Functional differences between D(1) and D(5) revealed by high resolution imaging on live neurons.
ABSTRACT The interaction between the dopaminergic and glutamatergic systems governs normal behavior and is perturbed in many psychiatric disorders including schizophrenia. Hypofunction of the D1 family of receptors, to which the D(1) and D(5) subtypes belong, is a typical feature of schizophrenia. Here we have used confocal live cell imaging of neurons to examine the distinct roles of the D(1) and D(5) receptors in the intra-neuronal interaction with the glutamatergic system. Using fluorescently tagged D(1) or D(5) expressed in cultured striatal neurons, we show that both receptor subtypes are primarily transported via lateral diffusion in the dendritic tree. D(1) is to a much larger extent than D(5) expressed in spines. D(1) is primarily expressed in the head whereas D(5) is largely localized to the neck of the spine. Activation of N-methyl-D-aspartic acid (NMDA) receptors slowed the diffusion rate and increased the number of D(1) positive spines, while no effect on D(5) diffusion or spine localization could be observed. The observed differences between D(1) and D(5) can be attributed to structural differences in the C-terminus and its capacity to interact with NMDA receptors and PSD-95. Identification of a unique role of D(1) for the intra-neuronal interaction between the dopaminergic and glutamatergic systems will have implications for the development of more specific treatments in many neuropsychiatric disorders.
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ABSTRACT: ABSTRACT Excessive activation of the N-Methyl-D-Aspartate (NMDA) receptor and the neurotransmitter dopamine (DA) mediate neurotoxicity and neurodegeneration under many neurological conditions, including Huntington's disease (HD), an autosomal dominant neurodegenerative disease characterized by the preferential loss of medium spiny projection neurons (MSNs) in the striatum. PSD-95 is a major scaffolding protein in the postsynaptic density (PSD) of dendritic spines, where a classical role for PSD-95 is to stabilize glutamate receptors at sites of synaptic transmission. Our recent studies indicate that PSD-95 also interacts with the D1 DA receptor localized in spines and negatively regulates spine D1 signaling. Moreover, PSD-95 forms ternary protein complexes with D1 and NMDA receptors, and plays a role in limiting the reciprocal potentiation between both receptors from being escalated. These studies suggest a neuroprotective role for PSD-95. Here we show that mice lacking PSD-95, resulting from genetic deletion of the GK domain of PSD-95 (PSD-95-ΔGK mice), sporadically develop progressive neurological impairments characterized by hypolocomotion, limb clasping, and loss of DARPP-32-positive MSNs. Electrophysiological experiments indicated that NMDA receptors in mutant MSNs were overactive, suggested by larger, NMDA receptor-mediated miniature excitatory postsynaptic currents (EPSCs) and higher ratios of NMDA- to AMPA-mediated corticostriatal synaptic transmission. In addition, NMDA receptor currents in mutant cortical neurons were more sensitive to potentiation by the D1 receptor agonist SKF81297. Finally, repeated administration of the psychostimulant cocaine at a dose regimen not producing overt toxicity-related phenotypes in normal mice reliably converted asymptomatic mutant mice to clasping symptomatic mice. These results support the hypothesis that deletion of PSD-95 in mutant mice produces concomitant overactivation of both D1 and NMDA receptors that makes neurons more susceptible to NMDA excitotoxicity, causing neuronal damage and neurological impairments. Understanding PSD-95-dependent neuroprotective mechanisms may help elucidate processes underlying neurodegeneration in HD and other neurological disorders.Journal of neurogenetics 04/2014; · 0.73 Impact Factor
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ABSTRACT: A state of low dopaminergic activity has been implicated in attention-deficit/hyperactivity disorder (ADHD). The clinical symptoms of ADHD include inattention, impulsivity and hyperactivity, as well as impaired learning; dopaminergic modulation of the functions in the hippocampus is important to both learning and memory. To determine dopamine receptor density in a well-established animal model for ADHD, we quantified the dopamine D5 receptors in the hippocampus in the spontaneously hypertensive rat. We used immunofluorescence microscopy and immunogold electron microscopy to quantify the dopamine D5 receptor density on CA1 pyramidal cell somas and dendrites and dendritic spines in stratum radiatum and stratum oriens. The density of the dopamine D5 receptors was significantly lower in the cytoplasm of pyramidal cell somas in the spontaneously hypertensive rat compared to the control, indicating a reduced reservoir for insertion of receptors into the plasma membrane. Dopamine receptors are important for long term potentiation and long term depression, hence the deficit may contribute to the learning difficulties in individuals with the diagnosis of ADHD.Neuroscience 03/2013; · 3.33 Impact Factor
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ABSTRACT: Dopamine is an inhibitory neurotransmitter involved in the pathology of schizophrenia.The revised dopamine hypothesis states that dopamine abnormalities in the mesolimbic and prefrontal brain regions exist in schizophrenia. However, recent research has indicated that glutamate, GABA, acetylcholine, and serotonin alterations are also involved in the pathology of schizophrenia. This review provides an in-depth analysis of dopamine in animal models of schizophrenia and also focuses on dopamine and cognition. Furthermore, this review provides not only an overview of dopamine receptors and the antipsychotic effects of treatments targeting them but also an outline of dopamine and its interaction with other neurochemical models of schizophrenia. The roles of dopamine in the evolution of the human brain and human mental abilities, which are affected in schizophrenia patients, are also discussed.Frontiers in Psychiatry 05/2014; 5.
