Molecular Psychiatry (2017) 00, 1–14
© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved 1359-4184/17
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ORIGINAL ARTICLE

Dopamine D1 receptor subtype mediates acute stress-induced
dendritic growth in excitatory neurons of the medial prefrontal
cortex and contributes to suppression of stress susceptibility
in mice
R Shinohara1,2,6, M Taniguchi1,3,6, AT Ehrlich2,7, K Yokogawa2, Y Deguchi2, Y Cherasse4, M Lazarus4, Y Urade4, A Ogawa2, S Kitaoka1,2,
A Sawa5, S Narumiya2 and T Furuyashiki1,2
Dopamine in prefrontal cortices is implicated in cognitive and emotional functions, and the dysfunction of prefrontal dopamine has
been associated with cognitive and emotional deﬁcits in mental illnesses. These ﬁndings have led to clinical trials of dopaminetargeting drugs and brain imaging of dopamine receptors in patients with mental illnesses. Rodent studies have suggested that
dopaminergic pathway projecting to the medial prefrontal cortex (mPFC) suppresses stress susceptibility. Although various types ofmPFC neurons express several dopamine receptor subtypes, previous studies neither isolated a role of dopamine receptor subtype
nor identiﬁed the site of its action in mPFC. Using social defeat stress (SDS) in mice, here we identiﬁed a role of dopamine D1
receptor subtype in mPFC excitatory neurons in suppressing stress susceptibility. Repeated social defeat stress (R-SDS) reduces theexpression of D1 receptor subtype in mPFC of mice susceptible to R-SDS. Knockdown of D1 receptor subtype in whole neuronal
populations or excitatory neurons in mPFC facilitates the induction of social avoidance by SDS. Single social defeat stress (S-SDS)
induces D1 receptor-mediated extracellular signal-regulated kinase phosphorylation and c-Fos expression in mPFC neurons.
Whereas R-SDS reduces dendritic lengths of mPFC layer II/III pyramidal neurons, S-SDS increases arborization and spines of apicaldendrites of these neurons in a D1 receptor-dependent manner. Collectively, our ﬁndings show that D1 receptor subtype andrelated signaling in mPFC excitatory neurons mediate acute stress-induced dendritic growth of these neurons and contribute tosuppression of stress susceptibility. Therefore, we propose that D1 receptor-mediated dendritic growth in mPFC excitatory neurons
suppresses stress susceptibility.
Molecular Psychiatry advance online publication, 19 September 2017; doi:10.1038/mp.2017.177

INTRODUCTION
Severe or persistent stress often causes harmful consequences,including emotional and cognitive abnormalities as well as neuralchanges, that promote systemic diseases.1,2 Epidemiologic studies
have suggested that stress during development and in adulthoodincreases the risk and severity of mental illnesses, such as
schizophrenia and mood disorders.3,4 Rodent studies haverevealed that dopamine in the medial prefrontal cortex (mPFC)
has a crucial role in suppressing stress susceptibility. Social defeat
stress (SDS) activates dopaminergic pathway projecting to mPFC,
and repetition of this stress attenuates dopaminergic function in
mPFC,5 leading to social avoidance. Pharmacological ablation5 or
optogenetic inhibition6 of this dopaminergic pathway facilitates
the induction of social avoidance by SDS in mice. Further,transgenic mice with neuronal expression of mutant DISC-1, a risk
factor for major mental illnesses, show social isolation-induced
deﬁcit in the dopaminergic pathway projecting to mPFC,7
1

suggesting reduced dopaminergic function in the mPFC associated with gene–environment interaction for mental illnesses.
Therefore, dopaminergic signaling in the mPFC may represent an
attractive target for the development of drugs and functional
biomarkers for stress-related pathophysiology in mental illnesses.
Dopamine exerts its functions through multiple dopamine
receptor subtypes, namely, D1-like (D1 and D5) and D2-like (D2,D3 and D4) receptors.8 Pharmacological studies have implicatedroles of D1-like receptors in prefrontal cortices in cognitive
functions, such as working memory and cognitive ﬂexibility, inboth primates9,10 and rodents.11,12 These studies have led topharmaceutical development of D1-like receptor agonists tonormalize cognitive dysfunction in mental illnesses.13 In addition,
positron emission tomography (PET) studies have shown that
reduced binding potential of D1-like receptors in the prefrontal
cortices is correlated to the levels of cognitive deﬁcits andnegative symptoms of schizophrenia.14 However, the therapeutic

Division of Pharmacology, Kobe University Graduate School of Medicine, Kobe, Japan; 2Medical Innovation Center, Kyoto University Graduate School of Medicine, Kyoto, Japan;
Department of Biological Sciences, Kyoto University Graduate School of Medicine, Kyoto, Japan; 4International Institute for Integrative Sleep Medicine, University of Tsukuba,
Tsukuba, Japan and 5Department of Psychiatry, Johns Hopkins University School of Medicine, Baltimore, MD, USA. Correspondence: Dr T Furuyashiki, Division of Pharmacology,
Graduate School of Medicine, Kobe University, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan or Dr S Narumiya, Medical Innovation Center, Graduate School of Medicine,
Kyoto University, 53 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto 606-8509, Japan
E-mail: tfuruya@med.kobe-u.ac.jp or snaru@mfour.med.kyoto-u.ac.jp
6
These authors contributed equally to this work.
7
Current address: Douglas Mental Health University Institute, McGill University, Montreal, Quebec, Canada.
Received 7 April 2017; revised 18 June 2017; accepted 28 July 20173

Role of dopamine D1 receptor in stress susceptibility
R Shinohara et al2effects of D1-like receptor agonists are varied among different
measures of cognitive functions,15,16 and the altered D1-likereceptor binding in schizophrenic patients described above is notconsistently found across previous studies.17,18 Rodent studies
have shown that pharmacological manipulation of D1-likereceptors in the mPFC affects spatial working memory andcognitive ﬂexibility in different dose–effect relationships,19
Molecular Psychiatry (2017), 1 – 14

suggesting that dopamine receptor subtypes in mPFC mediate
multiple cognitive functions through distinct mechanisms. It
should be noted that several dopamine receptor subtypes,
including D1 and D5, are expressed in various types of neurons
in the mPFC.20–22 Owing to lack of subtype-selective pharmacological probes,23 pharmacological studies cannot isolate a role ofeach receptor subtype. Pharmacological studies cannot© 2017 Macmillan Publishers Limited, part of Springer Nature.

Role of dopamine D1 receptor in stress susceptibility
R Shinohara et al

Figure 1. Dopamine D1 receptor subtype in medial prefrontal cortex (mPFC) neurons contributes to suppression of repeated social defeat
stress-(R-SDS) induced social avoidance. (a) mRNA levels of D1 (Drd1a), D2 (Drd2), D4 (Drd4) and D5 (Drd5) subtypes of dopamine receptors in
mPFC tissues obtained from naive, susceptible and resilient mice. The numbers of mice in respective groups are shown below bars. P-values
shown in the graph were calculated by Tukey’s post hoc test following one-way analysis of variance (ANOVA; for Drd1a, F(2, 14) = 11.44,
P = 0.0011; for Drd2, F(2, 14) = 11.25, P = 0.0012; for Drd4, F(2, 14) = 0.3839, P = 0.6881; for Drd5, F(2, 14) = 2.718, P = 0.1006). Results are shown asmean ± s.e.m. (b) mRNA levels of D1 receptor subtype (Drd1a) in nucleus accumbens (NAc) tissues obtained from naive, susceptible andresilient mice. The numbers of mice in respective groups are shown below bars. P-values shown in the graph were calculated by Tukey’s post
hoc test following one-way ANOVA (F(2, 14) = 1.471, P = 0.2631). Results are shown as mean ± s.e.m. (c) mRNA levels of D1 receptor subtype(Drd1a) in mPFC tissues obtained from mice without or with single social defeat stress (S-SDS) (Naive or Single, respectively). The numbers ofmice in respective groups are shown below bars. Results are shown as mean ± s.e.m. (d) Representative images of immunoﬂuorescent staining
for D1 receptor subtype and NeuN, a neuronal marker, in mPFC of naive and susceptible mice. Dashed lines indicate the boundaries ofrespective layers. Scale bar, 200 μm. (e–g) Quantiﬁcation of the immunoﬂuorescent signals for D1 receptor subtype in the layer II/III (e), layer V(f) and layer VI (g) in mPFC of naive and susceptible mice, as shown in panel (d). The numbers of mice in respective groups are shown below
bars. P-values shown in the graphs were calculated by unpaired t-test. Results are shown as the mean ± s.e.m. (h) A schedule of behavioral
experiments to test the effect of D1 knockdown in mPFC neurons on social interaction without stress (naive) or after S-SDS or R-SDS. Fourweeks after injection of adeno-associated virus (AAV) solutions to mPFC, the mice were isolated for a week and then were subjected to S-SDS
or R-SDS followed by the social interaction test. For naive mice, the mice that received AAV injection to mPFC were isolated for a week andthen were subjected to the social interaction test without SDS. (i) The effect of D1 knockdown in mPFC neurons on social avoidance without
prior stress. Mice without or with D1 knockdown in mPFC neurons (Ctrl-miR mice or D1-miR mice, respectively) were subjected to the social
interaction test without stress (Naive), and the proportion of time that each mouse spent in the avoidance zone was measured. The numbersof mice in the respective groups are shown below bars. Results are shown as mean ± s.e.m. (j) The effect of D1 knockdown in mPFC neurons
on the induction of social avoidance by SDS. Ctrl-miR mice or D1-miR mice were subjected to S-SDS or R-SDS, and the proportion of time thateach mouse spent in the avoidance zone was measured. The numbers of mice in the respective groups are shown below bars. P-values shownin the graph were calculated by Bonferroni’s post hoc test following two-way repeated-measure ANOVA. (F(1, 22) = 10.44, P = 0.0038 for the main
D1 knockdown effect). Results are shown as mean ± s.e.m. (k) The lack of the behavioral effect of D1 knockdown in mPFC neurons in thepresence of D1 microRNA (miRNA)-resistant D1 mutant. Mice without D1 knockdown (Ctrl-miR mice) or mice with D1 knockdown in mPFC
neurons in the absence (D1-miR mice) or presence (D1 miRNA+D1-mt9) of D1 miRNA-resistant D1 mutant were subjected to S-SDS or R-SDS.
The proportion of time that each mouse spent in the avoidance zone was measured. The numbers of mice in the respective groups are shown
below bars. P-values shown in the graph were calculated by Bonferroni’s post hoc test following two-way repeated-measure ANOVA (F(2,21) = 3.617, P = 0.0447 for the main D1 knockdown effect). Results are shown as mean ± s.e.m. NS, not signiﬁcant.

