Synaptotagmin-11 deficiency mediates schizophrenia-like behaviors in mice via dopamine over-transmission

Abstract

Schizophrenia is a severe neuropsychiatric disease, but the initiation mechanisms are unclear. Although antipsychotics are effective against positive symptoms, therapeutic interventions for negative symptoms are limited due to the lack of pathophysiological mechanisms. Here we identify synaptotagmin-11 (Syt11) as a potential genetic risk factor and dopamine over-transmission as a mechanism in the development of schizophrenia. Syt11 expression is reduced in individuals with schizophrenia but restored following the treatment with antipsychotics. Syt11 deficiency in dopamine neurons in early adolescence, but not in adults, leads to persistent social deficits and other schizophrenia-like behaviors by mediating dopamine over-transmission in mice. Accordingly, dopamine neuron over-excitation before late adolescence induces persistent schizophrenia-associated behavioral deficits, along with the structural and functional alternations in the mPFC. Notably, local intervention of D2R with clinical drugs presynaptically or postsynaptically exhibits both acute and long-lasting therapeutic effects on social deficits in schizophrenia mice models. These findings not only define Syt11 as a risk factor and DA over-transmission as a potential risk factor initiating schizophrenia, but also propose two D2R-targeting strategies for the comprehensive and long-term recovery of schizophrenia-associated social withdrawal.

Full text

Article https://doi.org/10.1038/s41467-024-54604-4
Synaptotagmin-11 deficiency mediates
schizophrenia-like behaviors in mice via
dopamine over-transmission
Yang Chen
1,10
,YuhaoGu
1,10
, Bianbian Wang
1,10
,AnqiWei
1,10
, Nan Dong
1
,
Yong Jiang
2
, Xiaoying Liu
1,3,4
, Li Zhu
5
,FengZhu
6
,TaoTan
7
,ZexinJing
1
,
Fenghan Mao
1
, Yichi Zhang
1
,JingyuYao
1
,YuxinYang
1,4
, Hongyan Wang
4
,
Hao Wu
1
,HuaLi
1
, Chaowen Zheng
1
,XuetingDuan
1
, Jingxiao Huo
1
, Xuanang Wu
1
,
Shaoqin Hu
1
, Anran Zhao
1
,ZiyangLi
1
, Xu Cheng
3
,YuhaoQin
3
, Qian Song
1
,
Shuqin Zhan
1
,QiuminQu
8
, Fanglin Guan
5
, Huadong Xu
1
,
Xinjiang Kang
2,3,4
& Changhe Wang
1,2,3,9
Schizophrenia is a severe neuropsychiatric disease, but the initiation
mechanisms are unclear. Although antipsychotics are effective against positive
symptoms, therapeutic interventions for negative symptoms are limited due
to the lack of pathophysiological mechanisms. Here we identify
synaptotagmin-11 (Syt11) as a potential genetic risk factor and dopamine over-
transmission as a mechanism in the development of schizophrenia. Syt11
expression is reduced in individuals with schizophrenia but restored following
the treatment with antipsychotics. Syt11 deficiency in dopamine neurons in
early adolescence, but not in adults, leads to persistent social deficits and
other schizophrenia-like behaviors by mediating dopamine over-transmission
in mice. Accordingly, dopamine neuron over-excitation before late adoles-
cence induces persistent schizophrenia-associated behavioral deficits, along
with the structural and functional alternations in the mPFC. Notably, local
intervention of D2R with clinical drugs presynaptically or postsynaptically
exhibits both acute and long-lasting therapeutic effects on social deficits in
schizophrenia mice models. These findings not only define Syt11 as a risk factor
and DA over-transmission as a potential risk factor initiating schizophrenia,
but also propose two D2R-targeting strategies for the comprehensive and
long-term recovery of schizophrenia-associated social withdrawal.
Schizophrenia is a chronic and disabling psychiatric disorder with a
prevalence of 1% worldwide. This disease is characterized by the mani-
festation of positive symptoms such as delusions and hallucinations,
negative symptoms such as social withdrawal and loss of motivation,
and cognitive dysfunction1–3. The onset of schizophrenia is typically
characterized by social withdrawal and cognitive decline, which usually
begins in early adolescence and precedes the psychotic episode by
several years. While antipsychotic medications are effective in treating
positive symptoms in ~70% of patients4–6, they fail to improve negative
symptoms and cognitive impairments, which are considered as core
features of the disorder throughout the lifetime7.Despiterecent
advancements in understanding the etiology of schizophrenia, the
underlying pathophysiological mechanisms responsible for negative
symptoms, particularly social withdrawal, remain largely unknown.
Received: 27 March 2024
Accepted: 15 November 2024
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A full list of affiliations appears at the end of the paper. e-mail: fanglingguan@163.com;hdxu@pku.edu.cn;kxj335@163.com;changhewang@xjtu.edu.cn
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Converging evidence suggests that aberrant dopamine (DA)
transmission is associated with the positive psychotic symptoms of
schizophrenia2,8–13. However, the concept of hyperactive DA transmis-
sion has been challenged due to inconsistent clinical observations4,14–17
andisproposedtobedownstreamofhyperactiveglutamatergicpro-
jections or the consequence of excitatory-inhibitory imbalance of
synaptic inputs4,8,15,18. Although most currently-licensed antipsychotic
agents (e.g., chlorpromazine, haloperidol, clozapine, phenothiazine,
and butyrophenone) are suggested to alleviate positive symptoms
mainly through antagonizing D2 receptors (D2Rs)2,8, there is little
direct evidence (real time recordings) demonstrating aberrant DA
transmission in schizophrenia. The role of DA dysfunction in the onset
of schizophrenia remains a topic of debate in this field.
Based on the inability of D2R antagonists in treating negative
symptoms, the ‘dual dysregulation’hypothesis has been proposed as a
reformulation of the DA hypothesis15,19. According to this hypothesis,
excess striatal DA transmission leads to positive symptoms of schizo-
phrenia, while cortical hypodopaminergicfunction is implicated in the
negative and cognitive aspects15,17,19. However, the evidence for extra-
striatal DA deficits primarily relies on the indirect measurements of DA
levels in response to amphetamine administration or depletion para-
digm in preclinical and postmortem observations14,17. The causal rela-
tionship between DA deficiency and negative symptoms is largely
speculative and has not been adequately assessed to date. Thus, it
remains unclear whether and how extrastriatal DA transmission con-
tributes to the manifestation of negative and cognitive symptoms in
schizophrenia.
Large-scale genetic studies have identified numerous genes
associated with schizophrenia, but only few of them have been shown
to be sufficient to independently trigger the onset of
schizophrenia20–22. Thus, ideal genetic mouse models for schizo-
phrenia study are still lacking. Recent studies have identified SYT11 as a
candidate gene implicated in both familial and sporadic schizophrenia
susceptibility23,24. Synaptotagmins (Syts) are primary Ca2+ sensors that
mediate SNARE-dependent vesicle fusion during neurotransmission25.
We have recently found that Syt11, a non-Ca2+-binding Syt26,actsasa
constitutive brake of endocytosis in neurons27–29, while Syt11 accumu-
lation mediates the neurodegenerative changes in DA neurons, con-
tributing to the pathogenesis of Parkinson’s disease by inhibiting
vesicle recycling and DA release30. However, it remains unknown
whether and how Syt11 dysfunction leads to neurodevelopmental
deficits and the pathogenesis of schizophrenia in mice.
In this study, we have identified Syt11 as a potential risk factor for
schizophrenia, established plasma Syt11 asa biomarker for diagnosing
schizophrenia, and developed Syt11 conditional knockout (cKO)
mouse asa genetic mousemodel for schizophrenia study. Importantly,
our findings elucidate a role of DA over-transmission before late ado-
lescence in the initiation of schizophrenia, particularly in relation to
negative symptoms. Local administration of a D2R agonist in the
ventral tegmental area (VTA, inhibiting presynaptic DA release) or an
antagonist in the medial prefrontal cortex (mPFC, stimulating post-
synaptic cortical neurons) during late adolescence yields similar long-
lasting therapeutic effects on schizophrenia-like behavioral changes.
Altogether, this work not only defines Syt11 as a potential risk factor
and DA over-transmission as a neural mechanism initiating the
pathogenesis of schizophrenia but also unveils a time window and a
couple of D2R-targeting strategies for potential clinical treatment of
schizophrenia.
Results
Syt11 deficiency is linked to schizophrenia
To investigate the potential association between Syt11 expression and
schizophrenia, we analyzed Syt11 expression in three datasets of
human brain tissues: the Lieber Institute for Brain Development(LIBD),
the CommonMind Consortium (CMC), and the Human Brain
Collection Core (HBCC). These datasets included mRNA transcription
profiles from the dorsolateral prefrontalcortex (dlPFC)of postmortem
brains from both schizophrenia patients and healthy controls. To
minimize site-specific technical variations, the CMC and HBCC data-
sets, which underwent RNA extraction and data generation at a single
facility, were combined31. Our analysis revealed a significant reduction
in Syt11 expression in prefrontal cortex tissues of schizophrenia
patients (Fig. 1a, b). This finding was further confirmed by qPCR and
Western blot analyses, which showed a significant decrease in both
Syt11 transcription and protein expression in the plasma of schizo-
phrenia patients in two independent case-control samples (Fig.1c, d).
Specifically, ~50% of schizophrenia patients showed a clear reduction
of Syt11 expression in the plasma (by setting the threshold at the 90th
percentile of the highest value in the healthy control group), with an
area under the curve ratio (AUC) of 0.737 in the receiver operating
characteristic (ROC) curve (Supplementary information, Fig. S1). These
results not only identify Syt11 deficiency as a potential risk factor for
the pathogenesis of schizophrenia, but also define plasma Syt11 as a
biomarker for the clinical diagnosis of schizophrenia.
To validate the close association between Syt11 expression and
schizophrenia, we next examined Syt11 expression in plasma samples
collected from healthy individuals and schizophrenia patients before
and after antipsychotic treatment. We found that the decreased
expression of Syt11 in the plasma of schizophrenia patients was
restored after the antipsychotic treatment with olanzapine, haloper-
idol, or risperidone (Fig. 1e). Specifically, patients who received the
haloperidol treatment showed the most substantial changes in symp-
tom scores when compared to those treated with olanzapine or ris-
peridone, and this was correlated with the highest restoration of Syt11
expression in the haloperidol-treated patients (Fig. 1f, g). Importantly,
Pearson’s correlation analysis demonstrated a positive correlation
between the overall changes in schizophreniasymptom scores and the
changes in Syt11 expression after antipsychotic treatment (Fig. 1h),
suggesting that the rescue of Syt11 deficiency in schizophrenia patients
is closely associated with the therapeutic effects of antipsychotic
medications. Collectively, these findings from human samples and
clinical treatment demonstrate the close association between Syt11
deficiency and schizophrenia.
DA neuron-restricted knockout of Syt11 leads to schizophrenia-
like behaviors
To investigate the potential role of SYT11 deficiency in initiating the
pathogenesis of schizophrenia, we generated DA neuron-restricted
Syt11 cKO mice by crossing homozygous floxed Syt11-null mice with
DAT-driven Cre recombinase (DAT-Cre) transgenic mice (Fig. 1i), as
previously described30. Immunostaining for tyrosine hydroxylase (TH)
confirmed the specific loss of Syt11 in midbrain DA neurons (Fig. 1j).
The three-chamber social interaction test was employed to assess the
social disability, a prominent negative symptom in schizophrenia, in
young (6-8 weeks old) male Syt11-cKO mice (Fig. 1k). As expected,
control mice (DAT-Cre) spent more time interacting with a stranger
mouse (M1) than a fake mouse (F) (Fig. 1k). In contrast, Syt11-cKO mice
showed reduced sniffing time with the M1 mouse, while their interac-
tion with the F mouse remained unchanged, resulting in the decreased
social preference index (Fig. 1k, l), suggesting impaired social pre-
ference upon Syt11 deficiency in DA neurons. Similarly, in the social
novelty test, Syt11-cKO mice showed the reduced social preference for
a new stranger mouse (M2) over a familiar one, in contrast to control
mice (Fig. 1m, n). Importantly, the total sniffing time with both M1 and
M2 mice decreased greatly in Syt11-cKO mice (Fig. 1o), confirming the
impaired social activity in the absence of Syt11 in DA neurons.
Furthermore, we applied the social approach test to further
evaluate the behavioral deficits in young Syt11-cKO mice (Fig. 1p).
