Molecular phenotype of rat striatal neurons expressing the
dopamine D
5
receptor subtype
Alicia Rivera,1,2,* Israel Alberti,1,* Ana B. MartõÂn,1 Jose A. NarvaÂez,3Adelaida de la Calle2andRosario Moratalla1 1Instituto Cajal, Consejo Superior de Investigaciones Cientõ®cas, Avda Dr Arce 37, 28002 Madrid, Spain
2Department of Cell Biology,3Department of Physiology, University of MaÂlaga, 29071 MaÂlaga, Spain.
Keywords: basal ganglia, cat, ChAT, D1/ D2 receptor synergism, striatal LTP
Abstract
Dopamine is one of the principal neurotransmitters in the basal ganglia, where it plays a critical role in motor control and cognitive function through its interactions with the speci®c dopamine receptors D1to D5. Although the activities mediated by most
dopamine receptor subtypes have already been determined, the role of the D5receptor subtype in the basal ganglia has still not
been established. Furthermore, it is often dif®cult to distinguish between dopamine D5and D1receptors as they are stimulated by
the same ligands, and they have a similar molecular structure and pharmacology. In an effort to understand the differences between these two receptor subtypes, we have studied the distribution of neurons containing D5receptors in the striatum, and
their molecular phenotype. As a result, we show that the D5receptor subtype is present in two different populations of striatal
neurons, projection neurons and interneurons. Overall, the abundance of this receptor subtype in the striatum is low, particularly in striatal projection neurons of both the direct and indirect projection pathways. In contrast, the expression of D5 receptors in
striatal interneurons (cholinergic, somatostatin- or parvalbumin-positive neurons) is high, while low to moderate expression was observed in calretinin-positive neurons. Our results demonstrate the presence of D5receptors in all the striatal cell populations so
far described, although at different intensities in each. The fact that a large number of striatal neurons express the D5receptor
subtype suggests that this receptor ful®ls an important function in the process of integrating information in the striatum.
Introduction
In the basal ganglia the striatum plays a pivotal role in the regulation of motor control, as well as in processing cognitive and emotional inputs. The integration of information within this neural system requires the precise coordination of the activities of striatal projection neurons and interneurons. Dopaminergic inputs from the substantia nigra play a crucial role regulating the activity of all striatal neurons. Dopamine (DA) acts on speci®c G protein-coupled receptors that are grouped into two families based on their biochemical, pharmaco-logical and physiopharmaco-logical pro®les: D1-class (D1and D5subtypes) and
D2-class (D2, D3and D4subtypes) (Missaleet al., 1998).
Not all striatal neurons are equally responsive to dopaminergic stimulation. Behavioural sensitization and drug addiction induced by chronic exposure to cocaine involves the activation of D1-class receptors and the expression of immediate early genes in speci®c striatal neurons that express dynorphin or somatostatin but not in cholinergic or enkephalin-positive neurons (Moratalla et al., 1992, 1996a; Uslaneret al., 2001; Centonzeet al., 2002). Striatal cholinergic neurons respond differently, as activation of D1-class receptors induces acetyl choline (ACh) release (Consolo et al., 1992; Acquas & Di Chiara, 1999). This is due both to depolarization of cholinergic interneurons and to increase in cholinergic synaptic potentials (Consolo
et al., 1992; Aosakiet al., 1998; Calabresiet al., 1998; Acquas & Di
Chiara, 1999; Pisaniet al., 2000; Suzukiet al., 2001; Koos & Tepper, 2002). In addition, these neurons respond to dopamine-dependent-sensory-motor conditioning, a response that is dependent on the stimulation of D1-class receptors (Aosakiet al., 1994a,b). Although it
is clear that these effects involve the participation of D1-class
receptors, it has not been possible to differentiate between D1- and
D5-dependent effects, as there are no speci®c pharmacological agents
currently available to discriminate between them.
While the D1receptor subtype is much more abundant than the D5
subtype, the presence of D5receptors in the striatum is not negligible
because their af®nity for dopamine is ®ve times higher than that of D1
receptors (Grandyet al., 1991). The phenotype of neurons that express D1receptors is known. These receptors are mainly localized in neurons
of the direct pathway and are coexpressed with the neuropeptides substance P and dynorphin (Gerfenet al., 1990; Le Moineet al., 1991; Moratalla et al., 1996b; Faucheyet al., 2000). However, it has also been suggested that D1receptors are signi®cantly expressed in neurons
of the indirect pathway (Surmeieret al., 1996; Aizmanet al., 2000). Furthermore, D1receptors are weakly expressed on a small number of
NOS-positive interneurons in the striatum (Le Moineet al., 1991). Although it is known that D5 receptors are expressed in large and
medium size striatal neurons (Luedtkeet al., 1999; Ciliaxet al., 2000; Khanet al., 2000), the molecular phenotype of these neurons remains unclear. To better understand dopaminergic function in the basal ganglia it is critical to characterize the striatal neurons that express D5
receptors. In this study, we have identi®ed the molecular phenotype of projection neurons and interneurons that express the D5 receptor
subtype in the rat striatum, and we have compared their distribution in the rat and cat striatum.
Correspondence: Dr Rosario Moratalla, as above. E-mail: [email protected]
*A.R. and I.A. contributed equally to this work
Received 4 August 2002, revised 10 September 2002, accepted 13 September 2002
Materials and methods Animals and tissue preparation
Adult male Wistar rats (n= 15) and cats (n= 2) were deeply anaesthetized with sodium pentobarbital (i.p. 60 mg/kg) and perfused intracardially with 4% paraformaldehyde, 75 mM L-lysine and 10 mM sodium meta-periodate in 0.1Mphosphate buffer (PB), pH 7.4. Their brains were removed and post®xed in the same solution overnight at 4°C. The ®xed brains were cryoprotected with 30% sucrose in 0.1M phosphate-buffered saline (PBS), frozen in dry ice and sectioned
(30mm thick) with a freezing microtome (CM 1325, Leica,
Germany). Coronal sections through the striatum were collected and stored in PBS with 0.02% sodium azide at 4°C for either single-or double-label immunocytochemistry. All the studies were approved by the appropriate animal care committee at the Cajal Institute, CSIC, and all efforts were made to minimize the number of animals used.
Antisera
To localize D5 dopamine receptors we have used a monoclonal
antibody against the D5 receptor (1 : 50 dilution, Chemicon,
Temecula, CA, USA; Luedtkeet al., 1999) and a polyclonal rabbit antiserum speci®c for the D5 receptor previously developed and
characterized in our laboratory (1 : 500 and 1 : 1000 dilutions; Khan
et al., 2000).
