (Jones et al., 1998;
Kaupmann et al., 1998; Kuner et al., 1999; Ng et al., 1999;
Charles et al., 2001). This led to the suggestion that
GABABR1 can either function as a monomer in some
discrete regions or interact with other partners, which
have not yet been identified in the mammalian brain
(Marshall et al., 1999).
GABABreceptors subserve a variety of functions within
the basal ganglia, ranging from presynaptic modulation of
neurotransmitter release to postsynaptic regulation of in-
hibitory neurotransmission (Seabrook et al., 1991; DeBoer
and Westerink, 1994; Shen and Johnson, 1997, 2001; Tep-
per et al., 1998; Stefani et al., 1999). Localization studies
have shown radioactive binding, mRNA expression, and
cellular immunolabeling for GABABR1 in various basal
ganglia nuclei in rats (Bowery et al., 1987; Bischoff et al.,
1999; Fritschy et al., 1999; Lu et al., 1999; Margeta-
Mitrovic et al., 1999; Yung et al., 1999; Liang et al., 2000;
Ng and Yung, 2001a,b; Boyes and Bolam, 2003) and mon-
keys (Charara et al., 2000). Furthermore, we recently
showed that GABABR1 immunoreactivity is expressed in
putative glutamatergic afferents throughout the monkey
basal ganglia (Charara et al., 2000). At present, very little
is known about the localization of GABABR2 receptor
immunoreactivity, except for recent light microscopic
studies showing the cellular distribution of this receptor
subtype in the rat (Durkin et al., 1999; Clark et al., 2000;
Ng and Yung et al., 2001a,b) and human (Billinton et al.,
2000) CNS. Subcellular localization analyses have been
restricted to the rat visual cortex, cerebellum, thalamus,
and substantia nigra (Gonchar et al., 2001; Kulik et al.,
2002; Boyes and Bolam, 2003).
To characterize further the potential sites where
GABABR1 and GABABR2 may functionally interact, we
performed an electron microscopic study of GABABR2
receptor localization and compared its overall pattern of
distribution with that of GABABR1 in the monkey basal
ganglia.
MATERIALS AND METHODS
Animals and preparation of tissue
For the immunocytochemical localization of GABABR2
receptors, tissue was collected from three adult male rhe-
sus monkeys (Maccaca mulatta, 3–5 kg; Yerkes National
Primate Research Center colony). The experiments were
performed according to the National Institutes of Health
guide for the care and use of laboratory animals. All ef-
forts were made to minimize animal suffering and reduce
the number of animals used.
The monkeys were deeply anesthetized with an over-
dose of pentobarbital and perfused transcardially with
cold oxygenated Ringer’s solution, followed by fixative con-
taining 4% paraformaldehyde and 0.1% glutaraldehyde in
phosphate buffer (PB; 0.1 M, pH 7.4). After fixative per-
fusion, the brains were washed with PB, taken out from
the skull, and cut into 10-mm-thick blocks in the frontal
plane. Tissue sections through the rostrocaudal extent of
the basal ganglia were then obtained with a Vibratome (60
?m thick), collected in cold phosphate-buffered saline
(PBS; 0.01 M, pH 7.4), and treated with sodium borohy-
dride (1% in PBS) for 20 minutes.
For the Western immunoblots, one rhesus monkey
(Yerkes National Primate Research Center colony) and
one Sprague-Dawley rat (Charles River Laboratories, Wil-
mington, MA) were used. The monkey was overdosed with
pentobarbital (100 mg/kg, i.v.), followed by rapid removal
of the brain from the skull and dissection of regions of
interest. The rat was rapidly decapitated, and the brain
was removed from the skull and dissected on ice.
Immunoblot analysis
A polyclonal guinea pig antiserum raised against the
C-terminal of the GABABR2 receptor (Chemicon Interna-
tional, Temecula, CA) was used in the present study.