FUNCTIONAL DIFFERENCES BETWEEN D1AND D5REVEALED BY
HIGH RESOLUTION IMAGING ON LIVE NEURONS
M. KRUUSMÄGI, S. KUMAR, S. ZELENIN, H. BRISMAR,
A. APERIA AND L. SCOTT*
Department of Women’s and Children’s Health, Karolinska Institutet,
Abstract—The interaction between the dopaminergic and glu-
tamatergic systems governs normal behavior and is perturbed
in many psychiatric disorders including schizophrenia. Hypo-
function of the D1 family of receptors, to which the D1and D5
subtypes belong, is a typical feature of schizophrenia. Here we
have used confocal live cell imaging of neurons to examine the
distinct roles of the D1and D5receptors in the intra-neuronal
interaction with the glutamatergic system. Using fluorescently
that both receptor subtypes are primarily transported via lateral
diffusion in the dendritic tree. D1is to a much larger extent than
D5expressed in spines. D1is primarily expressed in the head
whereas D5is largely localized to the neck of the spine. Activa-
tion of N-methyl-D-aspartic acid (NMDA) receptors slowed the
diffusion rate and increased the number of D1positive spines,
while no effect on D5diffusion or spine localization could be
observed. The observed differences between D1and D5can be
attributed to structural differences in the C-terminus and its
capacity to interact with NMDA receptors and PSD-95. Identifi-
cation of a unique role of D1for the intra-neuronal interaction
between the dopaminergic and glutamatergic systems will have
implications for the development of more specific treatments in
many neuropsychiatric disorders. © 2009 IBRO. Published by
Elsevier Ltd. All rights reserved.
Key words: D1, D5, NMDA receptor, lateral diffusion, spines,
The D1 family of dopamine receptors plays a key role for
cognition and for organization of normal behavior (Missale
et al., 1998). Activation of the D1 family of receptors trans-
lates into various downstream effects, which are to a large
extent dependent on an intra-neuronal interaction between
the dopamine D1 family of receptors and N-methyl-D-as-
partic acid (NMDA) receptors (Castner and Williams,
2007). Perturbation of this interaction has been implicated
in psychiatric disorders, such as schizophrenia (Laruelle et
al., 2003). There are two subtypes of the dopamine D1
family of receptors, D1and D5, which both couple to G?s.
The receptors share 80% homology in their transmem-
brane domains, while the C-termini exhibit much lower
homology (Missale et al., 1998). Currently there are no
pharmacological tools that can distinguish between D1and
D5. Consequently, it is not known whether they play dis-
tinct roles with regard to the intra-neuronal interaction with
the glutamatergic system.