Striosomes were identi®ed with a polyclonal rabbit antiserum against them-opioid receptor 1 (MOR1; 1 : 50 000 dilution, provided by R. Elde, University of Minnesota, MN, USA; Arvidsson et al., 1995; Tanet al., 2000). To identify striatal projection neurons, we used a polyclonal rabbit antiserum against dynorphin B for the neurons of the direct pathway (1 : 5000 dilution, Peninsula Laboratories, Belmont, CA, USA) and a polyclonal rabbit antiserum against met-enkephalin for the neurons of the indirect pathway (1 : 1000 dilution, Instar, Stillwater, MN, USA). To identify striatal interneurons, a polyclonal goat antiserum against choline acetyl-transferase was used to identify cholinergic neurons (ChAT; 1 : 1000 dilution; Chemicon, Temecula, CA, USA; Rico & Cavada, 1998). We also used a monoclonal rat antibody against somatostatin (SS; 1 : 50 dilution; provided by J. Rodrigo and A. C. Cuello, Cajal Institute, CSIC, Spain; Milstein & Cuello, 1983), a monoclonal mouse antibody against parvalbumin (PV; 1 : 1000 dilution; Sigma), and a polyclonal goat antiserum against calretinin (CR; 1 : 1000 dilution; Swant, Bellinzona, Switzerland). All primary antibodies were diluted
in 0.1MPBS, with 0.2% Triton X-100 (PBS-TX) and 0.1% sodium
azide.
Immunohistochemical procedures
Single antigen immunohistochemistry was performed to localize D5
dopamine receptors in sections of the rat and cat striatum. Endogenous peroxidase activity was quenched by incubation for 10 min with 3% hydrogen peroxide in PBS. After washing with PBS,
sections were incubated for 72 h at 4°C in anti-D5 polyclonal
antiserum or monoclonal antibodies. Control sections were incubated in PBS-TX only or with primary antibody previously preadsorbed with the immunizing peptide (Khan et al., 2000). To visualize the antibody distribution, the sections were washed in PBS, incubated for 1 h in either biotin conjugated goat anti-rabbit IgG diluted 1 : 500 (Vector, Burlingame, CA) or biotin conjugated horse anti-mouse IgG diluted 1 : 200 in PBS-TX. The sections were washed again and incubated for 1 h in a streptavidin-peroxidase complex (Sigma) diluted 1 : 2000 in PBS-TX. Peroxidase activity was visualized with 0.05% 3,3¢-diaminobenzidine (DAB, Sigma) and 0.002% H2O2, and
the staining was intensi®ed with 0.8% nickel ammonium sulfate. The sections were mounted on gelatin-coated slides, air dried, dehydrated with ethanol, cleared in xylene and coverslipped with DPX mounting medium. Similar results were obtained when the polyclonal D5 receptor antiserum was incubated overnight at a
dilution of 1 : 500.
Double-labelling immunostaining with MOR1 and D5
Two-colour dual antigen immunostaining was carried out to visualize D5immunoreactivity in cells of the striosomal compartment of the
striatum labelled with the MOR1 antiserum. We used the protocol described previously in which sequential staining was performed (Moratallaet al., 1996a; Riveraet al., 2002). Immunodetection of D5
receptors was performed ®rst and visualized with DAB and nickel ammonium sulfate, yielding a dark purplish precipitate. We then carried out immunostaining for the MOR1 antigen that was visualized with DAB yielding a brown precipitate.
Double-labelling immunocytochemistry with ¯uorescence secondary antibodies
Double-labelling immunocytochemistry was performed in the rat sections. Non-speci®c binding sites were blocked for 30 min with 5% bovine serum albumin in PBS-TX. Sections were washed and incubated with the polyclonal or monoclonal anti-D5antiserum 72 h
at 4°C and then washed again. The antibody distribution was
detected using an Alexa-conjugated secondary antibody diluted 1 : 1000 in PBS-TX and incubated for 1 h (see Table 1). The sections were then incubated for 1 or 2 nights, in the dark, with anti-Enk, anti-Dyn, anti-ChAT, anti-PV, anti-SS or anti-CR. After washing, the sections were further incubated for 1±2 h in the appropriate Alexa-conjugated secondary antibody (Molecular Probes, Eugene, OR, USA) diluted 1 : 1000 in PBS-TX (see Table 1). In each experiment, the speci®city of staining was controlled by incubating sections with one primary antibody and then with both secondary antibodies, to detect any cross-reaction between the antibodies. Sections were mounted in PBS : glycerol (1 : 1) and 2% DABCO (Sigma), coverslipped, and observed by laser confocal microscopy (Leica TCS-NT, Germany).
TABLE1. Combination of antisera used in each double immunolabelling reaction
Staining Primary antibodies Secondary antibodies*
D5/Enk Mouse anti-D5and rabbit anti-Enk Alexa 488-conjugated goat anti-mouse (green) and Alexa 568-conjugated goat anti-rabbit (red)
D5/Dyn Mouse anti-D5and rabbit anti-Dyn Alexa 488-conjugated goat anti-mouse (green) and Alexa 568-conjugated goat anti-rabbit (red)
D5/ChAT Rabbit anti-D5and goat anti-ChAT Alexa 488-conjugated donkey anti-rabbit (green) and Alexa 598-conjugated donkey anti-goat (red)
D5/SS Rabbit anti-D5and rat anti-SS Alexa 488-conjugated goat anti-rabbit (green) and Alexa 568-conjugated goat anti-rat (red)
D5/PV Rabbit anti-D5and mouse anti-PV Alexa 488-conjugated goat anti-rabbit (green) and Alexa 568-conjugated goat anti-mouse (red)
D5/CR Rabbit anti-D5and goat anti-CR Alexa 488-conjugated donkey anti-rabbit (green) and Alexa 598-conjugated donkey anti-goat (red)
*Secondary antibodies have been supplied by Molecular Probes, Eugene, OR, USA. Enk, enkephalin; Dyn, dynorphin; ChAT, choline acetyltransferase; SS, somatostatin; PV, parvalbumin; CR, calretinin.
Image analysis
The speci®c immuno¯uorescence of the ¯uorophores Alexa 488 or
Alexa 568/598 was visualized by excitation at the blue (4906
30 nm) or the green wavelength (545630 nm), respectively. The images are shown in Figs 3±5, D5immunoreactivity (IR) is illustrated
in green and Enk-, Dyn-, ChAT-, SS-, PV- and CR-immunoreactivity in red. A semiquantitative evaluation of the intensity of the
immuno¯uorescence for D5 receptors was carried out using an
image analysis system (Visiolog 5.1.1, Noesis, Paris). The intensity of speci®c D5IR was measured in a scale of 256 grey levels in each
double-labelling experiment. Non-speci®c levels of immunolabelling (background levels) were subtracted from the values obtained in D5
-positive cells. The neuronal phenotypes were identi®ed by the expression of speci®c markers for each neuronal population.
Differences in the intensity of D5 IR between each type of
interneuron and medium sized neurons (projection neurons), and between Enk- and Dyn-expressing neurons were expressed as the
mean percentage6SD.