Abbreviations
Ax
b
DEN
GABA
GABAA
GABAC
GABAB
GABABR1
GABABR2
GABABR1a
GABABR1b
GABABR1c
GABABR1d
GPe
GPi
SNc
SNr
SP
STN
VTA
axon
bouton
dendrite
?-aminobutyric acid
GABA receptor subtype A
GABA receptor subtype C
GABA receptor subtype B
GABABreceptor subunit R1
GABABreceptor subunit R2
GABABreceptor R1 isoform a
GABABreceptor R1 isoform b
GABABreceptor R1 isoform c
GABABreceptor R1 isoform d
globus pallidus, external segment
globus pallidus, internal segment
substantia nigra pars compacta
substantia nigra pars reticulata
dendritic spine
subthalamic nucleus
ventral tegmental area
66A. CHARARA ET AL.

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Specificity of this antiserum was determined by using
immunoblots of membrane samples from transfected cells
and brain tissues. Human embryonic kidney (HEK-293)
cells were maintained in complete medium (minimum es-
sential medium with 10% fetal bovine serum and 1%
penicillin/streptomycin) in 10-cm culture dishes at 37°C
with 5% CO2. At ?50–80% confluency, the cells were
transiently transfected with 2 ?g of plasmid for GABAB
R1, GABABR2, or both, mixed with LipofectAMINE (In-
vitrogen, Carlsbad, CA), followed by a 4-hour incubation
at 37°C with 5% CO2. After 6 ml of complete medium was
added, the cells were incubated for an additional 24 hours.
The cells were then harvested and completely homoge-
nized with a sonicator in ice-cold buffer solution contain-
ing 20 mM HEPES, 10 mM EDTA, and 2 mM sodium
vanadate. The membrane samples were prepared at 4°C.
The homogenate was then centrifuged for 5 minutes at
2,000 rpm to remove tissue debris, and the membrane was
isolated from the supernatant by subsequent centrifuga-
tion for 30 minutes at 14,000 rpm. The resulting pellet
was then solubilized in a buffer solution containing 20 mM
HEPES and 0.1 mM EDTA before total protein concentra-
tion was measured using a Bio-Rad (Hercules, CA) Protein
Assay. The homogenates were then centrifuged for 30
minutes at 14,000 rpm, and the pellet was solubilized in a
lysis buffer containing 10 mM HEPES, 50 mM NaCl, 0.1
mM EDTA, 1 mM benzamidine, 1.0% Triton X-100, 0.1%
sodium dodecyl sulfate (SDS), and protease inhibitor cock-
tail (1 tablet per 50 ml; Roche, Mannheim, Germany). The
lysates (6 ?g protein) were then eluted with 6X SDS-
polyacrylamide gel electrophoresis (PAGE) sample buffer,
resolved by SDS-PAGE, and subjected to Western blot
analysis with guinea pig polyclonal antisera against
GABABR2 (1:2,000 dilution; Chemicon International).
Immunoreactive bands were detected using the enhanced
chemiluminescence detection system (Pierce, Rockford,
IL) with horseradish peroxidase-conjugated goat anti-
guinea pig secondary antibody (Chemicon International).
All brain membrane samples were prepared, and 40 ?g of
protein was subjected to Western blot analysis in the same
manner as above.