There is an emerging concept, based on the use of live
cell imaging, that the mobility of receptors in the dendritic
tree and the confinement of receptors at functional sites
have a significant impact on neuronal plasticity (Triller and
Choquet, 2003; Groc and Choquet, 2008; Jaskolski and
Henley, 2009). We have in a recent study shown that D1
can be trapped in spines by ligand-occupied NMDA recep-
tors (Scott et al., 2006). Corresponding information about
D5is lacking. In this study we have compared the behavior
of D1and D5and their capacity to interact with the NMDA
receptor. For this purpose, D1or D5,tagged with a fluores-
cent protein, was expressed in cultured rat striatum. Using
live cell imaging, we show that both receptor subtypes
diffuse freely in the plasma membrane, but that only D1,
and not D5, is confined by interaction with the NMDA
receptor. Both live cell imaging and biochemical studies
support the notion that D1, but not D5receptor, is a partner
in the postsynaptic density (PSD). Since interaction be-
tween the dopaminergic and glutamatergic systems plays
an important role for normal behavior and is perturbed in
many psychiatric disorders, these results may have impli-
cations for the development of new treatments.
Neuronal cultures and transfection
Striatal organotypic cultures were prepared from 5–6-day-old
Sprague–Dawley rats (Scanbur AB, Sollentuna, Sweden), as pre-
viously described (Scott et al., 2006). Briefly, the striatum was
excised under a microscope and subsequently sliced in 250 ?m
thick coronal slices. The striatal slices were then attached to
cover-slips using thrombin-coagulated chicken plasma (Sigma,
Steinheim, Germany). Cultures were grown in Dulbecco’s Modi-
fied Eagle’s Medium (55%) and Hank’s balanced salt solution
(32%), 0.297% glucose, 0.01 M HEPES, 10% fetal calf serum and
1% antibiotic-antimycotic (Invitrogen, Cat.nr: 15240–096). Cul-
tures were kept in an incubator with a roller drum at 37 °C at an
approximate humidity of 95–98% with a CO2concentration of 5%,
and grown for 2?3 weeks before transfection. All media compo-
nents were from Invitrogen (Paisley, UK). During the time in
culture neurons continue to differentiate and form more cell to cell
contacts. After 2 weeks in culture the neurons display spontane-
ous calcium activity (recorded using Fluo-4 calcium sensitive dye
loading, data not shown) and express both NMDA and GABAA
shown). Biolistic transfection was performed as previously de-
scribed (Scott et al., 2006). Briefly, preparation of gold microcar-
riers was performed according to the manufacturer’s instructions
*Correspondence to: L. Scott, Department of Women’s and Children’s
Health, Research Laboratory Q2:09, Karolinska University Hospital,
s-17176, Stockholm, Sweden. Tel: ?46-8-51777326; fax: ?46-8-
E-mail address: Lena.Scott@ki.se (L. Scott).
Abbreviations: FRAP, fluorescence recovery after photobleaching;
GPCR, G-protein coupled receptor; GST, glutathione S-transferase;
NMDA, N-methyl-D-aspartic acid; PSD-95, postsynaptic density-95.
Neuroscience 164 (2009) 463–469
0306-4522/09 $ - see front matter © 2009 IBRO. Published by Elsevier Ltd. All rights reserved.
(Bio-Rad, Hercules, CA, USA), except for the use of polyvinylpyr-
rolidone, which was excluded. For each 8 mg of 1.0 ?m gold
microcarriers (Bio-Rad), 10 ?g of DNA was used. Transfection
was performed using helium gas at 90 psi. Electroporation was
performed using the Cellaxess CX 1 system (Cellectricon AB,
Mölndal, Sweden). Cultures were transferred to a Petri dish con-
taining Dulbecco’s Modified Eagle’s Medium and then subject to
electroporation by, 25 ms pulses at 270 V using 20 ?l of DNA at
a concentration of 0.3 ?g ?l?1dispensed at 20 ?l min?1. Follow-
ing transfection cultures were returned to the incubator and cul-
tured for another 24–48 h. Experiments were performed on me-
dium spiny neurons selected based on their morphology. All ex-
perimental procedures were painless for the animals, and care
was taken to minimize the number of animals used in this study.
All procedures comply with internationally approved standards for
cDNA encoding full-length rat D1, was cloned into pVenus–N3
vector with a CMV promoter (pD1–Venus). pD1–Venus was mod-
ified to obtain a mutant cDNA encoding rat D1receptor with
truncation of the C-terminus tail at L390 (D1
was created by cloning rat D5cDNA into pVenus–N3. The pD5–
Venus was further modified by removing the 54 last amino acids
of the D5(D5
ferase (GST) fusion proteins with D1C-terminal residues L387–
T446, and D5C-terminal residues F360–S415 were cloned in
pDEST15 vectors by using site-specific recombination (Gateway
Technology, Invitrogen). The D5C-terminal residues E421–A475
were amplified with specific primers and cloned in pGEX–KG (GE
Healthcare Life Science, Uppsala, Sweden). The structure of all
constructs was confirmed by DNA sequencing.