Quantitative and statistical analyses
The number of neurons containing D5 receptors was analyzed
quantitatively and compared between the striosomes and the matrix of the rat striatum. Samples were taken from coronal sections of ®ve different rats. Cell counts and measurements of striatal areas were performed for both compartments with an image analysis system (Visiolog 5.1.1, Noesis, Paris). Before counting, images were thresholded at a standardized grey scale level empirically determined by two independent observers to allow detection of stained cells from low to high intensity, with suppression of negative cells. The number of positive cells per mm2 was determined in the striosomes and
compared with that obtained in the matrix. Statistical differences were determined by Student'st-test (P< 0.05).
Quantitative analysis was also performed to determine the
percentage colocalization of ChAT, SS, PV or CR with the D5
receptor subtype in the rat striatum and nucleus accumbens. Quanti®cation was performed on a confocal microscope at a magni®cation of 203. All striatal interneurons positive for ChAT, SS, PV or CR were counted in two sections (four hemispheres), from the rostral, middle and caudal levels of the striatum from ®ve different rats. The presence of the D5receptor subtype was assessed
by the yellow colour under confocal microscopy. The quantative differences between the receptor colocalization at the three different levels of the striatum were analyzed with a percentage comparison test (Lamotte, 1988).
Results
Localization of D5dopamine receptor in the rat and cat striatum
Immunostaining using either the polyclonal antiserum produced in our laboratory (Khan et al., 2000) or the commercial monoclonal antibody against the D5receptor (Chemicon, Temecula, CA) yielded
an identical pattern of D5receptor expression in the rat striatum. In
both cases we detected weak staining of medium size striatal cells, whereas large cells were stained more intensely. The identical distribution patterns and similar differential staining intensity between medium and large cells re¯ects the speci®city of the antibodies, despite the fact that they were raised in different species and against different epitopes of the antigen (Luedtkeet al., 1999; Khanet al., 2000).
Dopamine D5 receptor immunoreactivity (D5 IR) was mainly
associated to neuronal cell bodies in the rat striatum, in both the caudoputamen and nucleus accumbens (Fig. 1A). In the latter, moderate labelling was also observed in the neuropil. A similar distribution and intensity of D5 IR was observed in the cat caudate
nucleus and putamen (Fig. 1C). In both species, D5-positive neurons
were homogeneously distributed throughout the rostro-caudal, dorso-ventral and latero-medial axis of the striatum. No differences were
observed in the density of D5 IR neurons between striosomes
(9006223 neurons/mm2) and the matrix (9396246 neurons/mm2;
Fig. 2).
The D5-positive striatal neurons displayed different morphologies
and two populations of neurons were readily distinguished. One
population was composed of medium-size neurons (around 10mm
diameter) that displayed weak staining. In these cells, D5 IR was
mainly found in the peripheral regions of the somata and occasion-ally, in the primary dendrites. The labelling was punctate, consistent with the typical staining for receptors (Fig. 1B and D). This population of neurons was the most abundant and represented approximately 90% of the total D5-positive neurons. The second
population corresponds to large-size neurons (around 25mm diame-ter). D5 IR in these neurons was very intense covering the entire
cytoplasm and the primary dendrites demonstrating a punctate distribution of D5 receptors (Fig. 1B and D). This population
represented approximately 10% of all D5-positive striatal neurons.
Both populations were observed in the rat and cat striatum with no apparent differences between species. Similar observations have been made in the mouse and in all these species, D5IR in the striatum was
weaker than in the cortex or the hippocampus (data not shown).
Expression of D5dopamine receptor subtype in identi®ed striatal projection neurons
We next set out to identify the molecular phenotype of the different striatal neurons that express D5 receptors in the rat striatum. To
determine if the medium size D5-positive neurons belong to the direct
or indirect striatal output pathways, we carried out double immunolabelling experiments to determine whether D5 receptors
FIG. 1. Dopamine D5 receptor distribution in rat and cat striatum.
Photomicrographs of the immunostaining of dopamine D5 receptors in the
rat striatum (A and B) and cat caudate nucleus (C and D) are shown. The majority of D5-positive neurons are medium-size, although a few of them
are larger (arrows). B and D are high power images illustrating the neurons that express D5 receptor in the rat (B) and cat (D) striatum. Note the
stronger labelling of cell bodies and primary dendrites in the larger neurons (arrows) compared to the weak immunostaining in medium-size neurons (arrowheads). Scale bar, 80mm (A and C) and 20mm (B and D).
colocalized with dynorphin or enkephalin. Analysis of these experi-ments by confocal microscopy revealed that almost all enkephalin-positive neurons were also enkephalin-positive for D5 receptors (Fig. 3A±C).
However, in these experiments, a substantial number of neurons only expressed D5receptors (Fig. 3C). With respect to dynorphin, a high
percentage of dynorphin-positive neurons were also immunoreactive
FIG. 2. Homogeneous D5 receptor expression in the striosomal and matrix compartments of the rat striatum. (A) Photomicrograph illustrating dual labelled
immunocytochemistry with MOR1 (shown in brown) and D5IR neurons (shown in dark purple). Medium-sized and large neurons are found in both striatal
compartments. Arrows show examples of large neurons located within and outside of the striosome (asterisk). (B) Quantitative analysis of D5IR neurons in
the two striatal compartments. Bars indicate the number of immunoreactive cells per mm2in the striosomes (black column) and in the matrix (white column).
Error bars indicate standard deviation of the mean. No differences were found in the number of D5IR cells between striosomes and matrix. Scale bar, 50mm.
FIG. 3. Expression of dopamine D5 receptors in striatal projection neurons. Confocal laser photomicrographs illustrating the colocalization of D5 receptors
with enkephalin and dynorphin neuropeptides, markers for the direct and indirect striatal pathways. A and D show neurons labelled for D5 receptors, B
neurons with enkephalin and E neurons with dynorphin. Paired images showing double-labelled cells are presented in C for D5/enkephalin and in F for
for D5receptors (Fig. 3D±F). No difference in D5receptor content, as
assessed by the intensity of the staining, was evident between dynorphin- and enkephalin-positive neurons (see below). Together, these experiments indicate that D5receptors are expressed in neurons
of the direct and indirect striatal output pathways at similar weak intensity.
Expression of D5dopamine receptors in striatal interneurons
To determine if the large-size neurons that contain D5 receptors
belong to one or more types of striatal interneurons, we carried out double immunolabelling experiments to determine whether the polyclonal D5 antiserum colocalized with antibodies speci®c for
each of the classes of striatal interneurons. We found that D5
receptors are expressed in all striatal interneurons, but differences were observed in the number and the pattern of colocalization between each interneuronal class. Moreover, differences in signal intensity for the various types of interneurons were also evident.