manipulate dopamine receptor subtypes in a speciﬁc neuronal
population in the mPFC that mediates each cognitive function
either.
To identify a subtype-speciﬁc role, systemic knockout mice ofeach dopamine receptor subtype have frequently been used.24
However, this technique cannot identify the site of action of
dopamine receptor subtypes and cannot rule out a possible roleof dopamine receptor subtypes during brain development.25 To
overcome these issues, conditional knockout mice of dopamine
receptor subtypes using mice expressing Cre recombinase in a
speciﬁc brain region and cell type of interest have beendeveloped and used for behavioral studies.26,27 However, miceexpressing Cre recombinase speciﬁcally in the mPFC are notavailable so far. The advent of recombinant viral vectors with highinfection efﬁciency to various types of neurons offers a novel
strategy to manipulate gene expression in any speciﬁc brain
region of interest,28 including mPFC. In the present study, using
mPFC injection of recombinant viral vectors combined with miceexpressing Cre recombinase speciﬁcally in excitatory or inhibitory
neurons or with a viral vector expressing Cre recombinase
speciﬁcally in mPFC excitatory neurons, we successfully manipulated expression of D1 receptor subtype selectively in each ofthese neurons in the mPFC and revealed that D1 receptor subtype
and related signaling in mPFC excitatory neurons contributeto suppression of susceptibility to repeated social defeat stress(R-SDS). We also found the evidence suggesting that single social
defeat stress (S-SDS) increases dendritic arborization and dendritic
spines through activation of D1 receptor subtype. To the best of
our knowledge, these morphological changes of mPFC excitatoryneurons are a novel neuronal correlate of suppressing stresssusceptibility through D1 receptor signaling in the mPFC.

Graduate School of Medicine. Materials and methods in detail aredescribed in Supplementary Materials and Methods.

SDS and social interaction test
SDS and social interaction test were performed as previously described29
with minor modiﬁcations.5 In this experiment, male C57BL/6N mice aged
13–14 weeks, that had received adeno-associated virus (AAV) injection to
the mPFC at the age of 8 weeks were used for all experiments, except inFigures 1a–c and 3d and e and Supplementary Figure 1, in which the mice
aged 8–9 weeks that had not received AAV injection were used. C57BL/6N
mice aged 8–9 weeks and 13–14 weeks showed similar degrees of social
avoidance after R-SDS. After isolation for a week, these mice were put tothe home cage of a male ICR mouse for 10 min daily for every day in the
10 day defeat. After the 10-min defeat episode, the mice were returned tothe home cages and kept isolated without any contact to the ICR mice
until SDS on the next day.
The social interaction test was performed 18 h after the ﬁrst (S-SDS) andtenth exposure to SDS (R-SDS). The second exposure to SDS was applied
about 2 h after the ﬁrst social interaction test. Behaviors in the open ﬁeld
chamber with a novel male ICR mouse enclosed in a metal meshwork wererecorded. The proportions of the time that each mouse spent in the social
avoidance zone and the social interaction zone (Supplementary Figure 1a)were measured post hoc. Stress-induced social avoidance from an ICR
mouse can be detected as an increase in the time for the social avoidancezone and a decrease in the time for the social interaction zone. As R-SDS
induces social avoidance in only half of the defeated mice,30 we chosesusceptible and resilient mice as follows: Susceptible mice refer to those inwhich the durations for both the interaction zone and the avoidance zone
were beyond respective s.d. from their averages of naive mice. Resilient
mice refer to those in which the durations for both the interaction zone
and the avoidance zone were kept within respective s.d. from theiraverages of naive mice.

Viral vectors
MATERIALS AND METHODSAll procedures for animal care and use were in accordance with the
National Institutes of Health Guide for the Care and Use of Laboratory
Animals and were approved by the Animal Care and Use Committees of
Kyoto University Graduate School of Medicine and Kobe University© 2017 Macmillan Publishers Limited, part of Springer Nature.

The AAVs generated in this study are as follows: AAV (serotype 2/rh10)
expressing artiﬁcial microRNA (miRNA) targeting dopamine D1 receptor orcontrol miRNA with Emerald Green Fluorescent Protein (EmGFP) only inthe presence of Cre recombinase (AAV-DIO-EmGFP-D1miR and AAV-DIOEmGFP-CtrlmiR, respectively); AAV2/rh10 expressing D1 miRNA-resistantD1 receptor mutant fused with FLAG tag at its N terminus (FLAG-D1-mt9)
Molecular Psychiatry (2017), 1 – 143

Role of dopamine D1 receptor in stress susceptibility
R Shinohara et al4only in the presence of Cre recombinase (AAV-DIO-D1-mt9); AAV2/rh10
expressing Cre recombinase fused with nuclear localization signal (NLS)
at its N terminus (NLSCre) only in the presence of Cre recombinase(AAV-DIO-Cre); and AAV2/rh10 expressing NLSCre under the CaMKIIαpromoter (AAV-CaMKIIα-Cre). AAV2/rh10 particles were produced, as previously described31 with minor modiﬁcations. A lentiviral vector expressing
mCherry only in the presence of Cre recombinase (Lenti-DIO-mCherry) was
produced, as described previously.32

Stereotaxic surgery for viral injection to the mPFC
Stereotaxic surgeries for viral injection to mPFC were performed aspreviously described5 with minor modiﬁcations. The stereotaxic coordinates were targeted to the ventral portion of mPFC (1.8 mm anterior fromthe bregma, 0.4 mm lateral from the midline and 2.8 mm ventral from theskull surface at the bregma) based on the Paxinos and Franklin mousebrain atlas.33 Mice were recovered for 4 weeks after the surgery, during
which the expression of miRNA or a protein of interest was fully induced
before behavioral experiments.

Immunoﬂuorescent staining
Immunoﬂuorescent staining in brain sections and AAV293 cells, except foranalyses of dendritic morphologies (shown below), was performed aspreviously described34,35 with minor modiﬁcations.

Analysis of dendritic morphology of mPFC neurons
For morphometric analyses of mPFC neurons, coronal brain sections of
100 μm thickness were made by cryostat (Leica, Wetzlar, Germany). Layer
V/VI was identiﬁed by the presence of Ctip2-positive neurons, and mPFC
pyramidal neurons at layer II/III or layer V/VI were separately analyzed.
Semiautomated tracing of dendrites was performed, and the lengths andbranching points of dendrites were measured, using the IMARIS software(Bitplane, Zurich, Switzerland). The Sholl analysis was also performed aspreviously described36 with minor modiﬁcations. As the depth of the cellbody from the midline varied considerably across neurons, the interval of
the concentric circles was normalized to the depth of the cell body foreach neuron.
Spine classiﬁcation was determined by the parameters of the spine
length and the diameter of spine neck and head. Spines with head/neckdiameter ratios 41.7 were classiﬁed as ‘Type 1’ spines, and the remains
were classiﬁed as ‘Type 2’ spines. Within spines with head/neck diameter
ratio 41.2, spines with the spine length o3 μm and the spine head
40.6 μm were categorized into mushroom spines, and the remains werecategorized into thin spines. Within spine with head/neck diameter ratio
no more than 1.2, spines with the spine length o 1 μm were categorized
into stubby spines, and the remains were into ﬁlopodia. This classiﬁcationof mushroom spines, thin spines, stubby spines and ﬁlopodia was
performed as previously described37 with minor modiﬁcations.

RNA extraction and quantitative reverse transcriptase PCR(RT-PCR)RNA extraction from brain tissues and quantitative RT-PCR were performed
as previously described30 with minor modiﬁcations.

Statistical analysis
All data are shown as mean ± s.e.m. Statistical analyses were performed
using unpaired two-tailed Student’s t-test, one-way analysis of variancefollowed by Tukey’s post hoc test and two-way analysis of variancefollowed by Bonferroni’s post hoc test, as described in the ﬁgure legends. A
value of Po0.05 was considered as statistically signiﬁcant.

RESULTS
Repeated SDS reduces the expression of dopamine D1 receptor
subtype in the mPFC
As dopaminergic projections in mPFC are critical for suppressing
the induction of social avoidance by SDS,5 we examined whether
R-SDS alters the mRNA levels of dopamine receptor subtypes in
mPFC in a manner correlated to social avoidance. For this purpose,
we punched out mPFC tissues after R-SDS or without stress (naivemice) and measured the mRNA levels of ﬁve subtypes of
Molecular Psychiatry (2017), 1 – 14