?CTtail). Constructs encoding glutathione S-trans-
Live measurements and fluorescence recovery after
All experiments were carried out using a Zeiss 510 LSM laser
scanning confocal microscope (Zeiss, Jena, Germany). The 514
nm laser line of a 40 mW argon laser was used for both imaging
and bleaching. A 40?0.8 NA water dipping lens was used. Cells
were continuously perfused with physiological phosphate buffer
(in mM: 140 NaCl, 5.4 KCl, 25 HEPES, 33 glucose, 2 MgCl2, and
1.3 CaCl2) through an inline-heater (Warner Instruments, Ham-
den, CT, USA) set at 37 °C. For live measurements of spines a
three-dimensional stack was recorded for a dendrite of interest
during control conditions and 15 min after NMDA exposure (100
?M for 30 s). Images were analyzed and the number of spines on
each dendrite was determined by examining all images in the
stack. For fluorescence recovery after photobleaching (FRAP), a
dendritic segment was selected and recorded at 0.2 Hz. The first
five images were recorded pre-bleach to establish a baseline, and
then a region of the dendrite was bleached, followed by a series of
recordings. Two separate recordings were performed; the first was
made under control conditions, and the second after NMDA expo-
sure. The data were background-subtracted, normalized to the in-
tensity before bleach, corrected for acquisition bleaching and
fitted to the Brownian diffusion model shown in Eqn. 1 (Feder et al.,
1996) with F0?fluorescent intensity before bleach, Fb?fluorescent
intensity directly after bleach, R?mobile pool, t?time and t½?
recovery half-time. The recovery half-time, t½, was then used to
calculate the diffusion coefficient for a dendritic segment as pre-
viously described (Scott et al., 2006).
GST affinity pull-down
GST-fusion proteins were produced in Escherichia coli (BL21–
A1), and purified using glutathione Sepharose 4 B beads (Amer-
sham Biosciences, Uppsala, Sweden). Rat striatal lysate was
prepared in a buffer containing 50 mM Tris HCl, 150 mM NaCl, 2
mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 0.25% sodium
deoxycholate, 1% Triton X-100 and protease inhibitors (Pierce,
Rockford, IL, USA). Striatal lysate (1 mg of total protein) was
added to the beads in a volume of 1 ml and incubated overnight at
?4 °C. The beads were then washed three times with PBS 1%
Triton X-100 and protease inhibitors. Proteins were eluted with 50
?l of 2? Laemmli buffer for 15 min at 65 °C. Samples were then
subjected to SDS–PAGE for Western blotting, using antibodies
for: PSD-95 (1:1000, Abcam, Cambridge, UK), NMDAR1 (1:1000,
Chemicon Int. Inc., Billerica, MA, USA), GABAA?1(1:1000, Novus
Biological, Littleton, CO, USA), GABAA?2(1:1000, Chemicon Int.
Inc.). The experimental procedure was repeated at a minimum of
Subcellular localization of D1and D5and the role of
To monitor the mobility and localization of D1and D5in
striatal neurons, we used striatal organotypic cultures
transfected with cDNA encoding D1or D5fused to the
fluorescent protein Venus (Nagai et al., 2002). D1and D5
exhibited a similar overall localization (Fig. 1A). Strong
fluorescent signals were observed in the plasma mem-
brane of the cell body, dendrites and in spines. We have
previously shown that the C-terminus tail of D1plays an
important role for the transport of D1to the dendritic tree
(Scott et al., 2006). Here we compared the sub-cellular
localization of D1and D5following truncation of their re-
spective C-terminus tails (D1
1B for illustration). The truncated receptors were ex-
pressed in striatal neurons in organotypic culture. The
localization of D5
A strong signal was observed in the plasma membrane of
the cell body and the dendritic tree, as well as in spines
and filopodia (Fig. 1B). In contrast, the D1
a strong signal in the plasma membrane of the cell body,
and a weak signal in dendrites. No D1
were detected (Fig. 1B).