Cholinergic interneurons
Double immunocytochemical studies showed that all ChAT
inter-neurons in the caudoputamen and nucleus accumbens express D5
receptors (Table 2). ChAT interneurons expressed D5IR in both cell
bodies and dendrites, and displayed the most intense levels when compared to other neuronal populations expressing D5 receptors
(Fig. 4A±C). Numerous double-labelled neurons (D5- and
ChAT-positive) neurons were also observed outside the striatum, especially in the diagonal band of Broca (data not shown). Thus, cholinergic neurons exhibit the highest dopamine D5 receptor content in the
striatum.
GABAergic interneurons containing SS, NPY and NOS
While in the striatum, a substantial number of SS interneurons were D5-positive (Fig. 4D±F), other SS neurons did not express D5
receptors (Fig. 4J±L). Because the morphology of SS interneurons varies, we analyzed whether these two types of neurons (SS-positive/ D5-positive and SS-positive/D5-negative) were also different with
respect to their morphology. No correlation between SS morphology and D5receptor expression was evident. Dense D5IR was observed in
the cell bodies of SS interneurons, and occasionally in primary dendrites. The percentage of SS cells expressing D5receptors was
69.4%66.0 in the caudoputamen and 83.2%613.8 in the nucleus accumbens (Table 2). This degree of colocalization was uniform throughout the rostro-caudal axis of the caudoputamen (Fig. 6A) and nucleus accumbens. While double-labelled cells were located preferentially in the medial striatum, SS interneurons lacking D5
receptors were scattered and mainly located in the lateral striatum (Fig. 6B). A graphical representation of the distribution of single
(SS-positive) and double (SS- and D5-positive) labelled-cells is depicted
in Fig. 6B.
GABAergic interneurons containing PV
A high degree of colocalization of PV IR and D5IR was observed in
striatal neurons (Fig. 5A±C). D5IR was seen only in cell bodies in
PV interneurons. Quanti®cation analysis of the double-labelled interneurons demonstrated that 73.1616.3% of PV-positive neurons express D5receptors in the caudoputamen and 85.267.4% in the
nucleus accumbens (Table 2). Rostral sections showed a higher percentage of colocalization (92.366.1%) than caudal sections (57.263.3%; Fig. 6C). Statistical analysis showed that differences in the number of D5- and PV-positive neurons between rostral,
middle, and caudal levels of the striatum were signi®cant (P< 0.01, percentage comparison test). Moreover, double-labelled cells were more abundant in the lateral striatum where a latero-medial gradient was observed (Fig. 6D).
GABAergic interneurons containing CR
Although GABAergic interneurons containing CR also express D5
receptors (Fig. 5D±F), colocalization of both proteins was less frequent than that observed for the other striatal interneurons. The percentage of colocalization was 47.669.1% in the caudoputamen
and 49.6612.4% in the nucleus accumbens (Table 2).
Double-labelled CR- and D5-positive cells were homogeneously distributed in
both regions (Fig. 6E and F). The percentages of colocalization of D5
receptors with each neuronal phenotype, as well as the distribution of double-labelled cells were consistent across the ®ve animals used in this experiment.
Finally, we carried out a semiquantitative analysis to evaluate the relative intensity of D5IR found in the different neuronal populations
studied. The intensity of D5IR in ChAT neurons was twofold higher
than that found in medium size neurons, while in SS and PV neurons it was approximately 7066.2% higher than that in medium size neurons. The intensity of D5IR in CR interneurons was similar to that
observed in medium sized neurons, differing only by 8.762.2%. D5
IR in dynorphin-positive cells was 664% more intense than in enkephalin-positive neurons. For each set of double-labelling experiments, a minimum of 10±15 interneurons and 20±30 medium size neurons were quanti®ed.
Discussion
Distribution of the D5dopamine receptor subtype in the striatum
We have shown here that D5-positive cells are distributed in a rather
homogeneous manner in the rat and cat striatum, in contrast to the pattern that we have reported for the dopamine D4receptors (Rivera
et al., 2002). This homogeneity was similar in both species, and is in agreement with previous reports of the immunocytochemical distri-bution of this receptor in the rat and monkey striatum (Bergsonet al., 1995a,b Arianoet al., 1997; Luedtkeet al., 1999; Ciliaxet al., 2000; Khanet al., 2000). However, in earlierin situhybridization studies and receptor autoradiography studies, authors were unable to detect D5receptors in the rat striatum (Tiberiet al., 1991; Meador-Woodruff
et al., 1992; Montague et al., 2001). These discrepancies are most probably due to the different techniques used to detect D5receptors or
to the low resolution of ®lm autoradiography. In addition, some species differences may exist, as there is strong evidence that D5
receptor transcripts are expressed in striatal cells in the monkey (Huntley et al., 1992; Rappaport et al., 1993; Choi et al., 1995).
TABLE2. Percentages of striatal interneurons * double-labelled for D5
receptor in the rat striatum
ChAT (%) SS (%) PV (%) CR (%)
CPu 100 69.4 70.5 46.9
(n= 1454) (n= 1712) (n= 1587) (n= 1384)
NAc 100 83.2 84.4 49.4
(n= 206) (n= 178) (n= 186) (n= 181)
*ChAT, choline acetyltransferase; SS, somatostatin; PV, parvalbumin; CR, calretinin. CPu, caudate putamen; NA, nucleus accumbens septi; n, number of neurons evaluated.
FIG. 6. Percentage and distribution of striatal interneurons that contain dopamine D5receptors. (A, C, and E) Bar graphs showing the percentage of SS- (A),
PV- (C) and CR- (D) positive interneurons that coexpress D5dopamine receptors. Mean percentages re¯ect the average of double-labelled cells counted in
sections corresponding to rostral, middle and caudal levels of the striatum. (B, D and F) Schematic representation of the distribution of single (white triangles) and double-labelled cells (black dots), SS (B), PV (D) or CR (F) and D5receptors. *P< 0.05, middle vs. caudal; **P< 0.05, rostral vs. middle and
caudal. Scale bar, 120mm (B, D and F).
FIG. 5. Expression of dopamine D5 receptors in striatal interneurons. Confocal laser photomicrographs illustrating the colocalization of D5 receptors with
parvalbumin (PV) and calretinin (CR). Neurons labelled with D5 receptors (A and D), parvalbumin (B), or calretinin (E). Paired images showing
double-labelled cells with D5/PV (C) and D5/CR (F). Arrows indicate double-labelled cells and arrowheads or asterisks single-labelled cells in the corresponding
images. Note that not all PV-positive neurons contain D5receptors (see asterisk in C). Scale bar, 30mm (A±C) and 20mm (D±F).
FIG. 4. Expression of dopamine D5 receptors in striatal interneurons. Confocal laser photomicrographs illustrating the colocalization of D5 receptors with
choline acetyltransferase (ChAT) and somatostatin (SS). Neurons labelled with D5receptors (A, D and G), ChAT (B) and SS (E and H). Paired images show
double-labelled cells with D5/ChAT (C) and D5/SS (F and I). Arrows indicate double-labelled cells and arrowheads or asterisks single-labelled cells in the
corresponding images. Note that all cholinergic neurons contain D5receptors (C), but not all SS-positive neurons contain D5receptors, see asterisk in I. Scale
bar 25mm (A±C) and 20mm (D±F).