Localization of GABABR2 immunoreactivity
at the light microscope level
The immunocytochemical localization of GABABR2 re-
ceptor subtype was performed by using the avidin-biotin
complex (ABC) method (Hsu et al., 1981). After blocking
nonspecific sites with 10% normal goat serum (NGS) and
1% bovine serum albumin (BSA) in PBS for 1 hour at room
temperature, the sections were incubated for 2 days at 4°C
in the primary antibody solution (1:500 dilution; Chemi-
con International). The sections were then washed in PBS,
incubated for 90 minutes in biotinylated goat anti-rabbit
IgG (1:200; Vector, Burlingame, CA), rinsed again in PBS,
and finally incubated for an additional 90 minutes in the
ABC solution (1:100, Vectastain Standard kit, Vector). All
immunoreagents were diluted in PBS containing 1% NGS,
1% BSA, and 0.1% Triton X-100. Sections were then
rinsed in PBS and Tris buffer (0.05 M, pH 7.6) before being
placedina solution
diaminobenzidine tetrahydrochloride (Sigma, St Louis,
MO), 0.01 M imidazole (Fisher Scientific, Norcross, GA),
and 0.006% H2O2for 10 minutes. The reaction was
stopped by repeated washes in PBS. Finally, the sections
were mounted on gelatin-coated slides, dehydrated in al-
containing 0.025%3-3?-
cohol, and immersed in toluene; then a coverslip was ap-
plied with Permount.
Localization of GABABR2 immunoreactivity
at the electron microscope level
The sections were placed in a cryoprotectant solution
(PB, 0.05 M, pH 7.4, containing 25% sucrose and 10%
glycerol) for 20 minutes, frozen at ?80°C for 20 minutes,
thawed, and washed in PBS before being processed for
immunocytochemistry. They were then processed for the
visualization of GABABR2 receptors according to the pro-
tocol described above, except that Triton X-100 was not
included in solutions. After immunostaining, they were
washed in PB (0.1 M, pH 7.4) and postfixed in osmium
tetroxide (1% in PB) for 20 minutes. This was followed by
washings in PB and dehydration in a graded series of
ethanol and propylene oxide. Uranyl acetate (1%) was
added to the 70% ethanol for 35 minutes to improve the
contrast in the electron microscope. The sections were
then embedded in resin (Durcupan ACM, Fluka, Ft.
Washington, PA) on microscope slides and placed in the
oven for 48 hours at 60°C. Areas of interest were selected,
cut out from the slides, and glued on the top of resin
blocks. Serial ultrathin sections were then cut on an ul-
tramicrotome(Leica Ultracut
Pioloform-coated single copper grids, stained with lead
citrate (Reynolds, 1963), and analyzed with the electron
microscope (Zeiss EM 10C).
Analysis of data
Transverse sections containing basal ganglia structures
were taken from each of the three monkeys for both light
and electron microscope analyses. At least one block was
taken from the striatum, external globus pallidus (GPe),
subthalamic nucleus (STN), and substantia nigra (SN) in
the three monkeys. The blocks from the striatum were all
cut from levels of putamen rostral to the anterior commis-
sure, whereas blocks from the SN were selected from the
ventral part of sections corresponding to the middle third
of the structure, which comprised perikarya and neuropil
elements of the pars reticulata (SNr) intermingled with
dendrites of pars compacta (SNc) neurons. The ultrastruc-
tural analysis was carried out on ultrathin sections col-
lected from the surface of each block where the staining
was optimal.
To compare the pattern of GABABR2 and GABABR1
immunolabeling, we made quantitative measurements of
the relative abundance of immunoreactive elements for
each receptor subtype in basal ganglia nuclei from two
monkeys with the best ultrastructural preservation. For
this part of the study, the GABABR1 measurements were
made from immunostained sections used in our previous
study of GABABR1 localization in monkey basal ganglia
(Charara et al., 2000). Quantitative data were collected
the same way in both materials, i.e., ulthrathin sections
from the most superficial sections of blocks were scanned
at 20,000–25,000?, and all immunoreactive elements
randomly encountered were photographed. To decrease
the likelihood of sampling immunoreactive elements more
than once, ultrathin sections used for quantification were
separated from each other by at least 0.5 ?m (i.e., 8-12
ultrathin sections). The labeled elements were then cate-
gorized in various groups based on ultrastructural fea-
tures (Peters et al., 1991), and their relative proportion
was calculated and expressed as percent of total labeled
T2),collected onto
67GABABR2 RECEPTORS IN THE PRIMATE BASAL GANGLIA

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elements for each receptor subtype. The total surface of
tissue examined in basal ganglia nuclei for each receptor
subtype is given in the legend of Figure 5. Statistical
differences in the pattern of distribution of the two recep-
tors subtypes were assessed with a Chi-square analysis.