Next, we compared the number of spines per unit
length of a dendrite in D5–Venus (n?17 dendrites) and in
D1–Venus (n?11 dendrites) expressing neurons. The
measurements were generally performed on a dendritic
segment between the first and second branching. The
number of D5expressing spines was significantly lower
than the number of D1expressing spines (0.25?0.03
spines??m?1and 0.42?0.03 spines??m?1respectively,
P?0.001). The D5signal was predominantly found in the
stubby and thin spines, as well as in filopodia (Fig. 2). D5
was, to a much lesser extent than D1, found in mushroom-
?CTtail, see Fig.
?CTtailwas similar to that of wild-type D5.
D1but not D5interacts with postsynaptic proteins
Because of the observed differences with regard to spine
localization of D1and D5, we wanted to compare the ability
M. Kruusmägi et al. / Neuroscience 164 (2009) 463–469464
of D1and D5to interact with the postsynaptic proteins
PSD-95 and NMDA-R. For this purpose GST pull-down
assays were performed using the C-terminus tail as bait.
The D5–CTtail (amino acids, 421–475) did not interact with
PSD-95 (Fig. 3), while the D1–CTtail (amino acids, 387–
446) strongly interacted with PSD-95 (Fig. 3). The C-ter-
minal tail of D1has been reported to interact with the
NMDA receptor, and this interaction was confirmed in the
present study (Fig. 3). In contrast, the D5–CTtail did not
interact with the NR-1 subunit of the NMDA receptor (Fig.
3). The residual part of the D5C-terminus (amino acids,
360–415) did not interact with NR-1 (data not shown).
D5receptors move by lateral diffusion
Recently we showed that D1moves by lateral diffusion in
the plasma membrane (Scott et al., 2006). Using FRAP
measurements in live striatal neurons expressing D5–Ve-
nus fusion protein, we found that D5, like D1, moves pri-
marily by lateral diffusion in the plasma membrane. Fur-
thermore the recovery of D5was homogenous and not
punctuated, and occurred symmetrically from both ends of
the bleached region (Fig. 4). The diffusion coefficient for
D5, 0.71?0.09 ?m2s?1, was similar to what was previ-
ously described for D1, 0.70?0.06 ?m2s?1.
D5mobility is, in contrast to D1mobility, not
regulated by NMDA receptor occupation
NMDA exposure decreases the diffusion rate of D1in the
plasma membrane and results in trapping of D1in spines
Fig. 1. Localization of D1and D5, role of the C-terminus. (A) Images
show typical neurons in striatal organotypic cultures expressing D1–
Venus (left) and D5–Venus (right), scale bar?20 ?m. Lower panel
shows higher magnification of dendrites with spines expressing D1–
Venus and D5–Venus. (B) The C-terminal tail (CTtail) displays a 28%
sequence homology between D1and D5, the cartoon show areas of
high homology in green and areas with low homology in red. The CTtail
corresponds to amino acids 387 through 446 in D1and amino acids
421 through 475 in D5. Images show typical neurons in striatal orga-
notypic cultures expressing C-terminal truncated receptors tagged
with Venus. The CTtail of each receptor is truncated, D1
spines, while D5
Scale bar?20 ?m.
?CTtail(right). Note that D1
?CTtaildoes not localize to dendrites and
?CTtailhave an appearance similar to wild-type D5.
Stubby MushroomThin Filopodia
# of structures / µm
Fig. 2. Quantification of D1and D5in different spine types. The
number of spines per ?m of dendrite length was quantified in D1–
Venus (n?11) and D5–Venus (n?17) expressing dendrites. The dif-
ferent types of spines were differentiated as follows; stubby spines
were defined as short (?1.5 ?m) stubby structures, mushroom spines
as having a large well defined head, and thin spines as thin structures
with a long thin neck. Filopodia were defined as long (?5 ?m) thin
structures without a head. A significantly larger amount of mushroom
spines express D1than D5(* P?0.001). For interpretation of the
references to color in this figure legend, the reader is referred to the
Web version of this article.
Fig. 3. D1but not D5interacts with postsynaptic proteins. Western
blots of PSD-95 and NR-1 NMDA receptor subunit after affinity pre-
cipitation by GST D1–CTtail but not by GST D5–CTtail or GST alone.