These authors also found that the levels of expression of D5mRNA in
the striatum were lower than in the cortex or in the lateral thalamus. This is in agreement with the lower levels of D5IR that we found in
the striatum when compared to the cortex or the thalamus.
The large difference that we observed in the intensity of D5receptor
IR between medium and large striatal cells is in agreement with that previously reported in several species, including rat, monkey, and humans (Rappaportet al., 1993; Luedtkeet al., 1999; Ciliaxet al., 2000; Khan et al., 2000). However, we have now extended these observations to the cat. Interestingly, this immunocytochemical data correlates with the differences in the number of D5receptor transcripts
found by in situhybridization in large neurons when compared to medium size neurons in the monkey striatum (Rappaportet al., 1993). Taken together these results indicate that the levels and the pattern of expression of the D5receptor subtype is conserved in the mammalian
brain, moreover, they support a similar functional role for D5receptors
in the basal ganglia complex of all these species.
Dopamine D5receptor expression in the direct and indirect striatal output pathways
The presence of D5 receptors in neurons of the direct and indirect
striatal output pathways demonstrated here has important implica-tions. Psychomotor behavioural synergism has been reported in several studies following D1 and D2 receptor stimulation, both in
normal and in dopamine-depleted animals. This phenomenon has been dif®cult to explain, as the available evidence indicates that each one of these receptor subtypes are segregated in different striatal output pathways. Therefore, it has been proposed that an indirect mechanism exists, mediated by inter±neuronal interactions, that involves the activation of D1 and D2receptors on separate
popula-tions of striatal neurons, striatonigral and striatopallidal neurons (Gerfenet al., 1995). Although these interactions are feasible through the synaptic connections that are established between these two neuronal populations (Yung et al., 1996), our ®nding that D5
receptors are expressed in enkephalinergic neurons that also express D2 receptors provides another possible explanation for the D1/D2
synergism. As such, this synergism might arise through the stimu-lation of D5and D2receptor subtypes within the same cell, given that
D1 and D5 receptors are stimulated by the same ligands and are
coupled to identical transduction pathways (Grandy et al., 1991; Sunahara et al., 1991; Tiberi et al., 1991). This direct mechanism would not exclude other possible interactions (see, Alonso et al., 1999) and at present, represents only a further possibility as our results do not allow us to reach any solid conclusion. Further work will be necessary to explore and consolidate this hypothesis.
A similar direct synergism between D5/D2 receptor subtypes has
been documented (Svenningsson et al., 2000; Svenningsson & Le Moine, 2002). These authors found that combined treatment with SKF-82958, a D1/D5agonist, and quinelorane, a D2receptor agonist,
induced c-fos mRNA expression in a large number of cholinergic interneurons, while treatment with either of these two agonists alone had only a minor effect. Because D2 and D5 dopamine receptors
colocalize in these neurons, these results provide further evidence that a synergistic effect mediated through these receptors could occur within the same neuron. Our results are consistent with the existence of an intracellular pathway that would mediate D5/D2synergism in
medium sized neurons.
It should also be taken into account that SKF-82958, a full agonist
of D1/D5 receptors, up-regulates c-fos mRNA expression in
striatonigral (dynorphin-positive) and in striatopallidal (enkephalin-positive) neurons (Svenningssonet al., 2000). This result correlates with our ®nding that D5 receptors are expressed in
enkephalin-positive neurons in the striatum. It is important to note that c-fos
mRNA is induced in both enkephalinergic and dynorphin neurons through the interaction with a full agonist of class D1 receptors as previous experiments using a partial agonist, SKF 38393, failed to detect c-fos expression in enkephalinergic neurons. Indeed, these results provide strong evidence that D5receptors are both present and
functional in striatal enkephalinergic neurons.
Certain controversy exists regarding the colocalization of the two major receptor subtypes, D1and D2, in the same cells (Gerfenet al.,
1990; Le Moineet al., 1991; Surmeieret al., 1992, 1996; Aizmanet al., 2000). Most of the experiments supporting the colocalization of these two receptors are heavily based on functional studies in isolated striatal neurons, where D1- and D2-class agonists were frequently found to modulate voltage dependent Ca2+channels in the same cell (Surmeier
et al., 1992, 1996; Aizman et al., 2000). These results have been interpreted as evidence for coexpression of D1 and D2 receptor
subtypes without taking into consideration the presence of other receptor subtypes such as the D5receptor, which is also able to respond
to D1-class receptor ligands. Our results indicate that the electro-physiological effects of these D1-class ligands might be mediated by stimulation of D5receptors. Performing similar electrophysiological
studies in the D1KO mice would help to clarify this important issue.
D5dopamine receptors modulate dopaminergic function in striatal interneurons
The presence of D5 receptors in large striatal neurons has been
described previously (Rappaportet al., 1993; Bergsonet al., 1995a,b; Khan et al., 2000). However, the cholinergic phenotype of these neurons had been inferred on the basis of their large size. The results described in this paper provide unequivocal evidence of the molecular identity of these D5 receptor-expressing neurons, con®rming their
cholinergic nature. In addition, we found that all the other types of chemically de®ned striatal GABAergic interneurons expressing parvalbumin, somatostatin or calretinin (Kawaguchi et al., 1995) also express this receptor. The precise regional distribution of the colocalization with D5 receptors, medially for SS/NPY/NOS and
dorsolateral for PV, may re¯ect the differential D5-mediated
dopaminergic modulation of neostriatal circuits. Alternatively, this distribution may simply be the result of the typical distribution of each class of interneurons with which the D5receptor colocalizes.
The functional role of D5 receptors in all these classes of
interneurons has not been established. Cholinergic and somatostatin-positive neurons receive inputs from the thalamus (Lapper & Bolam, 1992) and the cortex (Vuillet et al., 1989), respectively, and make contacts with projection neurons and interneurons (Bolam et al., 2000). For this reason, and due to the profuse arborization and the length of their axons, it has been proposed that these neurons may correspond to associative neurons previously thought not to be present in the striatum (Kawaguchiet al., 1995). Strikingly, somatostatin- and ChAT-positive neurons exhibit the highest levels of D5 receptor
expression among striatal interneurons, suggesting that the functions of these associative neurons could be mediated through D5receptors.