Compared to controls, Syt11-cKO mice spent significantly less time
approaching andinteracting with a cagedstranger mouse(Fig. 1p). The
Article https://doi.org/10.1038/s41467-024-54604-4
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home-cage social test also revealed reduced social interaction of Syt11-
cKO mice with a stranger intruder mouse (Fig. 1q), confirming that the
DA neuron-restricted KO of Syt11 is sufficient to induce social deficits
at early ages.
To examine whether these social deficits persist into adulthood,
we conducted the same social behavioral tests with adult (3 months
old) male Syt11-cKO mice. These mice also showed reduced sniffing
time with the M1 mouse and thus a decreased social preference index
(Supplementary information, Fig. S2a-c). Similarly, cKO mice per-
formed worse than controls in the social novelty test (Supplementary
information, Fig. S2d-f). Similar social deficits were observed in Syt11
cKO mice at 1 year old (Supplementary information, Fig. S2i,j), indi-
cating the long-lasting social withdrawal in the absence of Syt11.
Additionally, adult cKO mice also showed decreased social interaction
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Article https://doi.org/10.1038/s41467-024-54604-4
Nature Communications | (2024) 15:10571 3
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time with the stimulus mouse in the social approach test (Supple-
mentary information, Fig. S2g). Consistent with this, the social inter-
action time of adult Syt11-cKO mice with the stranger intruder mouse
was shorter than that of controls (Supplementary information, Fig.
S2h). Together, these results suggest an early-onset and enduring
social withdrawal phenotype in DA neuron-restricted Syt11-cKO mice.
To further confirm the association of social withdrawal in Syt11-
cKO mice with schizophrenia, we carried out a series of behavior
analyses to examine other schizophrenia-related symptoms. In the
locomotion test, Syt11-cKO mice showed the increased total travel
distance with intact travel speed at adolescence (Supplementary
information, Fig. S2k, l), indicating locomotion hyperactivity, which
corresponds to the psychomotor agitation observed in schizophrenia
patients. Furthermore, both adolescent and adult Syt11-cKO mice
exhibited aberrant prepulse inhibition (PPI) of the acoustic startle
response (Fig. 1r, s), a well-defined hallmark of sensorimotor gating
dysfunction manifested in early adulthood in patients with schizo-
phrenia. As a control, the intact startle amplitude indicated normal
gross auditory and motor ability in Syt11-cKO mice (Fig.1r, s). Although
Syt11-cKO mice did not show clear impairments in the T-maze test
during adolescence, adult cKO mice spent more time turning into the
goal arm (Fig. 1t), suggesting deficits in short-term working memory
upon Syt11 deficiency in DA neurons. In addition, the adult Syt11-cKO
mice also performed poorly in a spontaneous alternation Y-maze test
(Fig. 1u), consistent with the cognitive dysfunction observed in
patients with schizophrenia from ear ly adulthood. In contrast, the c KO
mice did not show enhanced marble-burying behavior (Supplementary
information, Fig. S2m) or excessive self-grooming inthe open field test
(Supplementary information, Fig. S2n), suggesting the absence of
repetitive behaviors associated with autism32. Overall, these results
demonstrate a role of Syt11 deficiency in DA neurons in mediating the
pathogenesis of schizophrenia and provide a mouse model for schi-
zophrenia study, particularly that related to social withdrawal and
other negative symptoms.
Syt11 deficiency in early adolescence mediates social deficits
We aimed to investigate whether there is a sensitive time-window
during which Syt11 deficiency leads to social deficits. To address this
issue, we generated DA neuron-restricted knockout of Syt11 during
early adolescence and adulthood, respectively. To generate DA
neuron-restricted knockout of Syt11 at early adolescence, we stereo-
taxically injected a TH-Cre expressing AAV9 virus into the VTA of
homozygous floxed Syt11-null mice on postnatal day 0–1(P0,Fig.2a).
Immunostaining confirmed the absence of Syt11 in virus-infected DA
neurons (GFP-positive) in the VTA 6 weeks after virus injection (Fig. 2b;
Supplementary information, Fig. S3a, b), and Western blot analysis
validated the decreased expression of Syt11 in the ventral midbrain
(Fig. 2c). Notably, DA neuron-restricted KO of Syt11 since P0 (P0-cKO)
exhibited decreased sniffing time with M1 mice and a reduced social
index in the three-chamber social interaction test during 6–8 weeks of
age (Fig. 2d-f). Similarly, these Syt11 P0-cKO mice also showed
decreased social preference for the stranger M2 mouse and reduced
total social time with both mice compared with controls (Fig. 2g-i). In
addition, Syt11 P0-cKO mice showed reduced social interaction with a
caged stranger mouse in the social approach test (Fig. 2j) and the
stranger intruder mouse in the home-cage social test (Fig. 2k). These
findings indicate that the absence of Syt11 at an early age can mediate
impairments in social a bility. Importantly, adult Syt11 P0-cKO mice also
showed impaired social behaviors in the three-chamber social test,
social novelty test, and social approach test (Supplementary infor-
mation, Fig. S3), suggesting that Syt11 deficiency since P0 is associated
with ongoing social deficits.
To test whether Syt11 knockout in adult mice also results in social
deficits, we generated Syt11 adult-cKO mice by injecting TH-Cre virus
into the VTA of adult (3-4 months) Syt11-flox mice (Supplementary
information, Figs. S4). Although Syt11-cKO led to increased DA release
in the nucleus accumbens (NAc, Supplementary information, Fig. S5a)
as revealed by electrochemical amperometric recordings, these mice
spent similar amounts of time interacting with the M1 mouse and thus
showed an unchanged social index in the three-chamber social inter-
action test (Supplementary information, Fig. S5b-d). In addition, the
social preference for theM2 mouse and the totalsocial time with both
mice remained intact in adult-cKO mice compared with controls
(Supplementary information, Fig. S5e-g). Syt11 adult-cKO mice also
showed similar social interaction time with the caged stranger mouse
in the social approach test (Supplementary information, Fig. S5h) and
the stranger intruder mouse in the home-cage social test (Supple-
mentary information, Fig. S5i). Collectively, these results demonstrate
that Syt11 adult-cKO mice exhibit normal social behaviors, suggesting
that there is a sensitive time-window before adult for Syt11 deficiency-
mediated schizophrenia-like social deficits, further supporting the
persistence of social deficits when Syt11 deficiency occurs at earlier
ages, and implying a potential role of Syt11 in neural development.
Syt11 deficiency leads to DA over-transmission via the acceler-
ated vesicle recycling and off-membrane trafficking of D2Rs
Our previous studies have shown that Syt11 serves as a clamp for
endocytosis and thus inhibits vesicle replenishment and DA release,
Fig. 1 | Syt11 deficiency in dopamine neurons leads to schizophrenia-like
behaviors. a,bTranscript expression levels of Syt11 in the dlPFC of postmortem
brainsfrom schizophrenia (SCZ) patients vs healthy controls (HC). RNA-sequenc ing
data set were obtainedfrom the Lieber Institute for Brain Development (LIBD), the
CommonMind Consortium (CMC), and the Human Brain Collection Core (HBCC).
The CMC and HBCC data sets were performed at a single facility with similar pro-
cesses and thus combined to minimize site-specific sources of technical variation.
cTranscript expression levels ofSyt11 in peripheral blood from SCZ patients vs HC.
dRepresentative western blots and expression levels of Syt11 in plasma from SCZ
patients vs HC. eTranscript expression levels of Syt11 in peripheral blood from HC
and SCZ patients before (SCZ-pre) and after (SCZ-post) antipsychotic treatment.
fChangesin SCZ symptomsscores of SCZ patients aftertreatment with olanzapine,
haloperidol, or risperidone. gTranscript expression changes of Syt11 in peripheral
blood from SCZ patients after treatment with olanzapine, haloperidol, or risper-
idone. hPearson correlation analysis between changes in Syt11 expression and
changes in SCZ symptom scores after antipsychotic treatment as in (e-g).
iSchematic of the generation of DA neuron-restricted Syt11 conditional knockout
(cKO) mice. jRepresentative micrograph showing the immunostaining of Syt11
(red) and TH(green) in a VTA-containing slice (enlarged insets in the lower panel).
Scale bars: 500 μm (upper), 100 μm (lower). Data from 3 mice. k, l Schematic,
representativeheat maps, and statistics ofthe three-chamber socialinteraction test
of juvenile (6–8 weeks) Syt11-cKO or DAT-Cre(Ctrl) mice. M1,a novel mouse; F,fake
toy mouse. Sniffingtime and social indexof Syt11-cKO vs control mice wereused for
analysis. mSchematic and representative heat maps of the three-chamber social
novelty test of juvenile Syt11-cKO vs control mice. M1, familiar mouse (the former
novel mouse in kand l); M2, new comer nov el mouse. n, o Statistics of sniffing time
(with M1 or M2) and total social time (sniffing with M1 and M2) of Syt11-cKO vs
controlmice. pLeft, schematic and representative heat maps of thesocial approach
test. Right, statistics of sniffing time with a caged novel mouse of Syt11-cKO vs
control mice (6–8weeks).qStatistics of sniffing time with an intruder mouse of
Syt11-cKO vs control mice (6–8 weeks) in the home-cage social test. r, s Statistics of
startleresponses and pre-pulse inhibition (PPI)of juvenile (6–8 weeks)and adult (3-
4 months) Syt11-cKO mice vs control mice. tStatistics of short-term memory (T-
maze) of juvenile and adult Syt11-cKO mice vs control mice. uStatistics of the
spontaneous alternation Y-maze test of juvenile and adult Syt11-cKO mice vs con-
trol mice. Data areshown as box-and-whiskerplots, with themedian represented by
the central line inside each box, the 25th and 75th percentiles represented by the
edgesof the box, and thewhiskers extending to the mostextreme datapoints. Two-
tailed Mann-Whitney test for (a-d, l, o-q, r-u), Pearson correlation analysis for (h),
one-way ANOVA for (f, g), or Ordinary two-way ANOVA followed by Bonferroni’s
multiple comparisons for (e, k, n), *P<0.05, **P< 0.01, ***P< 0.001, n.s. no sig-
nificant difference. Source data are provided as a Source Data file.
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while Syt11 cKO in midbrain DA neurons leads to excessive DA release
in the striatum27,29,30. Here, we further investigated whether Syt11 cKO
in VTADA neurons leads to social deficits via DA over-transmission. To
examine this, we applied amperometric recordings with electro-
chemical carbon fiber electrodes (CFEs) in the NAc and the medial
prefrontal cortex (mPFC), which are DA neuron-projecting regions
involved in social behaviors. Consistent with previous reports30,33,local
electrical pulse-stimulation induced a transient increase in ampero-
metric current (I
amp
), followed by a subsequent decay to the baseline,
representing transient DA release in the NAc (Fig. 2l). As expected, DA
neuron-restricted KO of Syt11 in the VTA led to increased DA release in
the NAc (Fig. 2l). To specifically assess DA vesicle recycling in the NAc,
we employed an optogenetic approach by injecting FLExloxP-based
Channelrhodopsin-2 (ChR2)-expressing AAV9 virus and TH-Cre virus
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into the VTA of Syt11-floxed null or control mice. Notably, the paired-
pulse ratio of DA release in NAc slices evoked by the 488-nm laser
stimulus increased substantially in Syt11-cKO mice compared with
control mice (Fig. 2m), validating accelerated vesicle recycling and
hence elevated DA release in the NAc in the absence of Syt11.
We next assessed DA release in the mPFC by utilizing a clozapine-
N-oxide (CNO)-based chemogenetic approach. TH-Cre and Cre-
dependent hM3Dq-expressing AAV2/9 viruses were stereotaxically
injected into the VTA of Syt11-floxed null or control mice (Fig. 2n).
Immunostaining confirmed that the expression of mCherry/hM3Dq
was restricted to DA neurons in the VTA (Supplementary information,
Fig. S6a, b). Patch-clamp recordings revealed an elevated firing rate of
DA neurons in the VTA upon CNO application (Supplementary infor-
mation, Fig. S6c), while CFE recordings demonstrated that CNO
application further elicited DA release in the mPFC (Fig. 2n). Similar
with that in the NAc (Fig. 2l), the CNO-evoked DA release in the mPFC
was higher in Syt11-cKO mice than in control mice (Fig. 2n). These
results validate the elevated DA transmission and accelerated DA
vesicle recycling upon Syt11 deficiency in DA neurons.