GST fusions contain amino acids 397?446 and 421–475 from the D1
and D5CTtail, respectively. Images show representative blots (n?3).
Input is 20 ?g of total protein.
M. Kruusmägi et al. / Neuroscience 164 (2009) 463–469465
(Scott et al., 2006). To test if this is also true for D5, we
performed FRAP measurements under control conditions
(vehicle) and following NMDA exposure (100 ?M for 30 s).
Two separate bleaching and recording sessions were
made from the same area. The first recording was per-
formed without treatment and the second recording was
performed following NMDA exposure, or vehicle as control.
We found no significant effect on the diffusion rate of D5
following NMDA exposure, while D1displayed a significant
reduction in the diffusion rate following NMDA exposure as
previously shown (Scott et al., 2006) (Fig. 5A).
There was no effect of NMDA exposure on the number
of D5positive spines. In accordance with our previous
results, we found that 15 min after NMDA exposure, the
number of D1positive spines was increased by 20% (Fig.
5C). Exposure to SKF81297, a non-selective D1/D5recep-
tor agonist, did not have any significant effect on the dif-
fusion rate of either D1or D5(Fig. 5B).
GABAAreceptor agonists have no effect on D5
mobility and spine localization
It has previously been reported that D5interacts with
GABAAreceptors (Liu et al., 2000). We were, however, not
able to find an interaction between the D5–CTtail and the
GABAA–?2 subunit or the GABAA–?1 subunit (Fig. 6A).
Nor did we find any interaction between D1-CTtail and
either of the GABAAreceptor subunits (Fig. 6A). We also
examined the effect of exposure to the GABAAreceptor
agonist muscimol on D5mobility. We found no effect of
muscimol on the diffusion rate of D5. We also tested si-
multaneous exposure to muscimol and SKF81297, but
found no effect on D5diffusion rate (Fig. 6B). Finally we
tested the effect of muscimol and SKF81297 exposure on
D5translocation to spines, but did not find a significant
effect on the number of D5positive spines 10–15 min
following wash out (Fig. 6C).
0 100 200300 400
Fig. 4. D5moves by lateral diffusion in the plasma membrane. (A) A
representative time series from a FRAP experiment performed on a
dendrite in a striatal organotypic neuron expressing D5-venus. Scale
bar?10 ?m. (B) A fluorescence recovery curve (filled circles) from a
typical FRAP experiment on a D5–Venus expressing dendrite. A dif-
fusion model based on Brownian diffusion (Eqn. 1 in Experimental
Procedures) was fitted to the data (red line). The diffusion coefficient
was calculated from the recovery half-time, t½, as described in exper-
imental procedures. F0?fluorescence intensity before bleach; Fb?
fluorescence intensity directly after bleach.
Fig. 5. D1but not D5mobility and spine localization is affected by
NMDA exposure. (A) The diffusion coefficient was calculated before
and after NMDA treatment for D5–Venus and D1–Venus. Treatment
with NMDA (100 ?M), 30 s followed by wash out, decreased the
diffusion rate of D1but not of D5. Graph shows average diffusion
coefficients before and after NMDA treatment, for D1n?16 (*P ?0.05)
and for D5n?12. (B) Diffusion rate for D5–Venus and D1–Venus
following SKF81297 treatment (10 ?M, 90 s followed by wash out, 15
min). Graph shows ratio of diffusion coefficient (after/before treat-
ment). SKF81297 has no significant effect on the diffusion of either
D5–Venus or D1.?Venus. (C) D5–Venus and D1–Venus positive
spines were counted before, and 15 min following the termination of
NMDA exposure (100 ?M, 30 s). NMDA exposure significantly in-
creased the number of D1–Venus positive spines but has no effect on
D5–Venus positive spines. Bars are expressed as % change in number
of spines following NMDA exposure, n?213 spines (D1, * P?0.001)
and 203 spines (D5).
M. Kruusmägi et al. / Neuroscience 164 (2009) 463–469 466
This study has demonstrated important differences be-
tween D5and D1in live neurons. In contrast to D1, D5does
not appear to be confined in the PSD or to interact with the
NMDA receptor. These results indicate that the functional
read-outs following D5and D1activation will be different,
and suggest that D5, in contrast to D1, is of less importance
for synaptic function. D5has a higher affinity for dopamine
than D1(Demchyshyn et al., 2000), which can be expected
for a receptor localized outside the synapse.