The micrographs were acquired with a 3.25 ?4-inch
Kodak EM plates (printed on Ilford paper) or with a CCD
camera (DualView 300W, Gatan, Pleasanton, CA) con-
trolled by DigitalMicrograph software (version 3.7.4., Ga-
tan). The digitally acquired micrographs were adjusted for
brightness and contrast, while maintaining the image res-
olution constant, with Photoshop software (version 7.0,
Adobe Systems, San Jose, CA).
RESULTS
Immunoblotting
Immunoblot analysis of transfected HEK-293 cell ex-
tracts showed that the anti-GABABR2 antisera recog-
nized GABABR2, with no detectable cross-reaction with
GABABR1 expressed in HEK-293 cells (Fig. 1). This an-
tibody also labeled a single band in monkey and rat brain
membranes with an estimated molecular weight of 120
kDa, which corresponds to the predicted molecular weight
of GABABR2 (Jones et al., 1998; Kaupmann et al., 1998;
White et al., 1998) (Fig. 1). Omission of primary antisera
resulted in total absence of labeling (not shown).
GABABR2 localization in monkey
basal ganglia
In all basal ganglia nuclei examined, the pattern of
GABABR2 immunostaining was quite similar, i.e., light to
moderate cellular and neuropil labeling was found at the
light microscopic level, whereas dendritic processes and
unmyelinated axons accounted for most labeled elements
in the electron microscope. There were no significant in-
terindividual differences in the pattern of cellular and
ultrastructural labeling among the three animals used in
this study. Omission of the primary GABABR2 antiserum
from incubation solutions completely abolished the immu-
nostaining.
The striatum displayed light GABABR2 immunoreac-
tivity made up of faintly labeled small cell bodies among
which were interspersed more darkly stained large-sized
neuronal perikarya (Fig. 2A). At the light microscope
level, the neuropil staining was rather discrete and
largely made up of small punctate structures. There was
no obvious patch/matrix pattern of GABABR2 neuropil
labeling in the caudate nucleus and putamen (Graybiel
and Ragsdale, 1978; Gerfen and Wilson, 1996). At the
electron microscope level, both large and small neuronal
cell bodies with ultrastructural features reminiscent of
those previously described for projection neurons and in-
terneurons (DiFiglia et al., 1976; DiFiglia and Aronin,
1982; Bolam et al., 1983, 1984) contained immunoreactiv-
ity. In either case, patchy peroxidase deposits were found
throughout the cytoplasm where they appeared to be as-
sociated preferentially with stalks of endoplasmic reticu-
lum (Fig. 2B). Dendrites of various sizes (0.25–1.0 ?m)
accounted for more than 50% of the total number of la-
beled elements in the striatum, whereas almost 20% of the
labeled structures were dendritic spines (Figs. 2C,D, 5). In
dendrites, the labeling was mainly associated with micro-
tubules, whereas spines contained a more diffuse deposit
that often filled the labeled element (Fig. 2C,D).
Other frequently encountered (20% of total labeled ele-
ments) GABABR2-immunoreactive structures were small
unmyelinated axons that often contained synaptic vesicles
(Figs. 2E, 5). In those sections that were cut in the appro-
priate plane, the labeled axons gave rise to terminals that
formed asymmetric axospinous synapses (Fig. 2E). Al-
though much more rarely seen (less than 5% total labeled
elements), a subset of axon terminals packed with round
synaptic vesicles and a few mitochondria forming asym-
metric synapses with dendritic spines were also labeled
(Figs. 2F, 5).