The existence of ACh/DA interactions within the striatum has been well documented (Di Chiara et al., 1994). Pharmacological studies demonstrate that D1- and D2-class receptors play different roles in ACh/DA interactions. Agonists of D2-class receptors inhibit ACh release (Bertorelli & Consolo, 1990; Acquas & Di Chiara, 1999), consistent with the presence of D2receptor subtypes in cholinergic
interneurons (Le Moineet al., 1990; Aubryet al., 1993; MacLennan
et al., 1994; Aubertet al., 2000). In contrast, ligands of D1-class receptors facilitate ACh release (Bertorelli & Consolo, 1990; Damsma
receptors are not present in cholinergic interneurons (Le Moineet al., 1991; Yan & Surmeier, 1997). Therefore, the dopamine-stimulated release of ACh from striatal cholinergic neurons could be due, as our data suggest, to D5receptor stimulation, as D5receptors are present at
high density and in all cholinergic neurons in the striatum. Further experimental work will be necessary to establish the role of D5
receptors in the release of ACh in the striatum. However, it is important to point out that a similar situation has been described in the hippocampus, where, using antisense oligonucleotides against D1and
D5receptors, it has been demonstrated that ACh release is regulated by
the stimulation of D5receptors (Hersiet al., 2000).
In addition, cholinergic neurons respond to classical conditioning paradigms, where a sensory stimuli is associated with a reward during the learning process (Aosaki et al., 1994b). This response is dependent upon dopaminergic input from the cell groups of the midbrain, and it is maintained for a long time after learning (Aosaki
et al., 1994b). This long-term maintenance of the conditioned response could be due to a dopamine-dependent long-term change in the synaptic ef®ciency of striatal neurons. In fact, it has recently been shown that high frequency stimulation induces long-term potentiation (LTP) in the striatum (Calabresiet al., 2000; Centonzeet al., 2001), and in particular in cholinergic neurons (Aosakiet al., 1998; Suzuki
et al., 2001). This change in the synaptic ef®ciency of cholinergic neurons induced by LTP is dependent on D1-class receptor stimu-lation but not on NMDA receptor stimustimu-lation. As these cells do not express D1type receptors, our results indicate that D5receptors must
be those that modulate this response. Therefore, an involvement of D5
receptors in the long term memory process coupled to associative learning can now be proposed.
Taking these results together, it appears that a segregation of neurons expressing D1and D5receptor subtypes exists in the striatum.
While D1 receptors are highly expressed in projection neurons, D5
receptors are mostly present in interneurons. Interneurons are a fundamental component for striatal function because they modulate the activity of projection neurons. The high percentage of D5receptors
in interneurons implies a relevant role for this receptor subtype in the regulation of striatal activity. In addition, molecular genetic approaches using antisense oligonucleotides against D1 and D5
receptors or KO mouse technology for D1receptors, have revealed a
different role for each receptor subtype in the control of locomotion (Dragoet al., 1994; Xuet al., 1994; Dziewczapolskiet al., 1997; Hersi
et al., 2000). In summary, our results together with those in the literature suggest that D1and D5receptor subtypes, by virtue of being
expressed in different neurons, may subserve different roles in the striatal dopaminergic transmission, despite being stimulated by the same ligands and sharing signal transduction pathways.
Acknowledgements
This work was supported by the Spanish Ministerio de Ciencia y TecnologõÁa, ref SAF00/122 and by FundacioÂn La Caixa to R.M., and by PM98/0223 and JA (CTS-0161) to A.C. A.R. is supported by a fellowship from MEC and I.A and A.B.M. by fellowships from CAM, Madrid, Spain. We thank JesuÂs SantamarõÂa and Concha BailoÂn for assistance in the confocal microscopy.
Abbreviations
ACh, acetyl choline; ChAT, choline acetyltransferase; CR, calretinin; DA, dopamine; D1-class, D1and D5receptor subtypes; D2-class, D2, D3and D4
receptor subtypes; Dyn, dynorphin; Enk, enkephalin; IR, immunoreactivity; NOS, nitric oxide synthase; NPY, neuropeptide Y; MOR1, mu opioid receptor 1; PB, phosphate buffer; PBS, phosphate-buffered saline; PV, parvalbumin; SS, somatostatin.
References
Acquas, E. & Di Chiara, G. (1999) Dopamine D1receptor-mediated control of
striatal acetylcholine release by endogenous dopamine.Eur. J. Pharmacol.,
383, 121±127.
Aizman, O., Brismar, H., Uhlen, P., Zettergren, E., Levey, A.I., Forssberg, H., Greengard, P. & Aperia, A. (2000) Anatomical and physiological evidence for D1 and D2 dopamine receptor colocalization in neostriatal neurons. Nature Neurosci.,3, 226±230.
Alonso, R., Gnanadicom, H., Frechin, N., La Fur, G. & SoubrieÂ, P. (1999) Blockade of neurotensin receptors suppresses the dopamine D1/D2
synergism on immediate early gene expression in the rat brain. Eur. J. Neurosci.,11, 967±974.
Aosaki, T., Graybiel, A.M. & Kimura, M. (1994a) Effect of the nigrostriatal dopamine system on acquired neural responses in the striatum of behaving monkeys.Science,265, 412±415.
Aosaki, T., Kiuchi, K. & Kawaguchi, Y. (1998) Dopamine D1-like receptor
activation excites rat striatal large aspiny neuronsin vitro.J. Neurosci.,18, 5180±5190.
Aosaki, T., Tsubokawa, H., Ishida, A., Watanabe, K., Graybiel, A.M. & Kimura, M. (1994b) Responses of tonically active neurons in the primate's striatum undergo systematic changes during behavioral sensorimotor conditioning.J. Neurosci.,14, 3969±3984.
Ariano, M.A., Wang, J., Noblett, K.L., Larson, E.R. & Sibley, D.R. (1997) Cellular distribution of the rat D1B receptor in central nervous system using anti-receptor antisera.Brain Res.,746, 141±150.
Arvidsson, U., Riedl, M., Chakrabarti, S., Lee, J.H., Nakano, A.H., Dado, R.J., Loh, H.H., Law, P.Y., Wessendorf, M.W. & Elde, R. (1995) Distribution and targeting of a mu-opioid receptor (MOR1) in brain and spinal cord.J. Neurosci.,15, 3328±3341.
Aubert, I., Ghorayeb, I., Normand, E. & Bloch, B. (2000) Phenotypical characterization of the neurons expressing the D1 and D2 dopamine
receptors in the monkey striatum.J. Comp. Neurol.,418, 22±32. Aubry, J.M., Schulz, M.F., Pagliusi, S. & Kiss, J.Z. (1993) Coexpression of
dopamine D2 and substance P (neurokinin-1) receptor messenger RNAs by a subpopulation of cholinergic neurons in the rat striatum.Neuroscience,53, 417±424.
Bergson, C., Mrzljak, L., Lidow, M.S., Goldman-Rakic, P.S. & Levenson, R. (1995b) Characterization of subtype-speci®c antibodies to the human D5
dopamine receptor: studies in primate brain and transfected mammalian cells.Proc. Natl Acad. Sci. USA,92, 3468±3472.
Bergson, C., Mrzljak, L., Smiley, J.F., Pappy, M., Levenson, R. & Goldman-Rakic, P.S. (1995a) Regional, cellular and subcellular variations in the distribution of D1and D5 dopamine receptors in primate brain.J. Neurosci.,
15, 7821±7836.