Our study was performed on striatal neurons in orga-
notypic culture. Subjects with schizophrenia have been
found to have increased glutamatergic input to striatal
neurons (Roberts et al., 2008). Single photon emission
computerized tomography (SPECT) studies have also
shown that schizophrenic patients display abnormal dopa-
mine function in the caudate putamen (Laruelle et al.,
Subcellular localization of transfected D1and D5is
similar to what has been reported for the
D1and D5are co-expressed in many brain regions, includ-
ing striatum, hippocampus and cortex (Zelenin et al.,
2002). In early electron microscopy studies, D1was ob-
served in both head and neck of spines in medium spiny
neurons (Levey et al., 1993; Hersch et al., 1995) and
cortical pyramidal neurons (Smiley et al., 1994; Muly et al.,
1998). In recent ultra-structural studies performed on the
primate prefrontal cortex, where D1and D5specific anti-
bodies were used, D1was found to be enriched in spines
to a much larger extent than D5, while D5was considered
to have a more dendritic profile (Bordelon-Glausier et al.,
2008). In the present study we confirm and extend these
findings to striatal medium spiny neurons in the rat, indi-
cating that dopamine receptors transiently expressed and
tagged with a fluorescent protein have a similar sub-cellu-
lar expression pattern as the endogenous receptors. We
found more D1expressing spines than D5expressing
spines. This is in line with the observation by (Bordelon-
Glausier et al., 2008) that D1has more of a spine profile,
while D5has more of a dendritic profile. In approximately
50% of D1expressing spines, the fluorescent signal was
observed in the head of the spine. In contrast, the D5signal
was mostly observed in protrusions that may have been
thin spines or spine necks. The PSD 95 protein, PSD-95, is
a scaffold protein enriched in the postsynaptic density,
where it serves as an anchor for many of the proteins
involved in synaptic transmission (Feng and Zhang, 2009).
The NMDA receptor is typically confined by PSD-95 (Feng
and Zhang, 2009), it has also been shown that D1can
interact with PSD-95 (Zhang et al., 2007). The finding that
D1, but not D5, interacts with both PSD-95 and the NR1
subunit of the NMDA receptor explains why D1accumu-
lates in the spine head to a higher extent than D5even
though both receptors are freely diffusing in the plasma
D5is transported in the dendritic tree via lateral
diffusion in the plasma membrane
There is emerging evidence that lateral diffusion is an
important means of regulation of the activity and plasticity
of synaptic receptors (Triller and Choquet, 2003; Heine et
al., 2008). The majority of studies that have been per-
formed with regard to lateral diffusion are on ionotropic
receptors. Once the receptors are inserted in the plasma
membrane, they will diffuse freely until they are confined to
a specific region, such as the PSD (Tardin et al., 2003), by
anchoring proteins (Bats et al., 2007). The interaction be-
tween the receptor and the anchoring protein can be dy-
namic and dependent on other surrounding proteins or
complexes. Live cell imaging is a powerful technique that
Fig. 6. Activation of GABAAreceptors does not influence D5diffusion or spine localization. (A) Western blots of GABAA–?1 and –?2 subunits show
no affinity precipitation by either GST D1–CTtail or GST D5CT–tail or GST alone. Images show representative blots (n?3). Input is 20 ?g total protein.
(B) Diffusion rate for D5–Venus before and after treatment, with muscimol (100 ?M) or muscimol (100 ?M) and SKF81297 (10 ?M), for 3 min followed
by wash out. No significant effect was found on the diffusion rate of D5–Venus for either treatment. Graph shows ratio of diffusion coefficients
(after/before treatment), n?6 (muscimol) and n?11 (muscimol?SKF81297). (C) D5–Venus positive spines were counted before and 15 min after
treatment with muscimol (100 ?M) and SKF81297 (10 ?M). Treatment did not have any significant effect on the number of D5–Venus positive spines.
Bars are expressed as % change in number of spines, where before treatment (control) is set to 100%, n?250 spines.