Moreover, electrophysiological patch-clamp recordings revealed
a pronounced increase in the firingrate of action potentials (APs) of DA
neurons in VTA slices from Syt11 P0-cKO mice (Fig. 2o), indicating the
over-excitation of midbrain DA neurons in the absence of Syt11. Con-
sistent with this, we also found the increased resting membrane
potential (RMP) in VTADA neurons, with the membrane capacitance
(Cm) and input membrane resistance (Rm) remained unchanged
(Supplementary information, Fig. S7a). We hypothesized that the sur-
face auto-inhibitory D2R receptor in DA neurons may undergo
alterations due to the accelerated vesicle recycling27,28,30,34. Consistent
with our expectation, the facilitatory effect of the D2R antagonist
haloperidol on AP firing of DA neurons was substantially diminished in
VTA slices from Syt11 P0-cKO mice (Fig. 2p; Supplementary informa-
tion, Fig. S7b). Similarly, we also observed a decreased inhibitory effect
of the D2R agonist quinpirole on AP firing (Fig. 2q; Supplementary
information, Fig. S7b), suggesting a reduction in functional D2R in DA
neurons in situ in the absence of Syt11. In line with this, we also
observed accelerated endocytosis with FM uptake (Supplementary
information, Fig. S7c) and decreased expression of membrane D2R
(Supplementary information, Fig. S7d, e) in Syt11 knockdown dopa-
minergic SY5Y cells, as well as the decreased total D2R expression in
the VTA in Syt11-cKO mice (Supplementary information, Fig. S7f).
Although we couldn’t fully exclude other possibilities (i.e. receptor
signaling adaptations, gene expression alterations), our recent
reports27,29,30 and the present findings collectively underscore that
Syt11 deficiency leads to elevated DA transmission via accelerated
endocytosis and vesicle recycling, as well as the hyperactivity of DA
neurons due to the increased off-membrane trafficking of surface
D2Rs (Fig. 2r).
DA neuron over-excitation during adolescence mediates
schizophrenia-related social deficits
To further determine whether the over-excitation of VTADA neurons,
which mimics elevated DA release, is sufficient to mediate
schizophrenia-like social withdrawal, we performed co-injections of
TH-Cre virus and Cre-dependent hM3Dq-expressing virus (with DIO-
mCherry blank virus as a control) into the VTA of 3-week-old mice.
These mice were then subjected to social behavior tests during ado-
lescence (6–8 weeks) following the chemogenetic activation of DA
neurons via intraperitoneal (i.p.) administration of CNO (Fig. 3a).
Notably, a single dose of CNO was capable of significantly reducing the
time spent sniffing the M1 mouse in the three-chamber social test by
hM3Dq-expressing mice (Fig. 3b, c). The social preference index was
also decreased greatly compared to control virus-injected mice
(Fig. 3d). Similarly, chemogenetic activation of VTADA neurons also
resulted in inferior performance of hM3Dq-expressing mice in the
social novelty test (Fig. 3e, f). Furthermore, the social interaction time
with the stimulus mouse in the social approach test and with the
stranger intruder mouse in the home-cage social test were both
reduced in mice following chemogenetic activation (Fig. 3g, h), con-
firming that hyperactivity of DA neurons during early development is
sufficient to cause socialdeficits. Importantly,mice with chemogenetic
activation also exhibited aberrant PPI in the acoustic startle response
and impaired short-term memory (Supplementary information, Fig.
S8), further supporting the association between DA over-transmission
and social withdrawal in schizophrenia. In contrast, similar chemoge-
netic activation of DA neurons failed to induce social deficits in adult
mice in the three-chamber social interaction test (Supplementary
information, Fig. S9a-c), social novelty test (Supplementary informa-
tion, Fig. S9d, e), and home-cage social test (Supplementary informa-
tion, Fig. S9f). These results demonstrate that transient over-excitation
of VTADA neurons only mediates social withdrawal before late adoles-
cence or young adulthood, suggesting a critical time window for DA
Fig. 2 | Syt11 deficiency at earlyages mediates social deficits via dopamine over-
transmission. a Schematic representation of virus injection (TH-Cre-EGFP, or TH-
EGFP served as a control) into the VTA of neonatal Syt11-flox/flox mice (P0) for the
generation of DA neuron-restricted Syt11-cKO mice from birth (Syt11 P0-cKO).
bRepresentative micrograph showing the immunostaining of Syt11 (magenta) and
TH (red) in a VTA-containing slice from a Syt11 P0-cKO mouse (6 weeks post virus
injection) as described in a. Enlarged insetsare shown on the right.n=3mice;Scale
bars: 400 μm (left), 50 μm(right).cRepresentative Western blots and statistics
showing the expression of Syt11 in the VTA of Syt11 P0-cKO mice compared to
controlmice. Scale bars, 400 μm. d–fRepresentative heat mapsand statisticsof the
three-chamber social interaction test of juvenile (6–8 weeks) Syt11 P0-c KO vs
control mice. g–iRepresentative heat maps and statistics of the socialnovelty test
of juvenile Syt11 P0-cKO vs control mice. jRepresentative heat maps and statistics
of sniffing time in the social approach test ofjuvenile Syt11 P0-cKO vs control mice.
kStatistics of investigation frequency and investigation time in the home-cage
social test of juvenile Syt11 P0-cKO vs control mice. lSchematic, representative
amperometric currents (I
amp
), and statistics showing DA release from DAergic
terminals in the NAc of Syt11 P0-cKO (n=3) vs control (n= 3) mice. Scale bars, 10
μm. mLeft, schematic showing the co-injection of TH-Cre and DIO-ChR2-mCherry
viruses into the VTA of juvenile (3 weeks) Syt11-flox/flox or wide-type mice to
generate Syt11 3W-cKO (n=3)orcontrol(n= 3) mice with C hR2 expressed in VTADA
neurons. Middle and right, representative paired-pulse stimulus (40 s)-evoked
amperometric signals and statistics of the paired-pulse ratio showing the recycling
of DA vesicles in NAc slices from Syt11 3W-cKO vs control mice. nSchematic
showingthe co-injectionof TH-Cre and DIO-hM3D-mCherryviruses into the VTA of
juvenile (3 weeks) Syt11-flox/flox or wide-type miceto generate Syt11 3W-cKO(n=3)
or control (n= 3) mice with hM3D expressed in VTADA neurons. Middle and right,
representativeamperometriccurrent (I
amp
) traces andstatistics of DA release in the
mPFC of Syt11 3W-cKO vs control mice following CNO application (5 μM).
oSchematic of virus injection (TH-Cre-EGFP or TH-EGFP) into the VTA of neonatal
Syt11-flox/flox mice(P0) for the generation of Syt11 P0-cKO(n=8)orcontrol(n=4)
mice. Middle and right, representative AP traces and statistics of the spontaneous
action potential firing rate of VTA DA neurons from Syt11 P0-cKO mice vs control
mice. pRepresentative AP traces and statistics showing the excitatory effect of the
D2R antagonist haloperidol (Halo, 50 nM) on DA neurons in VTA slices in situ from
Syt11 P0-cKO (n=4)vs co ntrol (n=4) mice.qRepresentative AP traces and statis-
tics showing the inhibitory effect of the D2R agonist quinpirole (Qp, 50 nM) on the
excitability of DA neurons in VTA slices in situ from Syt11 P0-cKO (n=5)vs control
(n=5)mice. rA working model showing that Syt11 deficiency increases DA trans-
mission via ①facilitating DA vesicle recycling, and ②decreasing surface auto-
receptor D2R expression, which leads to increased excitability of DA neurons.
Created in BioRender. Yang, C. (2023 ) https://BioRender.com/w74v548.Dataare
shown as box-and-whisker plots, with the median represented by the central line
inside each box, the 25th and75th percentiles representedby the edges of the box,
and the whiskers extending to the most extreme data points. Ordinary two-way
ANOVA followed by Bonferroni’s multiple comparisons for (e, h) or two-tailed
Mann-Whitney test for(c, f, i-q), *P< 0.05, **P< 0.01, ***P< 0.001, n.s. no significant
difference. Source data are provided as a Source Data file.
Article https://doi.org/10.1038/s41467-024-54604-4
Nature Communications | (2024) 15:10571 6
Content courtesy of Springer Nature, terms of use apply. Rights reserved

Investigation time (s)
Social approach
**
Social index
Ctrl hM3D
Social novelty
Control
hM3D
Social (6~8 W old)
M1 F*** ***
**
*** **
Sniffing time (s)
Social index
Total social time (s)
Ctrl hM3D Ctrl
Ctrl hM3D
hM3D
hM3D Ctrl hM3D
Sniffing time (s)
Control
hM3D
a
*
Ctrl hM3D
ef
g
Ctrl
h
P0 P7 P14 P42 P56
Virus
CNO
Behavior
C57 (3 W old)
VTA
TH-Cre
DIO-hM3D-mCherry
DIO-hM3D-mCherry
C57 (P0)
VTA
TH-Cre
(11)
(11)
(11)
(12)
(12) (12)
M1 M1
F
P21 P42 P56
Virus
CNO
(i.p.)
(i.p.)
Behavior
F
c
d
Ctrl
hM3D
Investigation time (s)
Ctrl hM3D
Sniffing time (s)
**
n.s.
Sniffing time (s)
Ctrl hM3D
0 s
M1
0 s
45 s
45 s
M1 M2
Total social time (s)
Ctrl hM3D
Ctrl hM3D
Investigation frequency
M1M2 M2
*
500
400
300
200
100
0
M1 F
M1
M1 M2 M2M1
F
n.s.
n.s.
300
250
200
150
100
50
0
250
200
150
100
50
0
1.2
1.0
0.8
0.6
0.4
0.2
0.0
400
300
200
100
0
***
Home cage social
(8)
(8)
(8)
(8)
(11) (9)
(9)
(12)
**
**
150
100
50
0
1.2
1.0
0.8
0.6
0.4
0.2
0.0
100
80
60
40
20
0
ijkl
m
** *
Investigation time (s)
120
100
80
60
40
20
0
Investigation time (s)
Investigation frequency
Home cage social
Social approach
Ctrl hM3D Ctrl hM3D
(12)
(10) (10)
(22) (22)
(12)
n
o
p
0
100
200
300
0
200
400
600
b
250
200
150
100
50
0
200
150
100
50
0
Social (6~8 W old)
M1 F
Control
hM3D
0 s
45 s
Social novelty
Control
hM3D
0 s
45 s
M1 M2
120
80
40
0
Fig. 3 | DA neuron over-excitation during adolescence mediates long-lasting
social deficits. a Schematic showing the co-injection of TH-Cre and DIO-hM3D-
mCherry/DIO-mCherry viruses into the VTA of juvenile (3 weeks) C57 mice.
b–dRepresentativeheat maps andstatistics ofthe three-chamber socialinteraction
test of juvenile (6–8 weeks) hM3D-expressing mice vs control mice following i.p.
administration of CNO (0.5 mg/kg). e,fRepresentative heat maps and statistics of
the social novelty test of juvenile hM3D-expressing mice vs control mice as
described in b–d.gStatistics of sniffing time in the social approach test of juvenile
hM3D-expressing mice vs control mice following i.p. administration of CNO.
hStatistics of investigation frequency and investigation time in the home-cage
social test of juvenile hM3D-expressing mice vs control mice following i.p. admin-
istration of CNO. iSchematic showing the co-injection of TH-Cre and DIO-hM3D-
mCherry/DIO-mCherry viruses into the VTA of neonatal C57 mice (hM3D, P0) and
the experimental procedure. j–lRepresentative heat maps and statistics of sniffing
time and social index in the three-chamber social interaction test of juvenile
repetitive CNO-treated (every second day during P7-P14) hM3D-expressing mice vs
control mice as described in i.m,nRepresentative heat maps and statistics of
sniffing time and total social time in the social novelty test of juvenile repetitive
CNO-treated hM3D-expressing mice vs control mice. oSniffing time of juvenile
repetitive CNO-treated hM3Dq-expressing mice vs control mice in the social
approach test with a caged novel mouse. pInvestigation frequency and investi-
gation time of juvenile repetitive CNO-treated hM3Dq-expressing mice vs control
mice in the home-cage social test. Data are shown as box-and-whisker plots, with
the median represented by the central line inside each box, the 25th and 75th
percentiles represented by the edges of the b ox, and the whiskers extending to the
most extreme data points. Ordinary two-way ANOVA followed by Bonferroni’s
multiple comparisons for (c, e, k, m), two-tailed Mann-Whitney test for (d, f–h, l,
n–p),*P<0.05,**P< 0.01, ***P< 0.001, n.s. no significant difference.Source data are
provided as a Source Data file.
Article https://doi.org/10.1038/s41467-024-54604-4
Nature Communications | (2024) 15:10571 7
Content courtesy of Springer Nature, terms of use apply. Rights reserved

hyperactivity in the pathogenesis of schizophrenia and implying a
development-dependent shift of DAergic circuit/pathway in social
behaviors.