dopamine receptors in mPFC. Quantitative RT-PCR analyses
detected the mRNA expression levels of D1, D2, D4 and D5
receptor subtypes (Figure 1a) but not of D3 receptor subtype (datanot shown). R-SDS reduced the mRNA expression of D1 receptor
subtype in susceptible mice, and this reduction was smaller inresilient mice (Figure 1a). Consistently, immunoﬂuorescent signals
for D1 receptor subtype were signiﬁcantly reduced in mPFC layer
II/III and layer V of susceptible mice compared with naive mice
(Figures 1d–g). In contrast to R-SDS, S-SDS did not alter the mRNA
expression of D1 receptor subtype in mPFC (Figure 1c). R-SDS did
not signiﬁcantly change the mRNA expression of D2 receptor
subtype in susceptible mice, although D2 mRNA was signiﬁcantly
elevated in resilient mice (Figure 1a). mRNA expression levels of
D4 and D5 receptor subtypes were not signiﬁcantly changed ineither susceptible or resilient mice (Figure 1a). We also measured
the mRNA levels of D1 receptor subtype in the nucleus
accumbens, a brain region that is also involved in the inductionof social avoidance by R-SDS.30 In contrast to mPFC, R-SDS did not
signiﬁcantly change the mRNA expression of D1 receptor subtype
in the nucleus accumbens (Figure 1b).
D1 receptor subtype in mPFC neurons contributes to suppressionof the induction of social avoidance by SDS
To examine a role of D1 receptor subtype in R-SDS, we generated
AAV (AAV2/rh10) expressing artiﬁcial microRNA targeting D1
receptor subtype (D1 miRNA) together with EmGFP under theconstitutive EF1α promoter (Supplementary Figure 2a). To achieve
neuron type-speciﬁc expression of D1 miRNA later, AAV wasdesigned to enable the expression of D1 miRNA and EmGFP onlyin the presence of Cre recombinase (AAV-DIO-EmGFP-D1miR). To
express D1 miRNA and EmGFP in all types of mPFC neurons, we
injected this viral vector to mPFC with AAV expressing Cre
recombinase under the constitutive CMV promoter (AAV-Cre)(Supplementary Figures 2b and c). Immunoﬂuorescent staining for
NeuN, a marker for neurons, showed that EmGFP is expressed
selectively in the neurons in mPFC (Supplementary Figure 2d). This
neuronal selectivity is probably due to the preference of this AAV
serotype for infecting neurons.31 mRNA expression of D1 receptor
subtype, but not of the other subtypes, was reduced with AAV
expressing D1 miRNA in the mPFC, compared with AAV
expressing negative control miRNA (Supplementary Figure 2e).
Immunoﬂuorescent signals for D1 receptor subtype in mPFC weredecreased with D1 miRNA, compared with the signals with control
miRNA (Supplementary Figures 4d and e). These ﬁndings
conﬁrmed that the expression of D1 miRNA induces knockdownof D1 receptor subtype.
We then examined whether knockdown of D1 receptor subtype
in mPFC neurons could affect the induction of social avoidance by
SDS (Figure 1h). Without SDS, either male mice expressing control
miRNA in mPFC neurons (Ctrl-miR mice) or male mice expressing
D1 miRNA in mPFC (D1-miR mice) similarly showed social
interaction with a novel ICR mouse enclosed in a metal meshwork(Figure 1i and Supplementary Figure 3a), suggesting that D1
receptor is not involved in social interaction without prior stress. In
contrast, D1-miR mice showed the facilitated induction of social
avoidance by SDS, compared with Ctrl-miR mice (Figure 1j andSupplementary Figure 3b). On the other hand, Ctrl-miR and D1miR mice showed similar durations of submissive posture, abehavioral sign of social defeat,38 during the ﬁrst and tenth SDS(Supplementary Figure 3c). Therefore, D1 receptor in mPFC
neurons is not critical for the perception of SDS.
To exclude a potential off-target effect of D1 miRNA, we
generated a D1 receptor mutant with nine synonymous substitutions in DNA sequence named D1-mt9, which are expected to
encode the same amino-acid sequence as wild-type D1 receptor
subtype (Supplementary Figure 4a). In cell lines, D1 miRNA
reduced immunoﬂuorescent signals for overexpressed wild-type© 2017 Macmillan Publishers Limited, part of Springer Nature.

Role of dopamine D1 receptor in stress susceptibility
R Shinohara et al

5
D1 receptor subtype but not the signals for the D1-mt9 mutant(Supplementary Figures 4b and c). These ﬁndings indicate that the
D1-mt9 mutant is resistant to D1 miRNA. We then injected AAV
expressing the D1-mt9 mutant (AAV-DIO-D1-mt9) with AAV-DIOEmGFP-D1miR and AAV-Cre and examined the level of social
avoidance induced by SDS. Simultaneous expression of theD1-mt9 mutant restored D1 receptor expression (Supplementary
Figures 4d and e) and normalized the level of social avoidance
induced by SDS even in the presence of D1 miRNA in mPFC
neurons (Figure 1k and Supplementary Figure 3d).
Taken together, these results indicate that D1 receptor subtype
in mPFC neurons contributes to suppression of the induction ofsocial avoidance by SDS.
mPFC excitatory neurons are the site of action of D1 receptor
subtype in suppressing the induction of social avoidance by SDS
Given that D1 receptor subtype is expressed in both excitatory
and inhibitory neurons in the mPFC,20 we performed neuron typespeciﬁc knockdown of D1 receptor subtype. To generate malemice expressing D1 miRNA selectively in mPFC excitatory neurons(CaMKIIα-D1-KD mice), we injected AAV-DIO-EmGFP-D1miR tomPFC of CaMKIIα-CreERT2 mice (Figure 2a), which express
tamoxifen-inducible Cre recombinase (CreERT2) under the CaMKIIα promoter.39 We ampliﬁed Cre expression by simultaneously
injecting AAV expressing Cre recombinase only in the presence ofCre recombinase (AAV-DIO-Cre) (see Supplementary Materials andMethods for the reason why we had to amplify Cre expression).
After postsurgical recovery for 3 weeks, tamoxifen was administrated for 5 consecutive days to induce Cre-mediated recombination. EmGFP (simultaneously expressed with D1 miRNA) was
induced in at least half of the mPFC neurons (Supplementary
Figure 5, right) and was largely restricted to CaMKIIα-expressing
excitatory neurons in the mPFC (Figure 2b, lower panels). As anegative control, AAV-DIO-EmGFP-D1miR and AAV-DIO-Cre were
injected to mPFC of wild-type littermates. EmGFP was notdetectable in these control mice (Figure 2b, upper panels).
CaMKIIα-D1-KD mice showed the facilitated induction of social
avoidance by S-SDS, compared with the control mice (Figure 2c
and Supplementary Figure 3e).
To equalize Cre expression and its potential toxicity in thecomparison groups, we performed mPFC injection of AAV-DIOEmGFP-D1miR or AAV expressing control miRNA only in thepresence of Cre recombinase (AAV-DIO-EmGFP-CtrlmiR) with AAV
expressing Cre recombinase under the CaMKIIα promoter (AAVCaMKIIα-Cre) (Figure 2d). This experiment showed that knockdown of D1 receptor subtype, rather than Cre expression,
selectively in mPFC excitatory neurons facilitates the inductionof social avoidance by SDS (Figure 2e and Supplementary
Figure 3f). In this experiment, the facilitated induction of social
avoidance was observed after R-SDS. This effect was not evident in
CaMKIIα-D1-KD mice after R-SDS in Figure 2c, as intraperitoneal
injection of tamoxifen appears to delay the induction of social
avoidance in both CaMKIIα-D1-KD mice and their control mice,compared with Figure 2e. By contrast, the facilitated induction ofsocial avoidance after S-SDS was observed in CaMKIIα-D1-KD micein Figure 2c, whereas this effect was not evident in the experimentwith AAV-CaMKIIα-Cre in Figure 2e, perhaps due to a low
expression level of D1 miRNA induced by Cre recombinase.
Despite these subtle differences, both of the two experiments
described above suggest that mPFC excitatory neurons are thesite of action of D1 receptor subtype for contributing tosuppressing stress susceptibility.
We next generated mice expressing D1 miRNA selectively in
mPFC inhibitory neurons (Viaat-D1-KD mice) by injecting AAV-DIOEmGFP-D1miR to mPFC of Viaat-Cre mice (Figure 2d), which
express Cre recombinase under the vesicular inhibitory aminoacid transporter (Viaat) promoter.40 EmGFP ﬂuorescent signals© 2017 Macmillan Publishers Limited, part of Springer Nature.

were restricted to GABA-positive and GAD67-positive neurons in
Viaat-Cre mice (Figure 2f). The signal intensities in individual
EmGFP-expressing neurons were comparable to those in CaMKIIαD1-KD and D1-miR mice (see Figure 2b and Supplementary
Figure 2d). In contrast to CaMKIIα-D1-KD mice, the response to
SDS of Viaat-D1-KD mice was comparable with the control mice
(Figure 2g and Supplementary Figure 3g).
S-SDS activates dopamine D1 receptor signaling in mPFC
excitatory neurons
As phosphorylation of extracellular signal-regulated kinase (ERK) is
coupled to dopamine receptor activation in many brain areas,41,42
we examined whether SDS induces ERK phosphorylation in mPFC
neurons through D1 receptor subtype. We ﬁrst examined theeffect of systemic injection of a D1-like receptor agonist SKF81297
on ERK phosphorylation in mPFC neurons. Ten minutes after
intraperitoneal injection of SKF81297 or saline, the mice were
killed for immunostaining for phosphorylated ERK (pERK) and
NeuN. The injection of SKF81297 increased not only the intensityof pERK in neurons but also the proportion of pERK-positive
neurons in multiple layers of mPFC compared with injection ofsaline (Supplementary Figure 6), suggesting that the activation of
D1-like receptors induces ERK phosphorylation in mPFC neurons.
We next examined whether SDS induces D1 receptordependent ERK phosphorylation in mPFC neurons. Immediately
after S-SDS, Ctrl-miR and D1-miR mice were killed for immunostaining for pERK and NeuN (Supplementary Figure 7). In Ctrl-miR
mice, S-SDS increased the intensity of pERK in mPFC neurons(Figures 3a and b) and the proportion of pERK-positive mPFC
neurons (Figure 3c). These changes induced by S-SDS wereattenuated in mPFC neurons in D1-miR mice (Figures 3a–c).
Double immunostaining with CaMKIIα or GABA, a marker for
excitatory or inhibitory neurons, respectively, showed that S-SDS
signiﬁcantly increases the number of pERK- and CaMKIIα-positive
excitatory neurons in the mPFC, and pERK was rarely observed in
GABA-positive inhibitory neurons without or with the stress(Figures 3d and e). D1 knockdown in mPFC neurons attenuated
S-SDS-induced increase in the number of pERK- and CaMKIIαpositive excitatory neurons (Figure 3f). These results indicate that
S-SDS induces ERK phosphorylation selectively in mPFC excitatoryneurons at least partly through D1 receptor subtype.
We also examined whether SDS induces D1 receptordependent expression of c-Fos and Zif268, immediate early genes
which have frequently been used as neuronal activation markers(for example, Jungenitz et al.43), in mPFC neurons. Ninety minutes
after S-SDS, the mice were killed for immunostaining for c-Fos orZif268 (Supplementary Figures 8 and 9). In Ctrl-miR mice, S-SDS
slightly increased the number of c-Fos-positive neurons in mPFC
layer II/III (Figures 3g and h), and this increase was less evident in
mPFC layer V/VI neurons (Figures 3g and i). In D1-miR mice, theinduction of c-Fos expression by S-SDS was abolished in mPFC
layer II/III neurons (Figures 3g and h), suggesting that D1 receptor
subtype and related signaling contribute to the response of theseneurons to S-SDS. The number of Zif268-positive mPFC neurons
was not affected by either S-SDS or D1 knockdown in mPFC
neurons (Supplementary Figure 9).
S-SDS increases dendritic arborization and spine density
selectively in apical dendrites of mPFC layer II/III pyramidal
neurons in a D1 receptor-dependent manner
Repeated restraint stress and chronic mild stress induce dendritic
shortening and reduced spine density in mPFC pyramidal
neurons,44,45 and these morphological changes are associatedwith stress-induced behavioral changes.46–48 We examined
whether such dendritic shortening could be induced by R-SDS
and whether D1 receptor subtype could be involved in thisprocess. To visualize dendritic morphology of mPFC pyramidal
Molecular Psychiatry (2017), 1 – 14