Bertorelli, R. & Consolo, S. (1990) D1 and D2dopaminergic regulation of
acetylcholine release from striata of freely moving rats.J. Neurochem.,54, 2145±2148.
Bolam, J.P., Hanley, J.J., Booth, P.A. & Bevan, M.D. (2000) Synaptic organisation of the basal ganglia.J. Anat.,196, 527±542.
Calabresi, P., Centonze, D., Gubellini, P., Pisani, A. & Bernardi, G. (1998) Endogenous ACh enhances striatal NMDA-responses via M1-like muscarinic receptors and PKC activation.Eur. J. Neurosci.,9, 2887±2895. Calabresi, P., Gubellini, P., Centonze, D., Picconi, B., Bernardi, G., Chergui, K., Svenningsson, P., Fienberg, A.A. & Greengard, P. (2000) Dopamine and cAMP-regulated phosphoprotein 32 kDa controls both striatal long-term depression and long-term potentiation, opposing forms of synaptic plasticity.J. Neurosci.,20, 8443±8851.
Centonze, D., Picconi, B., Baunez, C., Borrelli, E., Pisan, A., Bernardi, G. & Calabresi, P. (2002) Cocaine and amphetamine depress striatal GABAergic synaptic transmission through D2 dopamine receptors. Neuropsychopharmacology,26, 164±175.
Centonze, D., Picconi, B., Gubellini, P., Bernardi, G. & Calabresi, P. (2001) Dopaminergic control of synaptic plasticity in the dorsal.Eur. J. Neurosci.,
13, 1071±1077.
Choi, W.S., Machida, C.A. & Ronnekleiv, O.K. (1995) Distribution of dopamine D1, D2 and D5 receptor mRNAs in the monkey brain:
ribonuclease protection assay analysis. Brain Res. Mol. Brain Res., 31, 86±94.
Ciliax, B.J., Nash, N., Heilman, C., Sunahara, R., Hartney, A., Tiberi, M., Rye, D.B., Caron, M.G., Niznik, H.B. & Levey, A.I. (2000) Dopamine D5
receptor immunolocalization in rat and monkey brain.Synapse,37, 125± 145.
Consolo, S., Girotti, P., Russi, G. & Di Chiara, G. (1992) Endogenous dopamine facilitates striatalin vivoacetylcholine release by acting on D1
receptors localized in the striatum.J. Neurochem.,59, 1555±1557.
Damsma, G., Tham, C.S., Robertson, G.S. & Fibiger, H.C. (1990) Dopamine D1 receptor stimulation increases striatal acetylcholine release in the rat. Eur. J. Pharmacol.,186, 335±338.
Di Chiara, G., Morelli, M. & Consolo, S. (1994) Modulatory functions of neurotransmitters in the striatum: ACh/dopamine/NMDA interactions. Trends Neurosci.,17, 228±233.
Drago, J., Gerfen, C.R., Lachowicz, J.E., Steiner, H., Hollon, T.R., Love, P.E., Ooi, G.T., Grinberg, A., Lee, E.J., Huang, S.P., Bartlett, P.F., Jose, P.A., Sibley, D.R. & Westphal, H.I. (1994) Altered striatal function in a mutant mouse lacking D1A dopamine receptors.Proc. Natl Acad. Sci. USA,91, 12564±12568.
Dziewczapolski, G., Menalled, L.B., GarcõÁa, M.C., Mora, M.A., Gershanik, O.S. & Rubinstein, M. (1997) Opposite roles of D1 and D5 dopamine
receptors in locomotion revealed by selective antisense oligonucleotides. Neuroreport,9, 1±5.
Fauchey, V., Jaber, M., Caron, M.G., Bloch, B. & Le Moine, C. (2000) Differential regulation of the dopamine D1, D2 and D3 receptor gene
expression and changes in the phenotype of the striatal neurons in mice lacking the dopamine transporter.Eur. J. Neurosci.,12, 19±26.
Gerfen, C.R., Engber, T.M., Mahan, L.C., Susel, Z., Chase, T.N., Monsma, F.J. Jr & Sibley, D.R. (1990) D1and D2dopamine receptor-regulated gene
expression of striatonigral and striatopallidal neurons.Science,250, 1429± 1432.
Gerfen, C.R., Keefe, K.A. & Gauda, E.B. (1995) D1and D2dopamine receptor
function in the striatum: coactivation of D1- and D2-dopamine receptors on
separate populations of neurons results in potentiated immediate early gene response in D1-containing neurons.J. Neurosci.,12, 8167±8176.
Grandy, D.K., Zhang, Y.A., Bourier, C., Zhou, Q.Y., Johnson, R.A., Allen, L., Buck, K., Bunzow, J.R., Salon, J. & Civelli, O. (1991) Multiple human D5
dopamine receptor genes: a functional receptor and two pseudogenes.Proc. Natl Acad. Sci. USA,88, 9175±9179.
Hersi, A.I., Kitaichi, K., Srivasta, L.K., Gaudreau, P. & Quirino, R. (2000) Dopamine D5receptor modulates hippocampal acetylcholine release.Mol. Brain Res.,76, 336±340.
Huntley, G.W., Morrison, J.H., Prikhozhan, A. & Sealfon, S.C. (1992) Localization of multiple dopamine receptor subtype mRNAs in human and monkey motor cortex and striatum.Brain Res. Mol. Brain.,15, 181±188. Kawaguchi, Y., Wilson, C.J., Augood, S.J. & Emson, P.C. (1995) Striatal
interneurons: chemical, physiological and morphological characterization. Trends Neurosci.,18, 527±535.
Khan, Z.U., Gutierrez, A., Martin, R., PenÄa®el, A., Rivera, A. & de la Calle, A. (2000) Dopamine D5receptors of rat and human brain.Neuroscience,100,
689±699.
Koos, T. & Tepper, J.M. (2002) Dual cholinergic control of fast-spiking interneurons in the neostriatum.J. Neurosci.,22, 529±535.
Lamotte, M. (1988) EstadõÂstica bioloÂgica.Principios Fundamentales. Masson SA, Paris.
Lapper, S.R. & Bolam, J.P. (1992) Input from the frontal cortex and the parafascicular nucleus to cholinergic interneurons in the dorsal striatum of the rat.Neuroscience,51, 533±545.
Le Moine, C., Normand, E. & Bloch, B. (1991) Phenotypical characterization of the rat striatal neurons expressing the D1dopamine receptor gene.Proc. Natl Acad. Sci. USA,88, 4205±4209.
Le Moine, C., Tison, F. & Bloch, B. (1990) D2 dopamine receptor gene
expression by cholinergic neurons in the rat striatum.Neurosci. Lett.,117, 248±252.