We further explored whether over-excitation of DA neurons
before early adolescence leads to long-lasting social deficits resem-
bling those observed in individuals with schizophrenia. To this end, we
performed co-injections of TH-Cre virus and Cre-dependent hM3Dq-
expressing virus into the VTA during the early postnatal period (P0).
Subsequently, systemic administration of CNO (i.p.) every second day
from P7 to P14 was delivered to induce sustained over-excitation of DA
neurons during the synapse maturation phase (Fig. 3i). Notably,
compared with control mice, hM3Dq-expressing mice showed a sig-
nificant reduction in the time spent sniffing the M1 mouse, consistent
with the decreased social preference index in the three-chamber social
test 4–6 weeks after the CNO treatment (Fig. 3j-l). Furthermore, these
CNO-treated hM3Dq-expressing mice also displayed the reduced
social preference for the stranger mouse (M2) and consequently the
reduced total social time with both mice in the social novelty test
(Fig. 3m, n). In line with these results, CNO-treated hM3Dq-expressing
mice exhibited pronounced impairments in social interactions with the
stimulus mouse in the social approach test and the stranger intruder
mouse in the home-cage social test (Fig. 3o, p). These findings indicate
that the social deficits induced by repetitive chemogenetic activation
of VTADA neurons during the P7–P14 period can persist until at least
young adulthood. Interestingly, the over-excitation of VTADA neurons
was maintained at least 4 weeks following the repetitive chemogenetic
activation (Supplementary information, Fig. S10), suggesting a long-
lasting plastic change in the excitability of DA neurons. These findings
suggest that environmental disturbances leading to DA over-
transmission at an early age are sufficient to initiate persistent
schizophrenia-like behaviors.
Schizophrenia has been reported to be more prevalent and severe
in men than in women2,13. Therefore, we investigated whether the over-
excitation of DA neurons could similarly induce schizophrenia-like
social withdrawal in female mice. Interestingly, repetitive chemoge-
netic activation of DA neurons before early adolescence resulted in
persistent social deficits in female mice, ranging from late adolescence
(Supplementary information, Fig. S11) to adulthood (Supplementary
information, Fig. S12). These findings contradict the speculative ‘dual
dysregulation’of DA hypothesis15 and instead demonstrate that DA
over-transmission is indeed a mechanism underlying social with-
drawal. Considering the comprehensive schizophrenia-like behavioral
changes observed in Syt11-cKO and Syt11 P0-cKO mice (Figs. 1and 2;
Supplementary information, Figs. S1-S5) and mice with DA neuron
over-excitation (Figs. 2and 3; Supplementary information, Figs. S6-
S12), as well as the involvement of aberrant striatal DA release in the
positive symptoms2,thesefindings suggest a scenario in which DA
over-transmission may represent a shared pathway contributing to
different symptoms of schizophrenia during a critical time window
before late adolescence.
DA over-transmission in the mPFC during adolescence mediates
social deficits
Given that the NAc and the mPFC are primary DA neuron-projecting
regions involved in social behaviors, we used optogenetic manipula-
tion to further determine the specific brain region downstream of
VTADA neurons responsible for the social deficits in schizophrenia. The
FLExloxP-based ChR2-expressing AAV9 virus and TH-Cre virus were co-
injected into the VTA of 3-week-old mice (Fig. 4a). As expected, tran-
sient 473-nm light stimulation (L-stim, 1 ms duration) reliably elicited
AP firing in DA neurons (mCherry-positive) in current-clamp electro-
physiological recordings (Fig. 4b). Meanwhile, L-stim also triggered DA
release in the NAc and mPFC, as detected by electrochemical
amperometric recordings (Fig. 4b). Home-cage social test was carried
out during the P42–P56 time window to evaluate the possible
contribution of the mPFC and NAc to the social deficits.Notably, a train
of burst L-stim (5 ms, 8 pulses at 30 Hz; once every 5 s) on DA terminals
in the mPFC resulted in a pronounced reduction in the social interac-
tion with the intruder mouse (Fig. 4c). In contrast to the facilitatory
effect of VTA-NAc DA signals on social behaviors in adult mice35
(Supplementary information, Fig. S13), similar L-stim in the NAc failed
to induce detectable changes in social interaction in adolescent mice
(Supplementary information, Fig. S14), suggesting that the elevated DA
release in the mPFC, but not the NAc, during adolescence mediates the
social withdrawal downstream of VTADA neurons.
To further validate potential roles of DA over-transmission in the
mPFC in social withdrawal, we assessed social deficits with pharma-
cological intervention targeting postsynaptic D2Rs in the mPFC
(Fig. 4d). As expected36, the local application of quinpirole, a potent
D2R agonist, effectively reduced the activity of D2R-positive cortical
neurons in mPFC slices (Fig. 4e). Subsequently, we conducted social
behavior tests on adolescent mice following the stereotaxic injection
of quinpirole (bilateral, 1 µg/µl, 0.2 µl per side) into the mPFC to aug-
ment DA transmission by activating postsynaptic D2Rs (Fig. 4d). As
expected, quinpirole decreased the time spent in social interaction
with the M1 mouse and the social preference index in the three-
chamber social test (Fig. 4f–h). Similar inhibitory effects of quinpirole
were observed in the social novelty test (Fig. 4i–k) and the home-cage
social test (Fig. 4l). Conversely, the similar local infusion of quinpirole
into the NAc (Fig. 4m, n) failed to induce impairments in social activ-
ities in the three-chamber social test (Fig. 4o–q), the social novelty test
(Fig. 4r–t), and the home-cage social test (Fig. 4u). These findings
confirm that DA over-transmission in the mPFC, but not the NAc,
duringadolescence plays a central rolein mediating schizophrenia-like
social deficits. The time window-specific inhibition of social preference
by DA over-transmission in the mPFC suggests a role of DA transmis-
sion in the development and connectivity of mPFC neurons before late
adolescence.
To validate potential roles of DA transmission in the plastic
changes of mPFC neurons, we conducted assessments of morpholo-
gical changes in the mPFC of Syt11 cKO mice. Consistent with the
reduced cortical neurons and spine density observed in clinical
studies37–39 and post-mortem evidence40–42, we observed a significant
decrease of MAP2-positiveneurons in Layers I, II/III, and VI of the mPFC
from Syt11 cKO mice (Fig. 5a, b). Accordingly, the intensity of TH-
positive neurites in all Layers I-VI was substantially reduced (Fig. 5c, d).
Consistent with this, although the density of VTADA neurons in Syt11
cKO mice remained unchanged (Supplementary information, Fig.
S15a,b), the dendritic complexity of these neurons decreased greatly
(Supplementary information, Fig. S15c,d). In line with these findings,
repetitive chemogenetic activation of VTADA neurons during P7-P14
induced similar long-term changes in MAP2-positive neurons and TH-
positive neurites in the mPFC (Supplementary information, Fig.
S16a–d). These results suggest that DA over-transmission in the mPFC
atearlyagesissufficient to induce neurostructual alterations in the
mPFC. Subsequently, we performed patch-clamp electrophysiological
recordings to assess the functional changes of mPFC cortical neurons.
Consistent with the reduced cortical excitability observed in
schizophrenia43, we indeed found the decreased excitatory post-
synaptic current (Fig. 5e,f)andthedecreasedAPfiring rate of mPFC
cortical neurons in both adult Syt11 P0-cKO and CNO-treated (P7-P14)
hM3Dq-expressing mice (Fig. 5g, h). These findings suggest that DA
over-transmission before early adolescence leads to long-lasting
morphological and functional plastic changes in mPFC cortical
neurons.
To gain insights into mechanisms underlying the schizophrenia-
like behavioral changes observed in Syt11 cKO mice, we performed
genome-wide RNA-sequencing analysis to capture transcriptome-wide
alterations in the mPFC of adult (3 months) Syt11 cKO mice. By com-
paring the gene expression profile of control (DAT-Cre) mice, we
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Nature Communications | (2024) 15:10571 8
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identified 170 differentially expressed genes (DEGs) in Syt11-cKO mice
(Fig. 5i,j). Amongthese DEGs, 97 were upregulated and 73 were down-
regulated. Interestingly, some of the detected DEGs, such as Nrg1,Arc,
Ddc and Icam, are well-characterized schizophrenia risk ge nes that play
critical roles in neural development and/or are functionally involved in
the pathogenesis of schizophrenia44–47. Gene ontology (GO) analysis
revealed that DEGs were enriched in cellular components involved in
vesicle trafficking, such as the vesicle tethering complex, extracellular
vesicles, and extracellular exosomes (Fig. 5k). In the biological process
category, the enriched GO terms were strongly suggestive for pro-
cesses related to vesicular trafficking (e.g. vesicle transport, axonal
protein transport, post-synaptic retrograde transport, and vesicle-
mediated intercellular transport), neurotransmission (e.g. DA biosyn-
thetic process, membrane docking, synaptic transmission, synaptic
6
5
4
3
2
1
0
M1 M2
M2
M1
F
M1
M1 F
0 s
45 s
0 s
45 s
0 s
45 s
0 s
45 s
ab
eh
i
mPFC
NAc
TH-Cre
DIO-ChR2-mCherry
Ctrl ChR2 Ctrl ChR2
Investigation time (s)
Investigation frequency
mPFC
k
1s
l
mo
q
p
Saline Qp
Sniffing time (s)
120
100
80
60
40
20
0
Social (6~8 W old)
Saline Qp
Social index
(7)
(11)
n.s.
Saline Qp
Sniffing time (s)
Social novelty
(7)
(11)
n.s. n.s.
M1 M1M2 M2
M1 F
FM1
(7)
(11)
n.s.
n.s.
g
c
d
Social novelty
Control
Control
Qp
Control
Qp
Qp
Saline Qp
Sniffing time (s)
200
150
100
50
0
n.s. **
**
**
M1 M2 M1 M2
*
Saline
Total social time (s)
200
150
100
50
0
1.2
1.0
0.8
0.6
0.4
160
120
80
40
Qp
(14) (14)
(14)
(14)
Investigation time (s)
Saline Qp Saline Qp
400
300
200
100
0
Home cage social
NAc
Quinpirole
C57
P28 P42 P56
Cannule
Behavior
Investigation frequency
j
s
r
Saline Qp
Total social time (s)
60
40
20
0
VTA
VTA
(11) (7)
n.s.
Saline Saline
Qp Qp
Home cage social
Investigation frequency
Investigation time (s)
n.s. n.s.
(7)
(7) (7) (7)
tu
Home cage social
Quinpirole
mPFC
P28 P42
P42
P56
Cannule Optial fiber
Behavior
P21 P28 P56
Virus
Behavior
C57
Social (6~8 W old)
n
Control
Qp
M1 M1 F
F
n.s.
f
Saline Qp
Sniffing time (s)
1.0
0.8
0.6
0.4
0.2
0.0
***
Social index
200
150
100
50
0
Saline Qp
(14)
(14)
(14)
(14)
Qp
Qp
Qp
Pre
Qp
Pre
Frequency (Hz)
Frequency (Hz)
8
6
4
2
0
150
100
50
0
5 s
(11)
(10)
40 mV
mPFC
NAc
Action potential
DA release
20 mV
10 mV
100 ms
20 ms
20 Hz
L-stim L-stim
NAc
VTA
mPFC
**
(7)
(7)
(7)
(7)
(10)
(10)
(11) (11)
**
** **
70
60
50
40
30
20
10
0
350
300
250
200
150
100
50
0
Iamp
300
250
200
150
100
50
0
80
60
40
20
0
20 pA
**
5 s
40 mV 40 mV
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Nature Communications | (2024) 15:10571 9
Content courtesy of Springer Nature, terms of use apply. Rights reserved

plasticity, signal transduction, neurotransmitter receptor metabolic
process, receptor signaling pathway, and post-synapse organization),
and neural development (e.g. neural projection development,
embryonic brain development, and nervous system development)
(Fig. 5k). Consistently, repetitive chemogenetic activation of VTADA
neurons during P7-P14 induced similar long-term transcriptome-wide
changes in the mPFC (Supplementary information, Fig. S16e-g). Over-
all, the transcriptome sequencing analysis provides genetic insights
into the long-term changes in the mPFC that are linked with Syt11
deficiency and/or DA over-transmission.