In the external globus pallidus (GPe), subthalamic
nucleus (STN), and substantia nigra (SN), GABABR2
immunoreactivity was found in cell bodies, dendrites,
and small neuropil elements (Figs, 3, 4). Putative dopa-
minergic neuronal cell bodies in the pars compacta of
the SN (SNc) and the ventral tegmental area were more
strongly stained for GABABR2 than neurons in the SN
pars reticulata (SNr; Fig. 4A). At the electron micro-
scopic level the common feature to these brain regions
was the predominance of immunoreactive dendritic pro-
cesses (50–70% of total labeled elements; Figs. 3F,G,
4B, 5). In all cases, the immunoreactivity was preferen-
tially associated with microtubules, although patches of
peroxidase were occasionally seen along the plasma
membrane (Figs. 3F,G, 4B). A striking feature that
distinguished the labeling in GPe and SN from that in
STN was the abundance of immunoreactive small un-
myelinated axons (30% of total labeled elements in GPe
and SN; 10% in STN; Figs. 3C, 4C,D, 5). These axons
were often found in aggregates, although isolated la-
beled elements were also seen. When the labeled axons
were cut in the longitudinal plane and could be followed
to their terminals, the boutons were nonimmunoreac-
tive and always formed asymmetric axo-dendritic syn-
apses (Figs 3D, 4D). Although they were relatively rare
(1–10% total labeled structures), immunoreactive axon
Fig. 1.
plasmid (lane 1), GABABR1 (lane 2), GABABR2 (lane 3), and both
(lane 4) as well as membrane samples from the monkey putamen
(lane 5), the monkey cerebellum (lane 6), and the rat cerebellum (lane
7). The Chemicon anti-GABABR2 antibodies detected GABABR2
(lane 3) but not GABABR1 (lane 1). A band of about 120 kDa was also
detected in samples from the monkey putamen, monkey cerebellum,
and rat cerebellum (lanes 5–7, respectively). Note that expression of
GABABR2 is substantially lower in the monkey putamen (lane 5)
compared with the cerebellum (lane 6). Omission of primary antibod-
ies resulted in total absence of labeling (data not shown).
Western immunoblot of HEK-293 cells transfected with no
68 A. CHARARA ET AL.

Page 5

Fig. 2.
ous lightly labeled small GABABR2-immunoreactive perikarya (ar-
rowheads) and a large (arrow) darkly labeled neuron in the putamen.
B: Low-power electron micrograph of a GABABR2-immunoreactive
perikaryon (PER) in the putamen. The peroxidase deposit displays a
patchy distribution (open arrows and inset) associated with organelles
such as the endoplasmic reticulum and/or Golgi apparatus. No par-
ticular aggregation of peroxidase reaction product was found along
the plasma membrane. C: A GABABR2-immunoreactive dendrite
(DEN). Note the peroxidase reaction product associated with micro-
tubules. D: GABABR2-containing dendritic spines (SP) in the puta-
GABABR2 immunoreactivity in the striatum. A: Numer-
men. Note that one of the dendritic spines emerges from a nonimmu-
noreactivedendrite (DEN).The
nonimmunoreactive terminal forming an asymmetric synapse with a
labeled spine (arrowhead). E: Preterminal axonal segment that dis-
plays GABABR2 immunoreactivity and gives rise to a putative glu-
tamatergic terminal (b1) that contacts (arrowhead) a dendritic spine
(SP) in the putamen. F: A GABABR2-immunoreactive terminal (b1)
forming an asymmetric axospinous synapse (arrowhead) in the puta-
men. Note the presence of GABABR2-immunoreactive vesicle-filled
axons (ax). Scale bars ? 25 ?m in A; 2 ?m in B; 0.25 ?m in C; 0.5 ?m
in D; 0.5 ?m in E (applies to E,F).
asterisk (*)indicatesa
69GABABR2 RECEPTORS IN THE PRIMATE BASAL GANGLIA