Role of dopamine D1 receptor in stress susceptibility
R Shinohara et al6neurons, we sparsely labeled these neurons by mPFC injection of
low-titer lentivirus expressing mCherry in the presence of Cre
recombinase (Lenti-DIO-mCherry) with AAV-Cre (Supplementary
Figure 10). Ctrl-miR and D1-miR mice were subjected to R-SDS,
and the level of social avoidance was measured at 18 h later. Then
at 72 h after R-SDS, the mice were killed for morphologicalanalyses of mPFC neurons (Figure 4a). R-SDS signiﬁcantly reduced
dendritic lengths, but not dendritic branching points or spine
density, of apical dendrites of mPFC layer II/III pyramidal neurons
in the susceptible group, but not the resilient group, of Ctrl-miR
mice (Figures 4b and c and Supplementary Figures 11a–c),suggesting that R-SDS induces shortening of apical dendrites in
Molecular Psychiatry (2017), 1 – 14

mPFC layer II/III pyramidal neurons in a manner correlated withstress susceptibility. Such dendritic shortening appears to bemuch less in basal dendrites of mPFC layer II/III pyramidal neurons(Figures 4b and d) and apical and basal dendrites of mPFC layer V/
VI pyramidal neurons (Supplementary Figures 12a and b). In D1miR mice, the shortening of apical dendrites of mPFC layer II/III
neurons was not potentiated (Supplementary Figure 13a and b),
whereas the induction of social avoidance was facilitated (seeFigure 1j), suggesting the dissociation between the dendritic
shortening and the social avoidance in D1-miR mice. D1 knockdown in mPFC neurons did not apparently affect spine density ofmPFC layer II/III pyramidal neurons (Supplementary Figures 11d–f)© 2017 Macmillan Publishers Limited, part of Springer Nature.

Role of dopamine D1 receptor in stress susceptibility
R Shinohara et al

Figure 2. Medial prefrontal cortex (mPFC) excitatory neurons are the site of action of D1 receptor subtype for contributing to suppression of
repeated social defeat stress (R-SDS)-induced social avoidance. (a) A schedule of behavioral experiments to test the effect of D1 knockdown in
mPFC excitatory neurons on social interaction after single social defeat stress (S-SDS) or R-SDS. Three weeks after injection of adenoassociated virus (AAV) solutions to mPFC, the mice were administered with 1 mg of tamoxifen daily for 5 days. The mice were isolated for aweek and then were subjected to S-SDS or R-SDS followed by the social interaction test. (b) Representative images of Emerald Green
Fluorescent Protein (EmGFP) ﬂuorescence and immunoﬂuorescent staining for CaMKIIα in mPFC without or with D1 knockdown (‘Cre ( − )+Tamoxifen’ or ‘CaMKIIα-CreERT2+Tamoxifen’, respectively) in mPFC excitatory neurons. In the merged images, EmGFP and CaMKIIα are shownin green and red, respectively. Scale bar, 50 μm. (c) The effect of D1 knockdown in mPFC excitatory neurons on the induction of social
avoidance by SDS. Mice without or with D1 knockdown in mPFC excitatory neurons (Negative control mice or CaMKIIα-D1-KD mice,
respectively) were subjected to S-SDS or R-SDS, and the proportion of time that each mouse spent in the avoidance zone was measured. The
numbers of mice in the respective groups are shown below bars. A P-value shown in the graph was calculated by Bonferroni’s post hoc test
following two-way repeated-measure analysis of variance (ANOVA) (F(1, 21) = 4.348, P = 0.0495 for the main D1 knockdown effect). Results areshown as mean ± s.e.m. (d) A schedule of behavioral experiments to test the effect of D1 knockdown in mPFC excitatory neuron using AAVCaMKIIα-Cre (e) or mPFC inhibitory neurons using Viaat-Cre mice (g) on social interaction after S-SDS or R-SDS. Four weeks after injection of
AAV solutions to mPFC, the mice were isolated for a week and then were subjected to S-SDS or R-SDS followed by the social interaction test.
(e) The effect of D1 knockdown in mPFC excitatory neurons using AAV-CaMKIIα-Cre on the induction of social avoidance by SDS. Mice withoutor with D1 knockdown in mPFC excitatory neurons (‘CaMKIIα-Cre; Ctrl-miR’ or ‘CaMKIIα-Cre; D1 miR’, respectively) were subjected to S-SDS or
R-SDS, and the proportion of time that each mouse spent in the avoidance zone was measured. The numbers of mice in respective groups areshown below bars. A P-value shown in the graph was calculated by Bonferroni’s post hoc test following two-way repeated-measure ANOVA (F(1, 16) = 6.637, P = 0.0203 for the main D1 knockdown effect). Results are shown as mean ± s.e.m. (f) Representative images of EmGFP
ﬂuorescence and immunoﬂuorescent staining for GABA and GAD67 in mPFC of a Viaat-Cre mouse that was injected with AAV-DIO-EmGFPD1miR. In the merged image, EmGFP, GABA and GAD67 are shown in green, red and blue, respectively. Scale bar, 50 μm. (g) The effect of D1
knockdown in mPFC inhibitory neurons on the induction of social avoidance by SDS. Mice without or with D1 knockdown in mPFC inhibitory
neurons (Negative control mice or Viaat-D1-KD mice, respectively) were subjected to S-SDS or R-SDS, and the proportion of time that eachmouse spent in the avoidance zone was measured. The numbers of mice in respective groups are shown below bars. No signiﬁcant changes
are observed (F(1, 16) = 0.001491, P = 0.9697 for the main D1 knockdown effect by two-way repeated-measure ANOVA). Results are shown as
the mean ± s.e.m.

or the lengths of basal dendrites of mPFC layer II/III pyramidal
neurons (Supplementary Figure 13c) or apical or basal dendrites ofmPFC layer V/VI pyramidal neurons (Supplementary Figures 12c
and d) either.
As D1 receptor signaling in mPFC layer II/III excitatory neurons is
activated by S-SDS, we examined whether and how S-SDS could
affect neuronal morphology of mPFC pyramidal neurons andwhether D1 receptor subtype could be involved in this process. At
72 h after S-SDS, the mice were killed for morphological analyses
of mPFC neurons (Figure 4e). S-SDS increased the lengths andbranching points of apical dendrites of mPFC layer II/III pyramidal
neurons in Ctrl-miR mice (Figures 4f–h). By contrast, thesemorphological changes were not seen in D1-miR mice, and thelengths and branching points of apical dendrites of these neurons
were comparable to those of naive Ctrl-miR mice with or without
prior stress (Figures 4f–h). Using the Sholl analysis, we analyzed inwhich region(s) of apical dendrites S-SDS induces dendritic growththrough D1 receptor subtype. S-SDS appears to increase thecomplexity of apical dendrites within layer I and layer II/III in CtrlmiR mice but not in D1-miR mice (Supplementary Figure 15a). By
contrast, the morphological changes were not induced by S-SDS
in basal dendrites of mPFC layer II/III pyramidal neurons, where D1
knockdown slightly increased the lengths and branching points ofthese dendrites without or with S-SDS (Supplementary Figures 14
and 15b). In mPFC layer V/VI pyramidal neurons of Ctrl-miR andD1-miR mice, S-SDS did not induce dendritic growth in either
apical or basal dendrites (Supplementary Figure 16). We also
examined whether S-SDS affects the density of dendritic spines ofmPFC neurons in a manner dependent on D1 receptor subtype.
S-SDS increased the density of dendritic spines on both distal andproximal apical dendrites of mPFC layer II/III pyramidal neurons in
Ctrl-miR mice but not in D1-miR mice (Figures 5a–c). The increasein spine density after S-SDS was not evident on basal dendrites ofmPFC II/III pyramidal neurons in either Ctrl-miR or D1-miR mice
(Figure 5d).
We next examined whether and how S-SDS and D1 knockdown
could affect the morphology of dendritic spines. S-SDS appears toslightly increase the diameter of spine neck in Ctrl-miR mice but
not in D1-miR mice (Supplementary Figure 17a). In contrast, thediameter of spine head and the length of dendritic spine were less© 2017 Macmillan Publishers Limited, part of Springer Nature.

affected by S-SDS or knockdown of D1 receptor subtype(Supplementary Figures 17b and c). As there are several types ofdendritic spines according to their morphologies,49 we suspectedthat S-SDS might affect dendritic spines of a speciﬁc type throughD1 receptor subtype. According to the diameters of spine headand neck, we were able to identify two evident clusters ofdendritic spines: dendritic spines in which the diameter of spine
head is larger than that of a spine neck and those with a head anda neck of similar diameters (Supplementary Figures 18a and b). For
the purpose of statistical analyses, we separated these two clusters
by classifying spines with head/neck diameter ratios 41.7 as ‘Type
1’ spines and the remains as ‘Type 2’ spines. S-SDS signiﬁcantly
increased the density of Type 2 spines on distal apical dendrites ofmPFC layer II/III pyramidal neurons in Ctrl-miR mice, whereas thisincrease was less apparent in Type 1 spines (Figures 5e and f).
S-SDS-induced increase in the density of Type 2 spines were notobserved in D1-miR mice (Figure 5f). We also performed
a conventional classiﬁcation of dendritic spines, in which dendritic
spines have been classiﬁed to mushroom spine, thin spine, stubby
spine and ﬁlopodia, and the latter two types of spines areprominent during the early stage of synaptic formation.50 S-SDS
signiﬁcantly increased the densities of stubby spines andﬁlopodia in a D1 receptor-dependent manner, and this increase
was not evident in the other types of spines (Supplementary
Figures 18c–f).
Collectively, these results show that S-SDS activates D1 receptor
signaling and increases dendritic arborization and spine density,
especially that of immature types of spines, selectively in apicaldendrites of mPFC layer II/III pyramidal neurons, whereas theshortening of these dendrites induced by R-SDS is not potentiatedby D1 knockdown in mPFC neurons.
DISCUSSION
Roles for D1-like receptors in prefrontal cortices have been
extensively investigated in cognitive functions,19,51,52 andimpaired D1-like receptor signaling in the prefrontal cortices has
been implicated in cognitive impairments associated with mental
illnesses.53 However, as these studies relied on drugs or ligands
targeting D1-like receptors, they did not discriminate D1 and D5
Molecular Psychiatry (2017), 1 – 14

7

Role of dopamine D1 receptor in stress susceptibility
R Shinohara et al8

receptor subtypes and did not identify the site of action of
dopamine receptor subtypes in prefrontal cortices. Using mPFCand neuron type-speciﬁc manipulation of gene expression, here
we elucidated a novel role for D1 receptor subtype in mPFC
excitatory neurons, that is, suppressing stress susceptibility inmice. This newly described function of D1 receptor subtype mayunderlie stress-related pathophysiology of various mental illnesses
and represent an attractive target for the development oftherapeutic drugs and biomarkers of molecular imaging in mental
illnesses. Furthermore, we found that stress-evoked D1 receptor
signaling increases dendritic arborization and dendritic spine
density in mPFC excitatory neurons. This ﬁnding is, to the best of
our knowledge, the ﬁrst to demonstrate that D1 receptor signaling
induces dendritic growth of mPFC neurons in adult brains andpossibly exerts the long-lasting effect of D1 receptor signaling in
the mPFC. We propose that these morphological changes of mPFC
Molecular Psychiatry (2017), 1 – 14

excitatory neurons mediate the effect of D1 receptor subtype insuppressing stress susceptibility.
Reduced expression of D1 receptor subtype in mPFC after R-SDS
R-SDS reduces the mRNA level of D1 receptor subtype, but not
those of the other subtypes, in mPFC. As the immunoreactivity of
D1 receptor subtype is reduced broadly in the mPFC of thesusceptible mice compared with the naive mice, it is plausible thatthe protein level of D1 receptor subtype in mPFC is reduced after
R-SDS. Given the reduced mRNA level of D1 receptor subtype,
R-SDS could suppress its protein synthesis in mPFC neurons either
through reduced mRNA synthesis or reduced mRNA stability or
both. Although the mechanism underlying reduced expression of
D1 receptor subtype in the mPFC remains unclear, this reduction
is associated with the induction of social avoidance by R-SDS,© 2017 Macmillan Publishers Limited, part of Springer Nature.