Pathogenic effects of the D2R antagonist haloperidol on social
deficits
Haloperidol primarilyalleviates positive symptoms byantagonizing D2
receptors and reducing elevated DA transmission in the striatum48,but
its efficacy is limited and is often accompanied by adverse effects,
including the worsening of negative symptoms5.GiventhatD2Rsalso
act as inhibitory auto-receptors in DA neurons49, we hypothesized that
in addition to its therapeutic effect by antagonizing postsynaptic D2Rs,
haloperidol may have an additional pathogenic effect on negative
symptoms by removing the auto-inhibition of presynaptic/somatic
D2Rs in DA neurons (Fig. 6a). Our patch-clamp recordings indeed
showed that haloperidol application greatly increased the firing rate of
DA neurons in the VTA (Supplementary information, Fig. S17). To
further confirm the impact of haloperidol on DA release, we conducted
electrochemical CFE recordings in the mPFC in vivo following electric
stimulation of DA axons in the medial forebrain bundle. Notably, sys-
temic administration of haloperidol (0.4 mg/kg) substantially
enhanced evoked DA release in the mPFC (Fig. 6b), confirming the
drug’s ability to induce aberrant DA release in vivo. Thereby, the
increased DA levels led to a decrease in the firing rate of cortical
neurons in the mPFC (Fig. 6c), exhibiting an opposite effect on cortical
neuron excitability compared to the direct antagonism of postsynaptic
D2Rs. Consistent with these findings, local application of haloperidol
onto mPFC slices resulted inboth increased (55%, post-synaptic effect)
and decreased (20%, pre-synaptic effect) excitation of cortical neurons
(Fig. 6d), indicating a mixed effect of haloperidol in the mPFC with the
postsynaptic effect being predominant.
Regarding opposing roles of presynaptic and postsynaptic D2Rs
in DA transmission, we postulated that the lack of efficacy of halo-
peridol and other antipsychotic agents in treating negative symptoms
of schizophrenia could be attributed to their dual disinhibitory effects
on both presynaptic/somatic and postsynaptic D2Rs. To investigate
this, we tested whether the modulation of presynaptic/somatic D2Rs
by locally delivering the clinical drug haloperidol into the VTA (thereby
removing the auto-inhibition of DA neurons) could induce
schizophrenia-like social withdrawal in adolescent mice (Fig. 6e).
Consistent with chemogenetic manipulations, pharmacological acti-
vationof DA neurons with a single local-delivery ofhaloperidolinto the
VTA (b ilateral, 50 µM) resulted in impaired social behaviors, including
reduced social interaction time with the M1 mouse and a decreased
social preference index in the three-chamber social test (Fig. 6f-h).
Similarly, haloperidol infusion into the VTA also attenuated social
preference for the stranger mouse (M2) and total sniffing time with
both mice (Fig. 6i-k). Moreover, pretreatment with haloperidol in the
VTA impaired social interaction with a stranger intruder mouse in the
home-cage social test (Fig. 6l). Therefore, systemic treatment with
haloperidol or other antipsychotic agents targeting D2Rs may inad-
vertently exacerbate schizophrenia-associated symptoms by promot-
ing the over-excitation of DA neurons and thus the increased DA
release. These results further support a role of DA over-transmission
during adolescence in the pathogenesis of schizophrenia.
D2R as a dual therapeutic target for the treatment of
schizophrenia
Considering the opposing roles of presynaptic and postsynaptic D2Rs
in DA transmission, we propose that local administration of haloper-
idol or other D2R antagonists in the mPFC could be an effective
approach to alleviate negative symptoms by rectifying the aberrant DA
transmission postsynaptically (with a mixed effect, but the post-
synaptic effect predominated as shown in Fig. 6d). To test this
hypothesis, we locally infused haloperidol into the mPFC during the
period of P42–P56 and monitored its impact on the social behaviors of
Syt11 P0-cKO mice (Fig. 7a). Notably, a single administration of halo-
peridol into the mPFC restored the decreased social interaction with
the M1 mouse and the reduced social preference index in Syt11 P0-cKO
mice (Fig. 7b, c). Similarly, the diminished social preference for the
stranger mouse (M2) and the total sniffing time with both mice were
completely attenuated (Fig. 7d, e). Furthermore, compared with the
impaired social interaction observed in saline-control Syt11 P0-cKO
mice, the haloperidol-treated cKO mice exhibited a social interaction
time indistinguishable from that of control mice in the home-cage
social test (Fig. 7f). Therefore, local application of a D2R antagonist
into the mPFC before late adolescence(or early adulthood) can rescue
the schizophrenia-like social deficits. These findings not only provide a
reasonable explanation for the limited effectiveness of well-known
antipsychotics in alleviating negative symptoms in schizophrenia, but
also propose a potential therapeutic strategy for the clinical treatment
of the disease.
The present study has provided direct in vivo evidence that DA
over-transmission during preadolescence is a risk factor in initiating
the pathogenesis of schizophrenia (Figs. 1–4). Therefore, it is plausible
to reverse the social deficits by locally delivering a D2R agonist into the
VTA, which inhibits DA neurons by targeting somatic D2Rs, to rectify
Fig. 4 | DA over-transmission in the mPFC during adolescence mediates
schizophrenia-like social deficits. a Schematic of the co-injection of TH-Cre and
DIO-ChR2-mCherry viruses into the VTA of juvenile C57 mice (3 weeks). bUpper,
representative AP traces showing optogenetic activation of DA neurons in VTA
slices (mCherry-positive) by 473-nm light stimulation (L-stim). Lower, representa-
tive amperometric traces showing the L-stim induced DA release in the NAc and
mPFC slices. Data from 3 mi ce. cSchematic andstatistics of sniffing time/frequency
in the home-cage social test of the ChR2-expressing mice vs control mice following
L-stim trains in the mPFC. dSchematic of bilateral cannula application of the D2R
agonist quinpirole (Qp) into the mPFC and the experimental procedure.
eRepresentative AP traces and statistics showing the inhibitory effect of Qp
(50 nM) on theexcitabilityof D2R-positivecortical neuronsin the mPFC (Data from
3 mice and presented as mean ± SEM). f–hRepresentative heat maps and statistics
of the three-chamber social interactiontest of juvenile (6–8 weeks) mice following
the local applicationof Qp vs saline in the mPFC.i–kRepresentative heat mapsand
statistics of the social novelty test of the Qp- vs saline-treated juvenile mice as
described in f–h.lStatisticsof investigationfrequency andinvestigationtime in the
home cage social test of the Qp- vs saline-treated juvenile mice. mSchematic of
bilateral cannula application of Qp into the NAc and the experimental procedure.
nRepresentative AP traces and statistics showing the inhibitory effect of Qp
(50 nM) on the excitability of D2R-positivecortical neurons in the NAc (Data from 3
mice and presented as mean ± SEM).o–qRepresentative heat mapsand statisticsof
the three-chambersocial interactiontest of juvenile(6–8 weeks) mice following the
local application of Qp vs saline in the NAc. r–tRepresentative heat maps and
statistics of the three-chamber social novelty test of juvenile mice following the
local application of Qp vs saline in the NAc. uInvestigation frequency and inves-
tigation time in the home cage social test of juvenile mice following the local
application of Qp vs saline in the NAc. Data are shown as box-and-whisker plots,
with the median represented by the central line inside each box, the 25th and 75th
percentiles represented by the edges of the b ox, and the whiskers extending to the
most extreme data points. Ordinary two-wayANOVA followedby Bonferroni’spost-
hoc test for (g, j, p, s), paired two-tailed Student’st-test for (e, n), or two-tailed
Mann-Whitney test for (c, h, k, l, q, t, u), *P<0.05, **P< 0.01, ***P< 0.001, n.s. no
significant difference. Source data are provided as a Source Data file.
Article https://doi.org/10.1038/s41467-024-54604-4
Nature Communications | (2024) 15:10571 10
Content courtesy of Springer Nature, terms of use apply. Rights reserved

the hyperactivity of DA neurons during this period. Similar to the
effects of haloperidol in the mPFC, local delivery of the D2R agonist
quinpirole into the VTA also demonstrated a therapeutic effect in
adolescent Syt11 P0-cKO mice (Fig. 7g). Specifically, compared to the
impaired social preference of Syt11 P0-cKO mice injected with saline,
quinpirole-treated P0-cKO mice showed intact social interaction with
the M1 mouse and an unchanged social preference index (Fig. 7h, i). In
addition, local application of quinpirole in the VTA fully reversed the
pronounced social withdrawal of Syt11 P0-cKO mice in the social
novelty test (Fig. 7j, k). Moreover, social interaction with the stranger
intruder mouse in the home-cage social test was substantially atte-
nuated by the local administration of quinpirole (Fig. 7l).
To further validate the therapeutic effect of quinpirole in the VTA,
we utilized dizocilpine (MK-801)-induced schizophrenia-like mice.
Ctrl
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neurotransmission
neural development
vesicle trafficking
VTA
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regulation of neuronal synaptic plasticity
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sEPSC
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signal transduction
n.s.
cell surface receptor signaling pathway
regulation of transmission of nerve impulse
regulation of development process
neuron projection development
embryonic brain development
regulation of nervous system development
anterograde axonal protein transport
membrane to membrane docking
vesicle-mediated intercellular transport
neurotransmitter receptor metablic process
regulation of postsynapse organization
Human diseases
log2(FC)
Genetic information processing
Cellular processing
Others
Organismal systems
Metabolism
Environmental information processing
(20)
(25)
(26)
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(16)
(18)
(18)
P0 P7 P14
Virus
CNO
(i.p.)
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Tnfrsf25
Icam1
Ddc
Cell number/mm
(fold to control)
MAP2+
TH intensity
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b
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Nature Communications | (2024) 15:10571 11
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Consistent with our hypothesis, pre-treatment with MK-801 indeed
increased DA overflow in mPFC slices (Supplementary information,
Fig. S18a, b), while MK-801 administration during preadolescence led
to long-lasting social behavior deficits, which were fully reversed by
the local application of quinpirole in the VTA (Supplementary infor-
mation, Fig. S18c). Importantly, local application of quinpirole in the
VTA to block the elevated DA release or haloperidol in the NAc to
diminish the enhanced DA transmission to post-synaptic D2R neurons
reversed sensory gating dysfunction in the MK801-induced schizo-
phrenia mouse model (Supplementary information, Fig. S18d). These
results further confirm the role of DA over-transmission before late
adolescence in the development of schizophrenia and propose a
therapeutic strategy targeting D2Rs for the clinical treatment of the
disorders. Collectively, the present work suggests that both the pre-
synaptic/somatic application of D2R agonists in the VTA and the
postsynaptic delivery of D2R antagonists in the mPFC represent pro-
mising therapeutic approaches for the clinical treatment of
schizophrenia-associated social withdrawal.
Long-lasting rescue of social withdrawal by targeting pre-
synaptic or postsynaptic D2Rs
We have successfully demonstrated that the rectification of DA
transmission via the brain region-specific intervention of D2Rs during
adolescence can effectively reverse social deficits in schizophrenia. To
investigate the long-lasting effects of this therapeutic approach, we
conducted repeated local administration of haloperidol in the mPFC
every second day from P42 to P49. Notably, we observed sustained
rescue effects on social behaviors in Syt11 P0-cKO mice (Fig. 8a-h).
Specifically, the adult haloperidol-treated P0-cKO mice showed nor-
mal sniffing time with a stranger M1 mouse and an unchanged social
preference index in the three-chamber social test, similar to control
mice (Fig. 8b-d). In addition, they exhibited intact social preference for
the stranger M2 mouse and normal total social time with both mice in
the social novelty test (Fig. 8e-g). Consistent with these results, the
social interaction time with a stranger intruder mouse was effectively
restored in adult haloperidol-treated P0-cKO mice in the social
approach test (Fig. 8h). These findings confirm a complete and long-
lasting recovery of social deficits through the suppression of elevated
DA transmission via local application of a D2R antagonist in the mPFC.
Next, we determined whether local delivery of a D2R agonist into
the VTA during adolescence could also produce long-lasting rescue
effects on social deficits in Syt11 P0-cKO mice (Fig. 8i). Interestingly,
similar to the haloperidol treatment in the mPFC, repeated local
treatment with the D2R agonist quinpirole into the VTA restored the
impaired social interaction with the stranger M1 mouse and the
reduced social preference index in the three-chamber test in Syt11 P0-
cKO mice (Fig. 8j-l). Furthermore, the social preference for the M2
mouse and the total social time with both micewere indistinguishable
between adult quinpirole-treated Syt11 P0-cKO mice and control mice
(Fig. 8m-o). Finally, the social approach test confirmed that the ther-
apeuticeffects of quinpirole on social withdrawal in Syt11 P0-cKO mice
were maintained into adulthood (Fig. 8p). Taken together, these
findings provide valuable insights into long-lasting therapeutic stra-
tegies for schizophrenia by targeting D2Rs either presynaptically or
postsynaptically before late adolescence, offering potential benefitfor
the permanent recovery of schizophrenia in clinical treatment.