Luedtke, R.R., Grif®n, S.A., Conroy, S.S., Jin, X., Pino, A. & Sesack, S.R. (1999) Immunoblot and immunohistochemical comparison of murine monoclonal antibodies speci®c for the rat D1a and D1b dopamine receptor subtypes.J. Neuroimmunol.,101, 170±187.
MacLennan, A.J., Lee, N., Vincent, S.R. & Walker, D.W. (1994) D2dopamine
receptor mRNA distribution in cholinergic and somatostatinergic cells of the rat caudate-putamen and nucleus accumbens.Neurosci. Lett.,180, 214± 218.
Meador-Woodruff, J.H., Mansour, A., Grandy, D.K., Damask, S.P., Civelli, O. & Watson, S.J. (1992) Distribution of D5dopamine receptor mRNA in rat
brain.Neurosci. Lett.,145, 209±212.
Milstein, C. & Cuello, A.C. (1983) Hybrid hybridomas and their use in immunohistochemistry.Nature,305, 537±540.
Missale, C., Nash, S.R., Robinson, S.W., Jaber, M. & Caron, M.G. (1998) Dopamine receptors: from structure to function. Physiol. Rev., 78, 189±225.
Montague, D.M., Striplin, C.D., Overcash, J.S., Drago, J., Lawler, C.P. &
Mailman, R.B. (2001) Quanti®cation of D1B (D5) receptors in dopamine
D1A receptor-de®cient mice.Synapse,39, 319±322.
Moratalla, R., Elibol, B., Vallejo, M. & Graybiel, A.M. (1996a) Network-level changes in expression of inducible Fos-Jun proteins in the striatum during chronic cocaine treatment and withdrawal.Neuron,17, 147±156. Moratalla, R., Robertson, H.A. & Graybiel, A.M. (1992) Dynamic regulation
of NGFI-A (zif268, egr1) gene expression in the striatum.J. Neurosci.,12, 2609±2622.
Moratalla, R., Xu, M., Tonegawa, S. & Graybiel, A.M. (1996b) Cellular responses to psychomotor stimulant and neuroleptic drugs are abnormal in mice lacking the D1dopamine receptor.Proc. Natl Acad. Sci. USA,93,
14928±14933.
Pisani, A., Bonsi, P., Centonze, D., Calabresi, P. & Bernardi, G. (2000) Activation of D2-like dopamine receptors reduces synaptic inputs to striatal
cholinergic interneurons.J. Neurosci.,20, 1±6.
Rappaport, M.S., Sealfon, S.C., Prikhozhan, A., Huntley, G.W. & Morrison, J.H. (1993) Heterogeneous distribution of D1, D2and D5receptor mRNAs
in monkey striatum.Brain Res.,616, 242±250.
Rico, B. & Cavada, C. (1998) A population of cholinergic neurons is present in the macaque monkey thalamus.Eur. J. Neurosci.,10, 2346±2352. Rivera, A., Cuellar, B., Giron, F.J., Grandy, D.K., de la Calle, A. & Moratalla,
R. (2002) Dopamine D4 receptors are heterogeneously distributed in the
striosomes/matrix compartments of the striatum.J. Neurochem.,80, 219± 229.
Sunahara, R.K., Guan, H.C., OõÂDowd, B.F., Seeman, P., Laurier, L.G., Ng, G., George, S.R., Torchia, J., Van Tol, H.H.M. & Niznik, H.B. (1991) Cloning of the gene for a human dopamine D5 receptor with higher af®nity for
dopamine than D1.Nature,350, 614±619.
Surmeier, D.J., Eberwine, J., Wilson, C.J., Cao, Y., Stefani, A. & Kitai, S.T. (1992) Dopamine receptor subtypes colocalize in rat striatonigral neurons. Proc. Natl Acad. Sci. USA,89, 10178±10182.
Surmeier, D.J., Song, W.J. & Yan, Z. (1996) Coordinated expression of dopamine receptors in neostriatal medium spiny neurons.J. Neurosci.,16, 6579±6591.
Suzuki, T., Miura, M., Nishimura, K. & Aosaki, T. (2001) Dopamine-dependent synaptic plasticity in the striatal cholinergic interneurons. J. Neurosci.,21, 6492±6501.
Svenningsson, P., Fredholm, B.B., Bloch, B. & Le Moine, C. (2000) Co-stimulation of D1/D5 and D2dopamine receptors leads to an increase in c-fosmessenger RNA in cholinergic interneurons and a redistribution of c-fosmessenger RNA in striatal projection neurons.Neuroscience,98, 749± 757.
Svenningsson, P. & Le Moine, C. (2002) Dopamine D1/D5 receptor
stimulation inducesc-fosexpression in the subthalimic nucleus: possible involvement of local D5receptors.Eur. J. Neurosci.,15, 133±142.
Tan, A., Moratalla, R., Lyford, G.L., Worley, P. & Graybiel, A.M. (2000) The activity-regulated cytoskeletal-associated protein arc is expressed in different striosome-matrix patterns following exposure to amphetamine and cocaine.J. Neurochem.,74, 2074±2078.
Tiberi, M., Jarvie, K.R., Silvia, C., Falardeau, P., Gingrich, J.A., Gonidot, N., Bertrand, L., Yang-Feng, T.L. Jr, Fremeau, R.T. & Caron, M.G. (1991) Cloning, molecular characterization and chromosomal assignment of a gene encoding a second D1 dopamine receptor subtype: differential expression pattern in rat brain compared with D1A receptor.Proc. Natl Acad. Sci. USA,
88, 7491±7495.
Uslaner, J., Badiani, A., Norton, C.S., Day, H.E., Watson, S.J., Akil, H. & Robinson, T.E. (2001) Amphetamine and cocaine induce different patterns of c-fos mRNA expression in the striatum and subthalamic nucleus depending on environmental context.Eur. J. Neurosci.,13, 1977±1983. Vuillet, J., Kerkerian, L., Kachidian, P., Bosler, O. & Nieoullon, A. (1989)
Ultrastructural correlates of functional relationships between nigral dopaminergic or cortical afferent ®bers and neuropeptide Y-containing neurons in the rat striatum.Neurosci. Lett.,100, 99±104.
Xu, M., Moratalla, R., Gold, L.H., Hiroi, N., Koob, G.F., Graybiel, A.M. & Tonegawa, S. (1994) Dopamine D1 receptor mutant mice are de®cient in
striatal expression of dynorphin and in dopamine-mediated behavioural responses.Cell,79, 729±742.
Yan, Z. & Surmeier, D.J. (1997) D5 dopamine receptors enhance
Zn2+-sensitive GABA (A) currents in striatal cholinergic interneurons through a PKA/PP1 cascade.Neuron,19, 1115±1126.
Yung, K.K., Smith, A.D., Levey, A.I. & Bolam, J.P. (1996) Synaptic connections between spiny neurons of the direct and indirect pathways in the neostriatum of the rat: evidence from dopamine receptor and neuropeptide immunostaining.Eur. J. Neurosci.,5, 861±869.