Role of dopamine D1 receptor in stress susceptibility
R Shinohara et al

Figure 3. Single social defeat stress (S-SDS) induces extracellular signal-regulated kinase (ERK) phosphorylation and c-Fos expression in medial
prefrontal cortex (mPFC) neurons through D1 receptor subtype. (a) Representative images of immunoﬂuorescent staining for phosphorylated
ERK (pERK) in mPFC of mice without or with S-SDS (Naive or Single, respectively) and without or with D1 knockdown in mPFC neurons(Ctrl-miR mice or D1-miR mice, respectively). NeuN was co-stained to identify neurons in mPFC. The signals for pERK and NeuN are shown ingreen and red, respectively. The same images of respective ﬂuorescent channels and their magniﬁed images are shown in Supplementary
Figure 7. Scale bars, 200 μm. (b) Cumulative frequency of immunoﬂuorescent signals for pERK in mPFC neurons of Ctrl-miR mice or D1-miR
mice without or with S-SDS (Naive or Single, respectively). N = 35 345–41 283 neurons from 6–7 mice for each group. The inset shows theaverages of the immunoﬂuorescent signals for pERK shown in the cumulative frequency across neurons for respective mice. Each dot
represents the averaged values for an individual mouse. The numbers of mice in respective groups are shown below bars. Results are shownas mean ± s.e.m. (c) The proportions of pERK-positive neurons in mPFC of Ctrl-miR mice or D1-miR mice without or with S-SDS (Naive orSingle, respectively). Those neurons that showed pERK signals higher than the averaged values for naive Ctrl-miR mice by the s.d. (a dashedline in Figure 3b) are deﬁned as pERK-positive neurons. The numbers of mice in respective groups are shown below bars. P-values shown in
the graph were calculated by Bonferroni’s post hoc test following two-way analysis of variance (ANOVA) (F(1,22) = 3.406, P = 0.0785 for the main
RNAi effect, F(1,22) = 4.359, P = 0.0486 for the main Stress effect). Results are shown as mean ± s.e.m. (d) Representative images of
immunoﬂuorescent staining for pERK in mPFC of mice without or with S-SDS (Naive or Single, respectively). CaMKIIα or GABA was co-stained
as a marker for excitatory or inhibitory neurons in the mPFC, respectively. In the merged images, the signals for pERK and CaMKIIα or GABA are
shown in green and red, respectively. Arrows represent pERK- and CaMKIIα-positive neurons in mPFC. Scale bar, 50 μm. (e) The densities ofpERK-positive excitatory or inhibitory neurons in the mPFC without or with S-SDS (Naive or Single, respectively). The numbers of mice inrespective groups are shown below bars. P-values shown in the graph were calculated by Bonferroni’s post hoc test following two-way ANOVA(F(1,20) = 8.638, P = 0.0081 for the main Stress effect, F(1,20) = 99.48, P o0.0001 for the main Neuron-type effect). Results are shown as mean ± s.
e.m. (f) The densities of pERK-positive excitatory neurons in the mPFC of Ctrl-miR mice or D1-miR mice without or with S-SDS (Naive or Single,
respectively). The numbers of mice in respective groups are shown below bars. P-values shown in the graph were calculated by Bonferroni’s
post hoc test following two-way ANOVA (F(1,22) = 4.864, P = 0.0382 for the main RNAi effect, F(1,22) = 7.683, P = 0.0111 for the main Stress effect).
Results are shown as mean ± s.e.m. (g) Representative images of immunoﬂuorescent staining for c-Fos in mPFC of Ctrl-miR mice or D1-miR
mice without or with S-SDS (Naive or Single, respectively). NeuN was co-stained to identify neurons in the mPFC. The signals for c-Fos and
NeuN are shown in green and red, respectively. The same images of respective ﬂuorescent channels and their magniﬁed images are shown in
Supplementary Figure 8. Scale bars, 200 μm. (h) The densities of c-Fos-positive neurons in the mPFC layer II/III of Ctrl-miR mice or D1-miR mice
without or with S-SDS (Naive or Single, respectively). The numbers of mice in respective groups are shown below bars. P-values shown in thegraph were calculated by Bonferroni’s post hoc test following two-way ANOVA (F(1,19) = 3.438, P = 0.0793 for the main RNAi effect, F(1,19) = 2.000,
P = 0.1735 for the main Stress effect). Results are shown as mean ± s.e.m. (i) The densities of c-Fos-positive neurons in the mPFC layer V/VI of
Ctrl-miR mice or D1-miR mice without or with S-SDS (Naive or Single, respectively). The numbers of mice in respective groups are shown
below bars. No signiﬁcant changes are observed (F(1,19) = 4.381, P = 0.0500 for the main RNAi effect, F(1,19) = 2.942, P = 0.1026 for the main
Stress effect by two-way ANOVA). Results are shown as mean ± s.e.m.

because the reduction of mRNA was seen in susceptible mice, butmuch smaller in resilient mice. Importantly, reduced mRNA
expression of D1 receptor subtype was not observed in the
nucleus accumbens, suggesting that the expression of D1 receptor
subtype is regulated in a brain region-speciﬁc manner. Further,
R-SDS did not affect the mRNA expression of D5 receptor subtype
in both susceptible and resilient mice, despite its expression in
mPFC neurons.22 These ﬁndings provide a rationale for the use of
D1 receptor subtype in the mPFC as a functional biomarker ofstress-related pathophysiology in PET. PET studies of D1 receptors
have thus far relied on the use of D1/D5 mixed ligands,14,17,18
because no subtype-speciﬁc D1 receptor ligand is available so far.
Given the importance of D1 receptor subtype in suppressing
stress susceptibility, it is desirable to develop a PET radioligand
speciﬁc to D1 receptor subtype rather than D5 receptor subtype.
Previous PET studies have investigated the relationship between
D1-like receptor subtypes in prefrontal cortices and cognitive
impairments in patients with mental illnesses.14,18,54 However, theresults appear to be inconsistent across studies, and the reason forthis inconsistency remains elusive.55 As the expression of D1
receptor subtype in mPFC is susceptible to R-SDS in mice, thehistory of stressful life events of individuals should be taken into
consideration to decipher and analyze the data of PET studies of
D1 receptors in the prefrontal cortices.
In contrast to D1 receptor subtype, we found that R-SDS
increases the mRNA expression of D2 receptor subtype in themPFC only in resilient mice. Consistently, it has recently beenreported that deep brain stimulation of the medial forebrain
bundle increases the expression of D2 receptor subtype in themPFC along with antidepressant-like effects.56 In mPFC excitatoryneurons, expression levels of D1 and D2 receptor subtypes are
partially overlapped, and it has been suggested that D1 and D2
receptor subtypes form a D1–D2 heteromer coupled to Gq
signaling rather than Gs/Golf or Gi/Go signaling activated by D1
or D2 receptor subtype independently.23 Thus increased© 2017 Macmillan Publishers Limited, part of Springer Nature.

expression of D2 receptor subtype, but not of D1 receptor
subtype, in the mPFC of resilient mice would increase theproportion of D2 receptor subtype uncoupled from D1 receptor
subtype. It has been reported that pharmacological disruption of
the D1–D2 heteromer in the mPFC induces antidepressant-like
effects in the forced swim test and the learned helplessness test in
rats.57 Therefore, whether D2 receptor subtype uncoupled fromD1 receptor subtype is involved in stress resilience warrants future
investigation.
A role for D1 receptor subtype in mPFC excitatory neurons insuppressing stress susceptibility
In most of the previous studies, pharmacological agents havebeen used to examine the roles for D1 receptors in prefrontal
cortices.9–12 However, the pharmacological studies cannot discriminate respective functions of D1 and D5 receptor subtypes
and cannot identify their sites of action. Therefore, in this study,
we generated AAV expressing D1 miRNA and injected this viral
vector to mPFC. We conﬁrmed that the expression of D1 miRNA inmPFC neurons selectively reduced the mRNA expression of D1
receptor subtype, but not those of the other subtypes, including
D5 receptor subtype. We also developed neuron type-speciﬁc
expression of D1 miRNA in mPFC, using AAV expressing D1 miRNA
only in the presence of Cre recombinase and transgenic miceexpressing Cre recombinase in speciﬁc neuronal populations orusing AAV expressing Cre recombinase selectively in excitatoryneurons, and showed that D1 receptor subtype and related
signaling in mPFC excitatory neurons contribute to suppression ofstress susceptibility. By contrast, D1 receptor subtype in mPFC
inhibitory neurons appears not to be critical for suppressing stresssusceptibility, although we cannot fully exclude the possibility that
D1 knockdown in mPFC inhibitory neurons may not be sufﬁcient
to cause its behavioral phenotype. These results represent, to thebest of our knowledge, the ﬁrst to demonstrate a role for a
Molecular Psychiatry (2017), 1 – 149

Role of dopamine D1 receptor in stress susceptibility
R Shinohara et al10

dopamine receptor subtype in a speciﬁc neuronal population in
prefrontal cortices in vivo. Therefore, this study provides a novel
method to investigate the roles of individual dopamine receptor
subtypes in a speciﬁc brain area of interest with advantage overclassical pharmacological agents.
Distinct groups of mPFC excitatory neurons project to different
brain areas58 and may exert opposing actions on depression-likebehaviors. It has been suggested that mPFC neurons projecting to
the raphe nucleus59 and those projecting to the nucleus
Molecular Psychiatry (2017), 1 – 14

accumbens60 suppress depression-like behaviors, whereas thoseprojecting to the lateral habenula increases these behaviors.59
Distinct groups of mPFC excitatory neurons may be susceptible tostress in various degrees, as repeated restraint stress induces
dendritic shortening in mPFC layer II/III pyramidal neurons projecting
to the entorhinal cortex but not in those projecting to the basolateral
amygdala.61 Therefore, it is critical to identify a brain region projectedby mPFC excitatory neurons in which D1 receptor subtype andrelated signaling contribute to suppression of stress susceptibility.
© 2017 Macmillan Publishers Limited, part of Springer Nature.