Discussion
While there is ongoing debate, it has been reported that aberrant DA
transmission is involved in the manifestation of positive symptoms in
schizophrenia, which can be effectively alleviated by drugs antag-
onizing D2Rs in clinical treatments50. Nonetheless, the role of DA
transmission in schizophrenia and the underlying mechanisms of
negative symptoms remain largely unclear, making it challenging to
identify suitable therapeutic targets. In this study, we identify that
SYT11 deficiency is a potential risk factor causally linked to schizo-
phrenia, demonstrate plasma Syt11 as a potential biomarker for the
diagnosis of schizophrenia, and present the Syt11-cKO mouse as a
valuable genetic animal model for schizophrenia study. Importantly,
we further define periadolescent DA over-transmission as a neural
mechanism initiating the pathogenesis of schizophrenia (Supplemen-
tary information, Fig. S19). The chemogenetic excitation of VTADA
neurons before late adolescence induced both acute and long-lasting
social deficits, establishing a direct link between DA dysregulation and
schizophrenia-related symptoms. We also show that local manipula-
tion to tune down DA transmission with clinical drugs, either a D2R
agonist in the VTA or an antagonist in the mPFC, before late adoles-
cence can produce sustained rescue effects on social deficits in Syt11
cKO mice. Thus, this study not only provides a reasonable explanation
for the limited efficacy of well-known antipsychotics in restoring
negative symptoms of schizophrenia, but also offers two potential
D2R-targeting strategies for the clinical treatment of schizophrenia
(Supplementary information, Fig. S19).
Although numerous schizophrenia risk genes have been identi-
fied, few have been shown to individually mediate the pathogenic
pathway, pending the mechanisms of this disorder due to the lacking
of an ideal genetic animal model51.SYT11, encoding a non-Ca2+-binding
Syt, is located on chromosome locus 1q22 and has been identified as a
major susceptibility locus for both familiar and sporadic schizophrenia
based on genome-wide scanning and case-control studies23,24.Our
previous studies have demonstrated that Syt11 inhibits endocytosis,
vesicle recycling, and DA release, and its accumulation plays a central
role in parkin-associated Parkinson’s disease27,29,30.Thecurrentwork,
by combining evidences from human samples and clinical data, iden-
tifies Syt11 as a potential risk factor closely associated with schizo-
phrenia (Fig. 1a-h). Furthermore, we define plasma Syt11 as a valuable
biomarker for the clinical diagnosis of schizophrenia (Supplementary
information, Fig. S1). In our study,we employed a DA neuron-restricted
Fig. 5 | Syt11 cKO and DA over-transmission lead to long-lasting structural and
functional alterations in the mPFC. a,bRepresentative micrographs and statis-
tics of MAP2-positive neurons in the mPFC of adult Syt11-cKO (n=6) vscontrol
(n= 5) mice. Scale bars: 50 μm for left, 20 μm for right. c,dRepresentative
micrographs and statistics of TH-positive neurites in the mPFC of adult Syt11-cKO
(n=6)vs control (n=5) mice. eRepresentative sEPSC traces and statistics of the
amplitude and frequency of sEPSC in mPFC cortical neurons from adult Syt11-cKO
(n=6)vs control (n=6) mice.fRepresentative mEPSC traces and statistics of the
amplitude and frequency of mEPSC in mPFC cortical neurons of adult Syt11-cKO
(n=6)vs control (n= 6) mice. gLeft, schematic of virusinjection (TH-Cre-EGFP/TH-
EGFP) into the VTA of neonatal Syt11-flox/flox mice (P0) for the generation of Syt11
P0-cKO orcontrol mice. Middle and right, representative AP traces and statistics of
spontaneous AP firing rates in mPFC cortical neurons of adult (3 months) Syt11 P0-
cKO (n=4)vs control (n=3)mice.hLeft, schematic showingthe co-injection of TH-
Cre and DIO-hM3Dq-mCherry/DIO-mCherry viruses into the VTA of neonatal C57
mice (hM3D, P0) and theexperimental procedure.Middle and right, representative
AP traces and statistics of spontaneous AP frequency in mPFC cortical neurons of
adult repetitive CNO-treatedhM3Dq-expressing (n=5)vs control (n= 4) mice.iThe
heatmap showing gene expression profiling determined by genome-wide RNA
sequencing (RNA-Seq) of the mPFC in Syt11cKO (n=4)vs control (n=3)mice.Rows
represent differentially expressed genes (DEGs), and columns represent tran-
scriptomic profiles of individual animals. jVolcano plots showing gene expression
profiling of the mPFC in Syt11 cKO vs control mice. The x-axis represents log
2
fold
change (FC) between the two groups. kIngenuity gene ontology (GO) analysis
indicating significantly enriched GO terms in cellular components and biological
processes. Data are shown as box-and-whisker plots, with the median represented
by the central lineinside each box,the 25th and 75thpercentiles representedby the
edgesof the box, and thewhiskers extending to the mostextreme datapoints. Two-
tailed Mann-Whitney test, *P<0.05,**P< 0.01, ***P< 0.001, n.s. no significant dif-
ference. Source data are provided as a Source Data file.
Article https://doi.org/10.1038/s41467-024-54604-4
Nature Communications | (2024) 15:10571 12
Content courtesy of Springer Nature, terms of use apply. Rights reserved

knockout of Syt11 and found that this alone is sufficient to mediate
long-lasting social withdrawal (Fig. 1k-q; Supplementary information,
Fig. S2a-j). This phenotype was accompanied with locomotion hyper-
activity, sensorimotor gating disruption and cognitive decline without
clear repetitive behaviors (Fig. 1r-u; Supplementary information, Fig.
S2k-n), thus confirming roles of Syt11 in the pathogenesis of schizo-
phrenia. Although the locomotion hyperactivity may affect the per-
formance in some of these behavioral tests, some of the key issues,
such as sniffing/interaction time in multiple social tests and PPI are
believed to be less dependent on locomotion activity52–54. We also
identified a critical time window for Syt11 deficiency-mediated social
deficits, occurring before late adolescence or young adulthood
(Fig. 2a-k; Supplementary information, Figs. S3-S5), suggesting a
potential role of Syt11 in neural development, which aligns with the
well-documented brain developmental anomalies observed in schizo-
phrenia. Therefore, our findings provide important insights into the
pathogenesis of a subset of schizophrenia patients ( ~ 50%) with clear
downregulation of Syt11 and offer a mouse model for pre-clinical stu-
dies of schizophrenia.
Both our previous studies30 and the present work have shown
that Syt11 deficiency leads to an elevated firing rate of DA neurons
(via decreasing surface functional D2Rs) and enhanced DA release
(via accelerating vesicle recycling) in the striatum, NAc and mPFC
(Fig. 2l-r). Moreover, we found that over-excitation of VTADA neurons
through chemogenetic (Fig. 3), optogenetic (Fig. 4a-c), or pharma-
cological (Figs. 4d-l; 6e-l) approaches is sufficient to mediate similar
impairments in social behaviors. These results indicate that Syt11
deficiency impairs social behaviors and other schizophrenia-related
160
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Investigation time (s)
Home cage social
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Sniffing time (s)
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VTA
Haloperidol
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Behavior
C57
(6)
(8)
*
DA
Halo i.p.
20%
b
Fig. 6 | Pathogenic effects of the D2R antagonist haloperidol on social deficits.
aIllustration showing the facilitatory effect of the D2R antagonist haloperidol
(Halo) on DA transmission by targeting presynaptic D2Rs. bSchematic and repre-
sentative traces showing increased DA release (evoked by a burst of electric sti-
mulation [Estim, 50 pulses at 80 Hz] at the medial forebrain bundle) in the mPFC
in vivo following i.p.application ofHalo (0.4 mg/kg) in juvenile C57 mice.Data from
3mice.cRepresentative trace andstatistics of spontaneous APsof cortical neurons
in mPFC slices in response to DA application (Data are presented as mean ± SEM).
Data from 4 mice. dLeft, illustration showing the dual effects of Halo on DA
transmission by targeting presynaptic and postsynaptic D2Rs. Middle and right,
representativetraces and statistics of spontaneous APs of mPFC cortical neuronsin
response to Halo application. Data from 3 mice. eSchematic of bilateral cannula
application of Halo into the VTA and the experimental procedure. f-h
Representative heat maps and statistics of three-chamber socialinteraction time in
juvenile (6–8 weeks) C57 mice following local application of Halo vs saline control
in the VTA. i-k Representative heat maps and statistics of the social novelty test in
Halo- vs saline-treated juvenileC57 mice. lStatistics of investig ation time in juvenile
C57 mice following local application of Halo vs saline control in the VTA in the
home-cage social test. Data are shown as box-and-whisker plots, with the median
represented by the central line inside each box, the 25th and 75th percentiles
represented by the edges of the box, and the whiskers extending to the most
extreme data points. Ordinary two-way ANOVA followed by Bonferroni’s post-hoc
test for (g, j), two-tailed paired Student’st-test for (c), or two-tailed Mann-Whitney
test for (h, k, l), *P< 0.05, **P<0.01, ***P< 0.001, n.s. no significant difference.
Source data are provided as a Source Data file.
Article https://doi.org/10.1038/s41467-024-54604-4
Nature Communications | (2024) 15:10571 13
Content courtesy of Springer Nature, terms of use apply. Rights reserved

symptoms, most probably via enhanced DA transmission. These
results are at odds with a prior report that forebrain-specific Syt11-
cKO mice only showed the impaired synaptic plasticity with no
significant alteration in fast neurotransmitter release55. Considering
that we have validated pivotal roles of Syt11 in endocytosis and
neural secretion in a variety types of cells, ranging from primary
sensory neurons, hippocampal neurons, midbrain DA neurons, to
glia cells27,30,56, the lack of a clear impairment in synaptic
transmission is probably attributed to the relatively lower level of
Syt11 expression in forebrain cortical neurons57. Considering the
general role of Syt11 in endocytosis and neurotransmission and its
ubiquitous expression in the brain27,30,56, Syt11 expression in other
cell types may also contribute to the pathogenesis of schizophrenia.
Nonetheless, we have defined the enhanced DA transmission as a
mechanism mediating Syt11 deficiency-induced pathogenesis of
schizophrenia.
a
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Home cage social
Social (6~8 W old)
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Home cage social
Social novelty
Social (6~8 W old)
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Total social time (s)
***
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*** n.s.
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(8)
(8)
(8)
(8)
Ctrl P0-cKO P0-cKO
M1
M1 M1 M1 M1
F
FF F
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(13) (12)
(14)
(10)
Ctrl
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Ctrl
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P0-cKO
P0-cKO P0-cKO
P0-cKO
(11)
Ctrl
b
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Nature Communications | (2024) 15:10571 14
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Although excess striatal DA release has long been proposed to be
associated with positive symptoms of schizophrenia9,13,58–60,the
hyperfunction of the DA pathway has faced significant challenges and
is proposed downstream of hyperactive glutamatergic projections or
that of the excitatory-inhibitory imbalance of synaptic inputs8,11,60–62.
Importantly, pathophysiological mechanisms underlying negative and
cognitive symptoms, particularly that of social withdrawal, remain
unclear. As an alternative, the ‘dual dysregulation’of DA alteration has
been proposed as a reformulation of the DA hypothesis, in which the
hypofunction of DA transmission in the prefrontal cortex has been
implicated in negative and cognitive symptoms15,16,19,63.However,this
DA pathway has not been thoroughly assessed yet. The present work
provides direct evidence that the over-excitation of DA neurons and
the enhanced DA release in Syt11 cKO (Fig. 2) and MK801-induced
(Supplementary information, Fig. S18a,b) schizophrenia mouse mod-
els are paralleled by schizophrenia-like social deficits. Additionally,
chemogenetic activation of VTADA neurons before late adolescence is
sufficient to mediate schizophrenia-like social deficits in both male
(Fig. 3a-h) and female mice (Supplementary information, Figs. S11 and
S12). Interestingly, repeated activation of VTADA neurons before early
adolescence leads to long-lasting impairments in social behaviors up
to late adolescence and adulthood (Fig. 3i-p; Supplementary infor-
mation, Figs. S10-S12). These findings suggest that DA over-
transmission plays an essential role in the pathogenesis of schizo-
phrenia. Notably, over-excitation of DA neurons in adult mice fails to
induce schizophrenia-associated social withdrawal (Supplementary
information, Fig. S9), highlighting the importance of a critical time
window for DA over-excitation in mediating the pathogenesis of the
disorder. Furthermore, local pharmacological manipulation of DA
transmission that reconciles with the enhanced DA release before late
adolescence fully mitigates social disability in Syt11 P0-cKO mice
(Fig. 7). This challenges the speculative ‘dual dysregulation’of DA
hypothesis15 and provides direct evidence that DA over-transmission is
a mechanism initiating social withdrawal and other negative symptom-
like behavioral changes. Taken together with the overall
schizophrenia-like behavioral changes in Syt11-cKO and Syt11 P0-cKO
mice (Figs. 1and 2;Supplementaryinformation,Fig.S2)aswellasthe
involvement of aberrant striatal DA release in positive symptoms2,15,
these findings suggest that DA over-transmission may represent a
common pathway for different symptoms of schizophrenia, particu-
larly during a critical time window before late adolescence.