M. Kruusmägi et al. / Neuroscience 164 (2009) 463–469 467
can be used to identify dynamic interactions between such
protein complexes, and the information derived from such
studies should be of importance for future development of
combination therapies. Few studies have focused on the
mobility of G-protein coupled receptors (GPCRs). Our
group recently reported that D1moves by lateral diffusion
in the plasma membrane of striatal neurons and that ex-
posure to NMDA decreases the diffusion rate of D1and
traps D1in spines (Scott et al., 2006). In the present study
we show that both D5and D1are transported in the den-
dritic tree via lateral diffusion and that the diffusion rates for
D5and D1are similar. Notably, the diffusion rates of both
D1and D5are higher than the diffusion rates reported for
ionotropic receptors (Borgdorff and Choquet, 2002). This
higher rate of diffusion might partly be ascribed to the small
cross section of D1/D5, compared to the cross section of
AMPA or NMDA receptors. We found no effect of NMDA
exposure on the D5diffusion rate or on the expression of
D5in spines. This is in line with the biochemical findings
that D5does not exhibit the properties required for being
trapped in the PSD by the NMDA receptor.
Differences between D1and D5receptors can be
attributed to the structural dissimilarity between the
The functional differences between D1and D5observed in
this study can be attributed to structural differences in the
C-terminus tails of D1and D5. The C-termini of GPCRs
have been reported to play key roles for receptor targeting,
for assembly with other proteins, for receptor trafficking,
and for the fine-tuning of the receptor signaling (Bockaert
et al., 2003, 2004). The C-terminus tail of D5did not, in
contrast to the C-terminus tail of D1, interact with the
NMDA receptor and PSD-95. Deletion of the C-terminus
tail had no detectable effect on the localization of D5, while
deletion of the C-terminus tail of D1almost completely
abolished the expression of the receptor in the dendritic
tree. This suggests that the D5receptor contains targeting
information in other sites of its sequence. It will be an
important topic for further studies to identify these sites,
since it will contribute to a better understanding of the
overall function of D5.
Lack of interaction between D5and GABAAreceptor
in striatal neurons
It is well documented that the C-terminus of D1is important
for the interaction with NMDA receptors (Fiorentini et al.,
2003; Scott et al., 2006). The corresponding part of the D5
C-terminal tail has, in studies performed on hippocampus
tissue, been reported to interact with the GABAAreceptor
?2 subunit (Liu et al., 2000). Here we were unable to show
an interaction between the C-terminus of D5and the
GABAAreceptor ?2 subunit. Since our studies were per-
formed using striatal tissue, the differences in results might
be due to regional differences in the interaction between
the two proteins. In the Liu et al. study, an interaction was
only demonstrated when activating both D1/D5receptor
and GABAAreceptor in HEK 293 cells expressing D5and
GABAAreceptor. We were unable to detect any functional
interaction between GABAAreceptor and D5using ago-
nists for GABAAand D1/D5receptors. Exposure to musci-
mol, a GABAAreceptor agonist, and SKF81297, a D1/D5
nonselective agonist, did not have any effect on the mo-
bility and spine expression of D5.
Implication of results
Cocaine and other addictive substances that act to increase
dopamine availability are also known to alter glutamatergic
synaptic plasticity (for a review see (Kauer and Malenka,
hyperactivity induced effects of cocaine treatment are unal-
tered in D5knockout mice, but lost in D1knockout mice
(Levine et al., 1996; Karlsson et al., 2008). The results from
the present study offer a cellular and molecular explanation
for these observations. The interaction between the dopami-
nergic and glutamatergic systems plays a pivotal role for
normal behavior and is perturbed in many psychiatric disor-
ders (Laruelle et al., 2003; Castner and Williams, 2007). The
findings of the present study indicate that in medium spiny
neurons the D1, but not the D5, is involved in the intraneuro-
nal interaction between the dopaminergic and glutamatergic
systems. This information will be of importance for the design
Acknowledgments—The authors wish to thank Tove Önfelt
Tingvall for experimental assistance. This work was supported by
Swedish Research Council, Familjen Erling Perssons Stiftelse,
Jeanssons foundation, the Magnus Bergvall foundation and the
Märta and Gunnar V. Philipson Foundation.
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(Accepted 24 August 2009)
(Available online 29 August 2009)
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