Role of dopamine D1 receptor in stress susceptibility
R Shinohara et al

Figure 4. Single social defeat stress (S-SDS) induces dendritic growth selectively in apical dendrites of medial prefrontal cortex (mPFC) layer II/
III pyramidal neurons through D1 receptor subtype. (a) A schedule of behavioral experiments to test the effect of repeated social defeat stress(R-SDS) on dendritic morphology of mPFC pyramidal neurons without or with D1 knockdown in mPFC neurons. Four weeks after injection ofviral solutions to the mPFC, the mice were isolated for a week and then were subjected to R-SDS followed by the social interaction test. Threedays after R-SDS, the brains were collected and subjected to morphological analysis of mPFC neurons. (b) Three-dimensional reconstruction ofmPFC layer II/III pyramidal neurons in naive, susceptible and resilient mice without D1 knockdown in mPFC neurons (Ctrl-miR mice). Apicaland basal dendrites are shown in red and blue, respectively. Scale bar, 100 μm. (c) The effects of R-SDS on the lengths of apical dendrites ofmPFC layer II/III pyramidal neurons in Ctrl-miR mice. Each dot represents the value from an individual neuron (left graph) or its average acrossneurons from an individual mouse (right graph). The numbers of neurons (left graph) or mice (right graph) in respective groups are shown
below bars. P-values shown in the graphs were calculated by Tukey’s post hoc test following one-way analysis of variance (ANOVA) (F(2,55) = 5.490, P = 0.0067 for the left graph and F(2, 17) = 6.288, P = 0.0090 for the right graph). Results are shown as mean ± s.e.m. (d) The effects of
R-SDS on the lengths of basal dendrites of the same neurons analyzed in panel (c). The values were analyzed and are shown similarly. The
numbers of neurons (left graph) or mice (right graph) in the respective groups are shown below bars. P-values shown in the graphs were
calculated by Tukey’s post hoc test following one-way ANOVA (F(2, 55) = 2.408, P = 0.0994 for the left graph and F(2, 17) = 3.974, P = 0.0384 for theright graph). Results are shown as mean ± s.e.m. (e) A schedule of behavioral experiments to test the effect of S-SDS on dendritic morphology
of mPFC pyramidal neurons without or with D1 knockdown in mPFC neurons. Four weeks after injection of viral solutions to mPFC, the mice
were isolated for a week and then were subjected to S-SDS followed by the social interaction test. Three days after S-SDS, the brains werecollected and subjected to morphological analysis of mPFC neurons. (f) Three-dimensional reconstruction of mPFC layer II/III pyramidal
neurons without or with S-SDS (Naive or Single, respectively) and without or with D1 knockdown in mPFC neurons (Ctrl-miR mice or D1-miR
mice, respectively). Apical and basal dendrites are shown in red and blue, respectively. Scale bar, 100 μm. (g) The effects of S-SDS on thelengths of apical dendrites of mPFC layer II/III pyramidal neurons in Ctrl-miR mice or D1-miR mice. Each dot represents the value from anindividual neuron (left graph) or its average across neurons from an individual mouse (right graph). The numbers of neurons (left graph) or
mice (right graph) in the respective groups are shown below bars. P-values shown in the graphs were calculated by Bonferroni’s post hoc test
following two-way ANOVA (F(1, 40) = 4.895, P = 0.0327 for the left graph and F(1, 15) = 6.646, P = 0.0210 for the right graph for the main Stresseffect). Results are shown as mean ± s.e.m. (h) The effects of S-SDS on the numbers of branching points of apical dendrites of the sameneurons analyzed in Figure 4g. The numbers of neurons (left graph) or mice (right graph) in respective groups are shown below bars. P-values
shown in the graphs were calculated by Bonferroni’s post hoc test following two-way ANOVA (F(1, 40) = 4.355, P = 0.0433 for the left graph and F(1, 15) = 4.312, P = 0.0554 for the right graph for the main Stress effect).

A novel neuronal correlate for the action of D1 receptor subtype in
mPFC excitatory neurons in suppressing stress susceptibility
In this study, we found that S-SDS increases dendritic arborizationand spine density of apical dendrites of mPFC layer II/III pyramidal
neurons. This study is, to the best of our knowledge, the ﬁrst toshow that brief stress exposure induces dendritic growth of mPFC
pyramidal neurons. As this dendritic growth is abolished by D1
knockdown in mPFC neurons, D1 receptor subtype and related
signaling contribute to this morphological change. Indeed, S-SDS
induces ERK phosphorylation selectively in mPFC excitatoryneurons, which depends, at least in part, on D1 receptor subtype.
It has been reported that stimulation of D1-like receptors
enhances N-methyl-D-aspartate (NMDA) receptor-mediated excitatory synaptic inputs62 and that NMDA receptor activation can
induce dendritic growth.63,64 ERK phosphorylation occurs upon
NMDA receptor stimulation65 and is required for NMDA receptormediated dendritic growth.66,67 Therefore, D1 receptor-mediatedenhancement of glutamatergic inputs and consequent ERK
phosphorylation in mPFC excitatory neurons may be involved indendritic growth of mPFC pyramidal neurons induced by S-SDS.
By contrast, we found that R-SDS induces dendritic shorteningof apical dendrites of mPFC layer II/III pyramidal neurons only insusceptible mice. This ﬁnding is consistent with previous reportsthat repeated restraint stress and chronic mild stress induce
dendritic shortening of mPFC pyramidal neurons in a mannercorrelated to behavioral changes.44,45 However, as D1 knockdown
in mPFC neurons did not potentiate the dendritic shortening but
facilitated the induction of social avoidance, the dendritic shortening of mPFC pyramidal neurons after R-SDS does not accountfor the action of D1 receptor subtype in mPFC in suppressing
stress susceptibility. This ﬁnding all the more highlights the link of
S-SDS-induced dendritic growth of mPFC layer II/III pyramidal
neurons through D1 receptor subtype with suppressing stresssusceptibility.
Our ﬁnding that S-SDS-evoked signaling of D1 receptor subtype
induces dendritic growth in mPFC neurons suggests the presenceof long-lasting effects of D1 receptor activation in mPFC. It has
been reported that a rapid-acting antidepressant such as
ketamine rapidly increases glutamatergic synaptic connections© 2017 Macmillan Publishers Limited, part of Springer Nature.

of mPFC pyramidal neurons,68 thereby producing the long-lasting
antidepressant effects.69 In primates, repeated treatments with a
D1 receptor agonist can reverse working memory deﬁcits inducedby chronic haloperidol treatment, and this improvement issustained for more than a year after cessation of treatment with
D1-like receptor agonist.70 These pieces of evidence, including
ours, suggest that activation of D1 receptor subtype provides
long-lasting effects on prefrontal functions. Studies using in vivo
proton magnetic resonance spectroscopy have shown reduced
glutamate levels in the anterior cingulate cortex of depressive
patients,71 and successful electroconvulsive therapy improves
these levels.72 Therefore, glutamate levels in prefrontal cortices
may be a promising biomarker to estimate long-term therapeutic
effects of drugs targeting D1-like receptors in patients with mental
illnesses.
As S-SDS and R-SDS induce dendritic growth and dendritic
shortening, respectively, of mPFC pyramidal neurons, the proadaptive dendritic growth switches into the maladaptive dendritic
shortening with repetition of SDS. As D1 knockdown in mPFC
neurons did not potentiate R-SDS-induced dendritic shortening ofmPFC pyramidal neurons, reduced D1 expression in mPFC cannot
account for R-SDS-induced dendritic shortening. Rodent studies
have shown that repeated stress increases the levels ofinﬂammation-related molecules in the brain, and these molecules
or their receptors are critical for repeated stress-induced
behavioral changes.5,73–75 Therefore, it is intriguing to examine
whether inﬂammation-related molecules are involved in repeatedstress-induced dendritic shortening and, if so, how D1-mediateddendritic growth switches into dendritic shortening mediated byinﬂammation-related molecules.
In conclusion, our ﬁndings show that D1 receptor subtype andrelated signaling in mPFC excitatory neurons mediate S-SDSinduced dendritic growth and contribute to suppression of stresssusceptibility. In contrast to previous pharmacological studies, thisstudy has identiﬁed a dopamine receptor subtype contributing tosuppression of stress susceptibility and its site of action in mPFC.
This study also substantiates the presence of long-term effects of
D1 receptor subtype on structures and functions of mPFC at leastin this behavioral model. As expression of D1 receptor subtype in
Molecular Psychiatry (2017), 1 – 1411