The NAc and the mPFC are primary DA neuron-projecting brain
regions involved in social behavior. The activity of DA neurons pro-
jecting from the VTA to the NAc has been shown to be motivationally
relevant with social stimuli and enhance social interaction in adult
mice, probably through the reward circuit35,64. However, whether DA
release in the NAc also contributes to the social deficits in schizo-
phrenia remain unclear. Although VTADA-NAc transmission facilitates
social behaviors in adult mice (Supplementary information, Fig. S13),
neither optogenetic activation of DA release (Supplementary infor-
mation, Fig. S14) nor postsynaptic activation of D2Rs in the NAc
(Fig. 4m-u) is capable of mediating social changes before adulthood.
Instead, we found that either chemogenetic/pharmacological over-
excitation of VTADA neurons (Figs. 3and 6e-l) or postsynaptic
enhancement of DA transmission with the D2R agonist quinpirole in
the mPFC (Fig. 4d-l) before late adolescence is sufficient to mediate
social deficits. Importantly, optogenetic activation of DA release in the
mPFC leads to similar social withdrawal in adolescent mice (Fig. 4a-c),
while local application of the D2R antagonist haloperidol in the mPFC
during adolescence completely and probably permanently rescues
social deficits in Syt11 P0-cKO mice (Figs. 7a-f and 8a-h). Consistent
with this, both Syt11-cKO and chemogenetic activation-mediated DA
over-transmission before early adolescence led to similar long-lasting
morphological, functional, and transcriptional plastic changes in the
mPFC (Fig. 5; Supplementary information, Fig. S16), supporting an
essential role of VTA-mPFC DA transmission in the pathogenesis of
schizophrenia. Collectively, we have identified a time window-specific
inhibition of social preference by DA over-transmission in the mPFC
before late adolescence. Considering that local circuit in the mPFC is
very complex65–71, which specific type of D2R neurons and how they
can mediate social impairments remain open questions. Excitation of
DA neurons in the substantia nigra pars compacta (SNpc) has also been
reported to cause deficits in social interaction72, implying that different
DA circuits may be involved in the SCZ-onset mechanism. Nonetheless,
the development-dependent switch from inhibitory (via the mPFC) to
facilitatory (via the NAc) effects of DA transmission on social pre-
ference deserves systematic investigation in future.
Although antipsychotic reagents targeting D2Rs are effective for
treating positive symptoms of schizophrenia73, they have limited effi-
cacy for negative and cognitive symptoms2,74.Thismaybeduetothe
fact that D2R antagonists can affect behavior either by dampening DA
transmission in postsynaptic neurons in the mPFC or by enhancing the
activity of VTADA neurons via inhibitory auto-receptors. Although
haloperidol has been reported to block D2 auto-receptors for decades
and is part of the established model of antipsychotic actions75,76,
whether and how pre-synaptic D2R auto-receptor contributes to the
pathogenesis of and therapeutic effects on schizophrenia remain lar-
gely unknown. In the present work, we found that when the D2R
antagonist haloperidol is delivered directly into the VTA during ado-
lescence, it leads to the development of schizophrenia-like social
deficits but fails to rectify them (Fig. 6; Supplementary information,
Fig. S17). Alternatively, targeting DA transmission specifically in the
mPFC, where the postsynaptic effect is dominant, may be a more
effective approach to prevent social disorders in a broader manner.
Importantly, we have found both the acute (Fig. 7a-f) and long-lasting
(Fig. 8a-h) restoration of social withdrawal in Syt11 P0-cKO mice by
locally delivering haloperidol into the mPFC during late adolescence.
These findings offer a scenario explanation as to why traditional anti-
psychotic agents have not been successful in treating negative symp-
toms of schizophrenia and propose a potentialtherapeutic strategyfor
achieving complete and long-term recovery from the disease.
Based on our findings that DA over-transmission before late
adolescence initiates the pathogenesisof schizophrenia (Figs. 1–6), it is
plausible to alleviate social deficits by delivering a D2R agonist locally
to the VTA to rectify the excitation of DA neurons during this critical
period. Consistent with the hypothesis, we found that a single local
Fig. 7 | D2R serves as a dual therapeutic target presynaptically and post-
synaptically for reversing schizophrenia-related social deficits. a Schematic of
bilateral cannula application of the D2R antagonist haloperidol (Halo, 50μM) into
the mPFC of juvenile Syt11 P0-cKO or control mice (TH-Cre-EGFP or TH-EGFP AAV
injected into the VTA of neonatal Syt11 flox/flox mice) and the experimental pro-
cedure. b, c Statistics of the three-chamber social interaction test in Syt11 P0-cKO
and control mice following local application of Halo vs saline in the mPFC. d,
eStatistics of the social novelty test in Halo- vs saline-treated Syt11 P0-cKO and
control mice. fStatistics of investigation time in Halo- vs saline-treated Syt11 P0-
cKO and control mice inthe home-cage social test. gSchematic of bilateral cannula
application of the D2R agonist qunipirole (Qp, 1 μg/μl)into the VTA of juvenileSyt11
P0-cKO and control mice and the experimental procedure. h, i Statistics of the
three-chamber social interaction test in Syt11 P0-cKO and control mice following
local applicationof Qp vs saline in the VTA.j, k Statisti cs of the social novelty test in
Qp- vs saline-treated Syt11 P0-cKO and control mice. lStatistics of investigation
time in Qp- vs saline-treated Syt11 P0-cKO and control micein the home-cage social
test. Data are shown as box-and-whisker plots, with the median represented by the
centralline inside eachbox, the 25th and 75thpercentiles representedby the edges
of the box, and the whiskers extending to the most extreme data points. Ordinary
two-way ANOVA followed by Bonferroni’s post-hoc test, *P<0.05,**P<0.01,
***P< 0.001, n.s. no significant difference. Source data are provided as a Source
Data file.
Article https://doi.org/10.1038/s41467-024-54604-4
Nature Communications | (2024) 15:10571 15
Content courtesy of Springer Nature, terms of use apply. Rights reserved

1.2
1.0
0.8
0.6
0.4
0.2
0.0
120
100
80
60
40
20
0
120
80
40
0
160
120
80
40
0
abd
g
ij
Total social time (s)
Social index
l
mop
Investigation time (s)
eh
c
Social (3~4 Mon old)
Social (3~4 Mon old)
Quinpirole
Total social time (s)
Investigation time (s)
(15) (7)
(10)
M1 M2
0 s
45 s
M1 M2 M1 M2
Ctrl P0-cKO
Ctrl P0-cKO
n.s.
n.s. n.s.
(15)
n.s.
(8)
(8)
Sniffing time (s)
P28
P0
P42P49
P84 P98
Behavior
Cannule
Halo
Virus
Control
P0-cKO
Control
Control P0-cKO
P0-cKO
Social novelty
Social novelty
f
k
n
(11)
M1 F
M1 M2
0 s
45 s
0 s
45 s
M1 F M1 F
M1 M2 M1 M2
Ctrl P0-cKO
Ctrl
P0-cKO P0-cKO
P0-cKOCtrl
Ctrl Ctrl
P0-cKO
Ctrl
P0-cKO
n.s.
n.s.
n.s.
Social approach
Social approach
Social index
P28
P0
P42P49
P84 P98
Behavior
Cannule
Qp
Virus
VTA
Control
P0-cKO
(15)
M1 F
0 s
45 s
Ctrl Ctrl
M1 FM1 F
P0-cKO P0-cKO
n.s.
n.s.
n.s.
(8)
(8)
(15)
Sniffing time (s)
(11)
(11)
(12)
n.s.
(12)
(11) (12)
(11)
(12)
Sniffing time (s)
Sniffing time (s)
Haloperidol
n.s.
1.2
1.0
0.8
0.6
0.4
0.2
0.0
160
120
80
40
0
160
120
80
40
0
100
80
60
40
20
0
0
100
200
300
400
0
100
200
300
400
n.s.
n.s.
(11)
mPFc
Fig. 8 | D2Rserves as a dual therapeutic target exhibiting long-lasting rescue of
social deficits. a Schema tic of repetitive bilateral cannula appl ication of a D2R
antagonist (Halo) in the mPFC (every second day during P42-P49) and the experi-
mental procedure for assessing social behaviors in Syt11 P0-cKO vs control mice.
b–dRepresentative heat maps and statistics of three-chamber social interaction in
adult (3–4 months) Halo-treated Syt11 P0-cKO vs control mice as described in a.
e–gRepresentative heat maps and statistics of the three-chamber social novelty
test in adult Halo-treated S yt11 P0-cKO vs control mice. hStatistics of investigation
time of a dult Halo-treated S yt11 P0-cKO vs control mice in the social approach test.
iSchematic of repetitive bilateral cannula application of a D2R agonist (Qp) in the
mPFC (every second day during P42-P49) and the experimental procedure for
assessing social behaviors in Syt11 P0-cKO vs control mice. j–lRepresentative heat
maps and statistics of three-chamber social interaction test of adult (3–4months)
Qp-treated Syt11 P0-cKO vs control mice as described in i.m–oRepresentativeheat
maps and statistics of the three-chamber social novelty test of adult Qp-treated
Syt11 P0-cKO vs control mice. pStatistics of investigation time of adult Qp-treated
Syt11 P0-cKO vs control mice in the social approach test. Data are shown as box-
and-whisker plots,with the median represented bythe central line inside each box,
the 25thand 75th percentiles represented by the edges of the box,and the whiskers
extending to the most extreme data points. Ordinary two-way ANOVA followed by
Bonferroni’spost-hoctest(c, f, k, n) or two-tailed Mann-Whitney test (d, g, h, l, o,
p), *P<0.05, **P< 0.01, ***P< 0.001, n.s. no significant difference. Source data are
provided as a Source Data file.
Article https://doi.org/10.1038/s41467-024-54604-4
Nature Communications | (2024) 15:10571 16
Content courtesy of Springer Nature, terms of use apply. Rights reserved

delivery of the D2R agonist quinpirole to the VTA during adolescence
led to complete recovery from both so cial deficits and theimpaired PPI
in both Syt11 P0-cKO (Fig. 7g-l) and MK801-induced (Supplementary
information, Fig. S18) schizophrenia mouse models. Importantly,
repeated delivery of quinpirole specifically into the VTA during late
adolescence also results in long-lasting restoration of social with-
drawal (Fig. 8i-p). These findings confirm an essential role of DA
transmission before late adolescence in the development of schizo-
phrenia and suggest a potential D2R-targeting therapeutic strategy for
the clinical treatment of the disorder. Given that aberrant striatal DA
release underlies positive symptoms of schizophrenia, these findings
not only indicate a common role of DA transmission in positive
symptoms, negative symptoms, and cognitive aspects of schizo-
phrenia but also open possibilities for clinical treatment of schizo-
phrenia by targeting presynaptic DA release with D2R agonists.
In summary, we have identified Syt11 as a potential risk factor for
schizophrenia, developed a mouse model for systematic schizo-
phrenia study, and presented direct evidence demonstrating that DA
over-transmission during a sensitive time window before late adoles-
cence plays a pivotal role in initiating the pathogenesis of schizo-
phrenia (Supplementary information, Fig. S19). These findings not only
provide important inputs in understanding the onset and progression
of socialwithdrawal but also contribute to the mechanisms underlying
other negative symptoms and cognitive dysfunction, highlighting DA
over-transmission as a common upstream trigger for neurodevelop-
mental anomalies in schizophrenia. Importantly, this study not only
offers a reasonable explanation for the limited effectiveness of tradi-
tional antipsychotics in alleviating negative symptoms but also sug-
gests two D2R-targeting strategies as potential treatments for
schizophrenia.
Methods
Study approval
For human studies, prior to their participation, all subjects provided
written informed consent. Our study was conducted in accordance
with the ethical principles outlined in the 2002 Declaration of Helsinki
and was approved by the Medical Ethics Committees of Xi’an Jiaotong
University (NO. 2014-003). For all animal studies, the use and care of
animals were conducted in accordance with the guidelines and reg-
ulations approved by the Animal Care and Use Committee of Xi’an
Jiaotong University (NO.2016-10).