Role of dopamine D1 receptor in stress susceptibility
R Shinohara et al

12

Figure 5. Single social defeat stress (S-SDS) increases spine density selectively on apical dendrites of medial prefrontal cortex (mPFC) layer II/III
pyramidal neurons through D1 receptor subtype. (a) Representative images of dendritic spines on distal apical dendrites of mPFC layer II/III
pyramidal neurons without or with S-SDS (Naive or Single, respectively) and without or with D1 knockdown in the mPFC neurons (Ctrl-miR
mice or D1-miR mice, respectively). Scale bar, 5 μm. (b) The effects of S-SDS on spine densities on distal apical dendrites of mPFC layer II/III
pyramidal neurons in Ctrl-miR mice or D1-miR mice. Each dot represents the averaged value across multiple images from an individual neuron(left graph) or the averaged value across multiple neurons from an individual mouse (right graph). The numbers of neurons (left graph) or
mice (right graph) in the respective groups are shown below bars. P-values shown in the graphs were calculated by Bonferroni’s post hoc test
following two-way analysis of variance (ANOVA) (F(1, 40) = 2.712, P = 0.1074 for the left graph and F(1, 15) = 3.110, P = 0.0982 for the right graphfor the main Stress effect). Results are shown as mean ± s.e.m. (c) The effects of S-SDS on spine densities on proximal apical dendrites of thesame neurons analyzed in panel (b). The values were analyzed and are shown similarly. The numbers of neurons (left graph) or mice (rightgraph) in respective groups are shown below bars. P-values shown in the graphs were calculated by Bonferroni’s post hoc test following twoway ANOVA (F(1, 40) = 0.8648, P = 0.3580 for the left graph and F(1, 15) = 0.1428, P = 0.7108 for the right graph for the main Stress effect). (d) The
effects of S-SDS on spine densities on basal dendrites of the same neurons analyzed in panel (b). The values were analyzed and are shown
similarly. The numbers of neurons (left graph) or mice (right graph) in the respective groups are shown below bars. No signiﬁcant changes areobserved (F(1, 40) = 2.609, P = 0.1141 for the left graph and F(1, 15) = 2.223, P = 0.1567 for the right graph for the main Stress effect by two-way
ANOVA). (e) The effects of S-SDS on the densities of Type 1 spines on distal apical dendrites of the same neurons analyzed in panel (b). The
values were analyzed and are shown similarly. The numbers of neurons (left graph) or mice (right graph) in the respective groups are shown
below bars. No signiﬁcant changes are observed (F(1, 40) = 1.688, P = 0.2013 for the left graph and F(1, 15) = 0.7160, P = 0.4107 for the right graphfor the main Stress effect by two-way ANOVA). (f) The effects of S-SDS on the densities of Type 2 spines on distal dendrites of the sameneurons analyzed in panel (b). The values were analyzed and are shown similarly. The numbers of neurons (left graph) or mice (right graph) in
the respective groups are shown below bars. P-values shown in the graphs were calculated by Bonferroni’s post hoc test following two-way
ANOVA (F(1, 40) = 2.210, P = 0.1449 for the left graph and F(1, 15) = 2.719, P = 0.1199 for the right graph for the main Stress effect).

Molecular Psychiatry (2017), 1 – 14

© 2017 Macmillan Publishers Limited, part of Springer Nature.

Role of dopamine D1 receptor in stress susceptibility
R Shinohara et al

13mPFC is reduced after R-SDS, prefrontal functions to cope withstress and therapeutic effects of D1 receptor agonists on
prefrontal dysfunctions may be limited in susceptible subjects
after repeated stress. Furthermore, as R-SDS attenuates the activityof dopamine neurons projecting to mPFC,5,6 the recovery in D1
expression in mPFC excitatory neurons alone may not be sufﬁcient
to restore the action of D1 receptor subtype in suppressing stresssusceptibility. Therefore, the molecular pathway downstream of
D1 receptor subtype that is maintained after repeated stress may
offer a novel and more promising target for therapeuticinterventions for prefrontal dysfunctions of mental illnesses.
CONFLICT OF INTEREST
The authors declare no conﬂict of interest.

ACKNOWLEDGMENTS
We thank Dr Dai Watanabe for his helpful comments and discussions; Tae Arai, Akiko
Washimi, Yuki Nakanishi and Misako Takizawa for secretarial helps; and Nodoka
Asamoto for animal breeding and care. The present study was supported by a grantfrom AMED-CREST of JST (to SN), Grants-in-Aids for Scientiﬁc Research from the Japan
Society for the Promotion of Science (24689015 and 16H05132 to TF; 13J04246 and
16K18365 to RS), Grants-in-Aids for Scientiﬁc Research from the Ministry of Education,
Culture, Sports, Science and Technology in Japan (25116517, 25116715, 15H01289,
17H05572 and 17H06057 to TF), a grant from AMED (to TF), grants from the Uehara
Memorial Foundation (to TF), the Sumitomo Foundation (to TF), the Astellas
Foundation for Research on Metabolic Disorders (to TF) and the Takeda Science
Foundation (to TF), United States Public Health Service Grants MH-084018,
MH-094268, MH-069853, MH-085226, MH-088753 and MH-092443 (to AS) and grants
from Stanley (to AS), RUSK (to AS), S-R foundations (to AS), NARSAD (to AS) andMaryland Stem Cell Research Fund (to AS).

REFERENCES
1 McEwen BS, Bowles NP, Gray JD, Hill MN, Hunter RG, Karatsoreos IN et al.
Mechanisms of stress in the brain. Nat Neurosci 2015; 18: 1353–1363.
2 Duman RS, Aghajanian GK, Sanacora G, Krystal JH. Synaptic plasticity anddepression: new insights from stress and rapid-acting antidepressants. Nat Med
2016; 22: 238–249.
3 Lee RS, Sawa A. Environmental stressors and epigenetic control of the
hypothalamic-pituitary-adrenal axis. Neuroendocrinology 2014; 100: 278–287.
4 Tost H, Champagne FA, Meyer-Lindenberg A. Environmental inﬂuence in thebrain, human welfare and mental health. Nat Neurosci 2015; 18: 1421–1431.
5 Tanaka K, Furuyashiki T, Kitaoka S, Senzai Y, Imoto Y, Segi-Nishida E et al. Prostaglandin E2-mediated attenuation of mesocortical dopaminergic pathway is
critical for susceptibility to repeated social defeat stress in mice. J Neurosci 2012;
32: 4319–4329.
6 Chaudhury D, Walsh JJ, Friedman AK, Juarez B, Ku SM, Koo JW et al. Rapid
regulation of depression-related behaviours by control of midbrain dopamine
neurons. Nature 2013; 493: 532–536.
7 Niwa M, Jaaro-Peled H, Tankou S, Seshadri S, Hikida T, Matsumoto Y et al. Adolescent stress-induced epigenetic control of dopaminergic neurons via glucocorticoids. Science 2013; 339: 335–339.
8 Beaulieu JM, Gainetdinov RR. The physiology, signaling, and pharmacology of
dopamine receptors. Pharmacol Rev 2011; 63: 182–217.
9 Vijayraghavan S, Wang M, Birnbaum SG, Williams GV, Arnsten AF. Inverted-U
dopamine D1 receptor actions on prefrontal neurons engaged inworking memory. Nat Neurosci 2007; 10: 376–384.
10 Puig MV, Miller EK. The role of prefrontal dopamine D1 receptors in the neuralmechanisms of associative learning. Neuron 2012; 74: 874–886.
11 Zahrt J, Taylor JR, Mathew RG, Arnsten AF. Supranormal stimulation of D1
dopamine receptors in the rodent prefrontal cortex impairs spatial working
memory performance. J Neurosci 1997; 17: 8528–8535.
12 Floresco SB, Magyar O, Ghods-Shariﬁ S, Vexelman C, Tse MT. Multiple dopamine
receptor subtypes in the medial prefrontal cortex of the rat regulate set-shifting.
Neuropsychopharmacology 2006; 31: 297–309.
13 Arnsten AF, Girgis RR, Gray DL, Mailman RB. Novel dopamine therapeutics forcognitive deﬁcits in schizophrenia. Biol Psychiatry 2016; 81: 67–77.
14 Okubo Y, Suhara T, Suzuki K, Kobayashi K, Inoue O, Terasaki O et al. Decreased
prefrontal dopamine D1 receptors in schizophrenia revealed by PET. Nature 1997;
385: 634–636.

© 2017 Macmillan Publishers Limited, part of Springer Nature.

15 George MS, Molnar CE, Grenesko EL, Anderson B, Mu Q, Johnson K et al. A single
20 mg dose of dihydrexidine (DAR-0100), a full dopamine D1 agonist, is safe andtolerated in patients with schizophrenia. Schizophr Res 2007; 93: 42–50.
16 Rosell DR, Zaluda LC, McClure MM, Perez-Rodriguez MM, Strike KS, Barch DM et al.
Effects of the D1 dopamine receptor agonist dihydrexidine (DAR-0100A) on
working memory in schizotypal personality disorder. Neuropsychopharmacology
2015; 40: 446–453.
17 Karlsson P, Farde L, Halldin C, Sedvall G. PET study of D(1) dopamine receptor
binding in neuroleptic-naive patients with schizophrenia. Am J Psychiatry 2002;
159: 761–767.
18 Abi-Dargham A, Mawlawi O, Lombardo I, Gil R, Martinez D, Huang Y et al. Prefrontal dopamine D1 receptors and working memory in schizophrenia. J Neurosci
2002; 22: 3708–3719.
19 Floresco SB. Prefrontal dopamine and behavioral ﬂexibility: shifting from an"inverted-U" toward a family of functions. Front Neurosci 2013; 7: 62.
20 Santana N, Mengod G, Artigas F. Quantitative analysis of the expression of
dopamine D1 and D2 receptors in pyramidal and GABAergic neurons of the rat
prefrontal cortex. Cereb Cortex 2009; 19: 849–860.
21 de Almeida J, Mengod G. D2 and D4 dopamine receptor mRNA distribution in
pyramidal neurons and GABAergic subpopulations in monkey prefrontal cortex:
implications for schizophrenia treatment. Neuroscience 2010; 170: 1133–1139.
22 Oda S, Funato H, Adachi-Akahane S, Ito M, Okada A, Igarashi H et al. Dopamine D5
receptor immunoreactivity is differentially distributed in GABAergic interneurons
and pyramidal cells in the rat medial prefrontal cortex. Brain Res 2010; 1329:
89–102.
23 Rashid AJ, O'Dowd BF, Verma V, George SR. Neuronal Gq/11-coupled dopamine
receptors: an uncharted role for dopamine. Trends Pharmacol Sci 2007; 28:
551–555.
24 Holmes A, Lachowicz JE, Sibley DR. Phenotypic analysis of dopamine receptor
knockout mice; recent insights into the functional speciﬁcity of dopamine
receptor subtypes. Neuropharmacology 2004; 47: 1117–1134.
25 Stanwood GD, Parlaman JP, Levitt P. Anatomical abnormalities in dopaminoceptive regions of the cerebral cortex of dopamine D1 receptor mutant mice. J
Comp Neurol 2005; 487: 270–282.
26 Ikegami M, Uemura T, Kishioka A, Sakimura K, Mishina M. Striatal dopamine D1
receptor is essential for contextual fear conditioning. Sci Rep 2014; 4: 3976.
27 Sarinana J, Kitamura T, Kunzler P, Sultzman L, Tonegawa S. Differential roles of the
dopamine 1-class receptors, D1R and D5R, in hippocampal dependent memory.
Proc Natl Acad Sci USA 2014; 111: 8245–8250.
28 Betley JN, Sternson SM. Adeno-associated viral vectors for mapping, monitoring,and manipulating neural circuits. Hum Gene Ther 2011; 22: 669–677.
29 Golden SA, Covington HE 3rd, Berton O, Russo SJ. A standardized protocol for
repeated social defeat stress in mice. Nat Protoc 2011; 6: 1183–1191.
30 Krishnan V, Han MH, Graham DL, Berton O, Renthal W, Russo SJ et al. Molecular
adaptations underlying susceptibility and resistance to social defeat in brain
reward regions. Cell 2007; 131: 391–404.
31 Lazarus M, Shen HY, Cherasse Y, Qu WM, Huang ZL, Bass CE et al. Arousal effect of
caffeine depends on adenosine A2A receptors in the shell of the nucleus
accumbens. J Neurosci 2011; 31: 10067–10075.
32 Hioki H, Kuramoto E, Konno M, Kameda H, Takahashi Y, Nakano T et al. High-leveltransgene expression in neurons by lentivirus with Tet-Off system. Neurosci Res
2009; 63: 149–154.
33 Paxinos G, Franklin KBJ. The Mouse Brain in Stereotaxic Coordinates, 2nd edn.
Academic Press: London, 2001.
34 Shinohara R, Thumkeo D, Kamijo H, Kaneko N, Sawamoto K, Watanabe K et al. A
role for mDia, a Rho-regulated actin nucleator, in tangential migration of interneuron precursors. Nat Neurosci 2012; 15: 373–380, S371–S372.
35 Ehrlich AT, Furuyashiki T, Kitaoka S, Kakizuka A, Narumiya S. Prostaglandin E
receptor EP1 forms a complex with dopamine D1 receptor and directs D1induced cAMP production to adenylyl cyclase 7 through mobilizing G(betagamma) subunits in human embryonic kidney 293T cells. Mol Pharmacol 2013; 84:476–486.
36 Sholl DA. Dendritic organization in the neurons of the visual and motor cortices of
the cat. J Anat 1953; 87: 387–406.
37 Cahill ME, Bagot RC, Gancarz AM, Walker DM, Sun H, Wang ZJ et al. Bidirectional
synaptic structural plasticity after chronic cocaine administration occurs throughRap1 small GTPase signaling. Neuron 2016; 89: 566–582.
38 Tornatzky W, Miczek KA. Long-term impairment of autonomic circadian rhythms
after brief intermittent social stress. Physiol Behav 1993; 53: 983–993.
39 Madisen L, Zwingman TA, Sunkin SM, Oh SW, Zariwala HA, Gu H et al. A robust
and high-throughput Cre reporting and characterization system for the whole
mouse brain. Nat Neurosci 2010; 13: 133–140.
40 Chao HT, Chen H, Samaco RC, Xue M, Chahrour M, Yoo J et al. Dysfunction in
GABA signalling mediates autism-like stereotypies and Rett syndrome phenotypes. Nature 2010; 468: 263–269.

Molecular Psychiatry (2017), 1 – 14

Role of dopamine D1 receptor in stress susceptibility
R Shinohara et al

14
41 Nagai T, Takuma K, Kamei H, Ito Y, Nakamichi N, Ibi D et al. Dopamine D1
receptors regulate protein synthesis-dependent long-term recognition memory
via extracellular signal-regulated kinase 1/2 in the prefrontal cortex. Learn Mem
2007; 14: 117–125.
42 Xue B, Mao LM, Jin DZ, Wang JQ. Regulation of synaptic MAPK/ERK phosphorylation in the rat striatum and medial prefrontal cortex by dopamine and muscarinic acetylcholine receptors. J Neurosci Res 2015; 93: 1592–1599.
43 Jungenitz T, Radic T, Jedlicka P, Schwarzacher SW. High-frequency stimulationinduces gradual immediate early gene expression in maturing adult-generated
hippocampal granule cells. Cereb Cortex 2014; 24: 1845–1857.
44 Duman RS, Aghajanian GK. Synaptic dysfunction in depression: potential therapeutic targets. Science 2012; 338: 68–72.
45 Davidson RJ, McEwen BS. Social inﬂuences on neuroplasticity: stress and interventions to promote well-being. Nat Neurosci 2012; 15: 689–695.
46 Liston C, Miller MM, Goldwater DS, Radley JJ, Rocher AB, Hof PR et al. Stressinduced alterations in prefrontal cortical dendritic morphology predict selective
impairments in perceptual attentional set-shifting. J Neurosci 2006; 26:
7870–7874.
47 Dias-Ferreira E, Sousa JC, Melo I, Morgado P, Mesquita AR, Cerqueira JJ et al.
Chronic stress causes frontostriatal reorganization and affects decision-making.
Science 2009; 325: 621–625.
48 Li N, Liu RJ, Dwyer JM, Banasr M, Lee B, Son H et al. Glutamate N-methyl-Daspartate receptor antagonists rapidly reverse behavioral and synaptic deﬁcits
caused by chronic stress exposure. Biol Psychiatry 2011; 69: 754–761.
49 Hering H, Sheng M. Dendritic spines: structure, dynamics and regulation. Nat Rev
Neurosci 2001; 2: 880–888.
50 Harris KM. Structure, development, and plasticity of dendritic spines. Curr Opin
Neurobiol 1999; 9: 343–348.
51 Williams GV, Castner SA. Under the curve: critical issues for elucidating D1
receptor function in working memory. Neuroscience 2006; 139: 263–276.
52 Takahashi H, Yamada M, Suhara T. Functional signiﬁcance of central D1 receptors
in cognition: beyond working memory. J Cereb Blood Flow Metab 2012; 32:
1248–1258.
53 Goldman-Rakic PS, Castner SA, Svensson TH, Siever LJ, Williams GV. Targeting the
dopamine D1 receptor in schizophrenia: insights for cognitive dysfunction. Psychopharmacology (Berl) 2004; 174: 3–16.
54 Kosaka J, Takahashi H, Ito H, Takano A, Fujimura Y, Matsumoto R et al. Decreased
binding of [11C]NNC112 and [11C]SCH23390 in patients with chronic schizophrenia. Life Sci 2010; 86: 814–818.
55 Takahashi H. PET neuroimaging of extrastriatal dopamine receptors and prefrontal cortex functions. J Physiol Paris 2013; 107: 503–509.
56 Dandekar MP, Luse D, Hoffmann C, Cotton P, Peery T, Ruiz C et al. Increased
dopamine receptor expression and anti-depressant response following deepbrain stimulation of the medial forebrain bundle. J Affect Disord 2017; 217: 80–88.
57 Pei L, Li S, Wang M, Diwan M, Anisman H, Fletcher PJ et al. Uncoupling the
dopamine D1-D2 receptor complex exerts antidepressant-like effects. Nat Med
2010; 16: 1393–1395.
58 Vertes RP. Differential projections of the infralimbic and prelimbic cortex in
the rat. Synapse 2004; 51: 32–58.

59 Warden MR, Selimbeyoglu A, Mirzabekov JJ, Lo M, Thompson KR, Kim SY et al. A
prefrontal cortex-brainstem neuronal projection that controls response to behavioural challenge. Nature 2012; 492: 428–432.
60 Christoffel DJ, Golden SA, Walsh JJ, Guise KG, Heshmati M, Friedman AK et al.
Excitatory transmission at thalamo-striatal synapses mediates susceptibility tosocial stress. Nat Neurosci 2015; 18: 962–964.
61 Shansky RM, Hamo C, Hof PR, McEwen BS, Morrison JH. Stress-induced dendritic
remodeling in the prefrontal cortex is circuit speciﬁc. Cereb Cortex 2009; 19:2479–2484.
62 Gonzalez-Islas C, Hablitz JJ. Dopamine enhances EPSCs in layer II-III pyramidal
neurons in rat prefrontal cortex. J Neurosci 2003; 23: 867–875.
63 Cline HT, Constantine-Paton M. NMDA receptor antagonists disrupt the retinotectal topographic map. Neuron 1989; 3: 413–426.
64 Sin WC, Haas K, Ruthazer ES, Cline HT. Dendrite growth increased by visual activity
requires NMDA receptor and Rho GTPases. Nature 2002; 419: 475–480.
65 English JD, Sweatt JD. Activation of p42 mitogen-activated protein
kinase in hippocampal long term potentiation. J Biol Chem 1996; 271:
24329–24332.
66 Wu GY, Deisseroth K, Tsien RW. Spaced stimuli stabilize MAPK pathway activationand its effects on dendritic morphology. Nat Neurosci 2001; 4: 151–158.
67 Wayman GA, Impey S, Marks D, Saneyoshi T, Grant WF, Derkach V et al.
Activity-dependent dendritic arborization mediated by CaM-kinase I activationand enhanced CREB-dependent transcription of Wnt-2. Neuron 2006; 50:
897–909.
68 Li N, Lee B, Liu RJ, Banasr M, Dwyer JM, Iwata M et al. mTOR-dependent synapse
formation underlies the rapid antidepressant effects of NMDA antagonists. Science 2010; 329: 959–964.
69 Autry AE, Adachi M, Nosyreva E, Na ES, Los MF, Cheng PF et al. NMDA receptor
blockade at rest triggers rapid behavioural antidepressant responses. Nature
2011; 475: 91–95.
70 Castner SA, Williams GV, Goldman-Rakic PS. Reversal of antipsychotic-induced
working memory deﬁcits by short-term dopamine D1 receptor stimulation. Science 2000; 287: 2020–2022.
71 Auer DP, Putz B, Kraft E, Lipinski B, Schill J, Holsboer F. Reduced glutamate in theanterior cingulate cortex in depression: an in vivo proton magnetic resonancespectroscopy study. Biol Psychiatry 2000; 47: 305–313.
72 Pﬂeiderer B, Michael N, Erfurth A, Ohrmann P, Hohmann U, Wolgast M et al.
Effective electroconvulsive therapy reverses glutamate/glutamine deﬁcit in theleft anterior cingulum of unipolar depressed patients. Psychiatry Res 2003; 122:
185–192.
73 Koo JW, Duman RS. IL-1beta is an essential mediator of the antineurogenic andanhedonic effects of stress. Proc Natl Acad Sci USA 2008; 105: 751–756.
74 Goshen I, Kreisel T, Ben-Menachem-Zidon O, Licht T, Weidenfeld J, Ben-Hur T et al.
Brain interleukin-1 mediates chronic stress-induced depression in mice via
adrenocortical activation and hippocampal neurogenesis suppression. Mol Psychiatry 2008; 13: 717–728.
75 Wohleb ES, Hanke ML, Corona AW, Powell ND, Stiner LM, Bailey MT et al. betaAdrenergic receptor antagonism prevents anxiety-like behavior and microglial
reactivity induced by repeated social defeat. J Neurosci 2011; 31: 6277–6288.

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