Subjects
To investigate Syt11 expression changes in schizophrenia, we obtained
RNA-sequencing data from three datasets: LIBD dataset (175 schizo-
phrenia patients and 318 healthy controls), CMC dataset (264 schizo-
phrenia patients and 294 healthy controls), and HBCC dataset
(97 schizophrenia patients and 220 healthy controls), as previously
described by Hoffman et al31.
To validate the Syt11 expression changes observed in the RNA-
sequencing data, we conducted qPCR and Western blot analyses using
two independent sporadic case-control samples. The qPCR samples
comprised 23 schizophrenia patients (12 females and 11 males) from
the Xi’an Mental Health Center and 40 healthy controls (20 females
and 20 males) from the Health Examination Center of the Second
Affiliated Hospital of Xi’an Jiaotong University. To ensure the integrity
of RNA for subsequent experiments, peripheral blood was collected
using PAXgene tubes (BD Biosciences, USA) to prevent RNA degrada-
tion. The Western blot samples consisted of 30 schizophrenia patients
(all males) and 30 age-matched healthy controls (all males) from the
Psychiatric Unit and Health Examination Center of the First Affiliated
Hospital of Xi’an Jiaotong University. Peripheral blood was collected
using EDTA-containing tubes to prevent coagulation. All schizophrenia
patients underwent standard diagnostic procedures and were con-
firmed by at least two experienced psychiatrists using the Structured
Clinical Interview for DSM-IV Axis I Disorders (SCID) and the Diagnostic
and Statistical Manual of Mental Disorders, Fifth Edition (DSM-V).
Patients were excluded from the study if they exhibited substance
abuse, suicidal tendencies, abnormal laboratory results or ECG/EEG
readings, or had a significant medical history such as brain surgery,
unstable somatic conditions, or viral infections. Patients who had
taken antipsychotic medication within one month prior to the
recruitment were also excluded. Healthy controls were individually
interviewed using the Structured Clinical Interview for DSM-IV-TR Axis
I Disorders Non-Patient Edition (SCID-NP) to ensure the absence of any
mental disorders. They self-reported no physical illness or personal/
family history of psychiatric disorders.
In accordance with the aforementioned diagnostic and exclusive
criteria, we recruited inpatients diagnosed with schizophrenia from
the Yulin Mental Health Center to form our third independent sample.
These inpatients had been receiving conventional antipsychotic
treatment (Haloperidol, Olanzapine, or Risperidone) for a minimum of
eight weeks, and treatment efficacy was assessed using the Positive
and Negative Syndrome Scale (PANSS), as documented in their medi-
cal records. The choice of anti-schizophrenia medication was deter-
mined by the attending physician based on the individual patient’s
condition. In our study, we collected peripheral blood samples from
each patient using PAXgene tubes (BD Biosciences, USA) at two time
points (before and after treatment). It is important to note that halo-
peridol is no longer considered a first-line treatment option for schi-
zophrenia in China. It is now reserved for cases where patients exhibit
severe positive symptoms and initially show uncooperative with the
treatment. For clarity, those patients who received a combination of
haloperidol and olanzapine in our study are referred to as the ‘halo-
peridol group’. Finally, a total of 20 schizophrenia inpatients (10
females and 10 males) were included and were assigned to one of the
three treatment groups (haloperidol, olanzapine, or risperidone)
based on individualized treatment plans prescribed by their attending
physicians, taking into consideration their respective symptom
conditions.
Animals
The floxed Syt11-null mice used in this study were obtained from The
Jackson Laboratory (strain B6.129-Syt11tm1Sud/J). DAT-Cre transgenic
mice (strain B6.SJL-Slc6a3tm1.1(cre) Bkmn/J) were kindly provided by
Dr. Minmin Luo (National Institute of Biological Science, China)77. Male
or female C57BL/6 J (B6) mice were sourced from Charles River
Laboratories. Heterozygous DAT-Cre mice were used as control of
Syt11 cKO mice. Control viral infection in floxed Syt11- null mice was
used as a control of Syt11 P0-cKO or Syt11 adult-cKO mice. Both male
and female mice were used as indicated in the study. All mice were
housed in the animal facility, maintained under a 12-h light/dark cycle
at 22 ± 2 °C and 40–60% humidity, and provided with ad libitum access
to food and water. The mice were finally euthanized with CO
2
, followed
by cervical dislocation.
Cell culture and transfection
Human neuroblastoma SH-SY5Y cells (ATCC® CRL-2266TM) were ori-
ginally sourced from the ATCC. They were cultured in Dulbecco’s
modified Eagle’s medium-F12 supplemented with 10% fetal bovine
serum at 37 °C with 5% CO
2
. For Syt11 knockdown, Syt11-shRNA car-
rying or scrambled control AAV virus (Shanghai OBiO Technology
Corp., Ltd.) were used to infect SH-SY5Y cells when cells reached ~75%
confluence. Immunoblotting was performed 3 days after transfection.
FM uptake
FM1-43 uptake was performed as described previously27. Cells were
washed 3 times with standard extracellular bath solution and then
incubated with 10 μM FM1-43 in standard or high K+(100 mM) -con-
taining external solution at room temperature. Then washed out with
Article https://doi.org/10.1038/s41467-024-54604-4
Nature Communications | (2024) 15:10571 17
Content courtesy of Springer Nature, terms of use apply. Rights reserved

the standard bath solution after incubation. The external solution
contained (in mM) 150 NaCl, 5 KCl, 2.5 CaCl
2
,1MgCl
2
, 10 H-HEPES, and
10 D-glucose, pH 7.4. In 100 mM K+external solutions, the NaCl con-
centration was reduced to maintain the same ionic strength. Fluores-
cence images were captured on a Leica TCS SP8 STED inverted
confocal microscope (Leica, Germany). The FM1-43 fluorescence
intensity was calculated with the software of Image J.
Stereotaxic cannulation surgery
Male adolescent mice (3–4 weeks old for cannula implantation and
5–6 weeks old for optical fiber implantation) were anesthetized with
avertin (10 mg/kg, i.p.). To maintain their body temperature at a con-
stant 37°C, a heating pad (KEL-2000, Nanjing, China) was used. The
mice were then carefully secured in a stereotaxic apparatus (Narishige
Inc., Japan), and the head position was adjusted to realize the same
height of the skull surfaces of Bregma and Lambda. Craniotomies were
meticulously performed using a cranial drill (RWD Instruments,China)
for the implantation of guide cannulas or optical fibers. The cannulas
were bilaterally implanted over the NAc (in mm: AP + 1.25, ML + /–0.75,
DV –4.3), the mPFC (in mm: AP + 2.8, ML + /–0.5, DV –1.7), or the VTA
(in mm: AP –3.4, ML + /–0.5, DV –4.4). In juvenile or adolescent mice,
these coordinates were correspondingly adjusted based on the pro-
portional relation between the measured distance from the bregma
and lambda and its default value (4.2mm) in adult mice. For optical
stimulation of VTA projections inthe NAc or mPFC, anoptical fiber was
carefully implanted into the lateral NAc and the prelimbic region of
the mPFC.
Stereotactic virus injection
Both adolescent(3–4 weeks old) and adult (3–4 months old) male mice
were anesthetized with avertin (10 mg/kg, i.p.), and their body tem-
perature was carefully maintained at a constant 37 °C using a heating
pad (KEL-2000, Nanjing, China). Then, they were securely placed in a
stereotaxic apparatus equipped with a mouse adapter (Narishige Inc.,
Japan), and the head position was adjusted to realize the same height
of the skull surfaces of Bregma and Lambda. Meticulous craniotomies
were performed using a cranial drill (RWD Instruments, China) to
minimize any potential damage to the cortical tissue. The virus
(2–5×10
12 vg/ml, 500 nl) was stereotaxically injected into the middle
region of the VTA (AP: -3.2mm, ML: 0 mm, DV: -4.5mm) according to
the coordinates described above with a glass micropipette. The
injection site in juvenile mice was adjusted nearby (AP -2.8 mm, ML
0 mm, DV -3.7 mm) according to the distance between bregma and
lamda. The infusion rate (100 nl/min) was precisely controlled using a
nanoliter injection pump (RWD, China). The micropipette was kept
stationary for 15 min before slow withdrawal. The micropipettes were
fabricated by glass capillary tubes (Narishige Inc., Japan) with a tip
diameter of ~20 μm. For postnatal day 0-1 (P0, male or female as
indicated) mice,the overall procedure of stereotaxic injections was the
same with some modifications. The pup mice were anesthetized by
deep hypothermia with ice and securely positioned in a stereotaxic
frame (Narishige, Japan) with a pair of soft faceplates. The dosage of
virusis150nl(2–5×1012 vg/ml) for each pup mouse. The coordinate of
VTA for injection was adjusted correspondingly (0.1 mm anterior, and
3.7 mm ventral to lambda).
Acute slice preparation
The mice were anesthetized with avertin (10 mg/kg, i.p.) and trans-
cardially perfused with ~5 ml ice-cold cutting artificial cerebrospinal
fluid (aCSF). The cutting aCSF contained the following components (in
mM): 110 C
5
H
14
NClO, 2.5 KCl, 0.5 CaCl
2
,7MgCl
2
, 1.3 NaH
2
PO
4,
25 NaCO
3
, 25 glucose (saturated with 95 O
2
and 5% CO
2
). Following
perfusion, the brain was carefully dissected and sliced into 300 µm
thick coronal sections by a vibratome (Leica VT 1200 s) in cutting
solution. Coronal slices containing the mPFC and NAc were collected
for recording. These slices were incubated in recording aCSF at 37 °C
for 30 min, followed by an additional 30 min at room temperature. The
recording aCSF contained the following components (in mM): 125
NaCl, 2.5 KCl, 2 CaCl
2
,1.3MgCl
2
,1.3NaH
2
PO
4
,25NaCO
3
, 10 glucose.
Subsequently, the slices were transferred to a recording chamber and
continuously perfused with recording aCSF at a rate of 2 mL/min.
Neurons were visualized by a microscope (BXWI51, Olympus) equip-
ped with infrared-differential interference contrast and an infrared
camera (IR-1000), and the fluorescence was imaged with U-HGLGPS
(Olympus).
Electrophysiology patch-clamp recording
Whole-cell patch-clamp recordings were performed as described
previously36. Pipettes were produced by a micropipette puller (Nar-
ishige Inc., Japan), and the pipette resistance was controlled between 3
and 5 MΩ. The intracellular pipette solution contained the following
components: 115 mM K-methylsulphate, 20 mM NaCl, 1.5 mM MgCl
2
,
10 mM HEPES(K), 10 mM BAPTA-tetrapotassium, 1 mg/mL ATP,
0.1 mg/mL GTP, and 1.5 mg/mL sodium phosphocreatine, with a pH of
7.4. Spontaneous and miniature excitatory postsynaptic currents
(sEPSCs and mEPSCs) were recorded by using the whole-cell voltage-
clamp recordings with an EPC10/2 amplifier controlled by Patchmaster
software (HEKA Elektronik, Germany). VTADA neurons and mPFC cor-
tical neurons were identified by cell-specific expression of EGFP/
mCherry, and their AP firing were recorded under current-clamp model
in whole-cell configuration. Series conductance and membrane con-
ductance were used to monitor the seal condition during patch-clamp
recordings. Signals were sampled at 20 kHz and low-pass filtered at
2.9 kHz. Pharmacological compounds, such as CNO (5 μM), D2R agonist
quinpirole (50 nM), D2R antagonist haloperidol (50 nM), were delivered
by a gravity-fed perfusion system (MPS-2, Yibo Inc., Wuhan, China). For
optogenetic activation of ChR2-expressing neurons/terminals, a blue
light pulse was emitted from a collimated light-emitting diode (473 nm)
driven by a T-Cube LED Driver (Beijing Viasho Technology Co., Ltd,
China) under the control of Pulse software (HEKA Elektronik, Germany).
All recordings were conducted at room temperature, and off-line data
analysis was performed by Igor software (Wavemetrics).
Amperometric DA recording in brain slices
Amperometric DA recordings in slices were conducted as described in
previous studies30,33. CFEs with a diameter of 7 μm and a sensor tip of
~200 μm were employed to measure DA release in the NAc and mPFC.
The exposed tip of the CFE was completely inserted into the subsur-
face of the slice at an angle of ~30°. An EPC10/2amplifier, controlled by
Pulse software (HEKA Electronic, Germany), applied a holding poten-
tial of 780 mV to the electrode. Single electrical field stimulus pulses
(0.2 ms, 0.6 mA) were delivered through a bipolar platinum electrode
(150 μm in diameter) using a Grass S88K stimulator (Astro-Med). In
chemogenetic experiments, DA release was triggered by treating the
slice with CNO (5 μM) for 10 s. The amperometric current (I
amp
)was
low-pass filtered at 100 Hz and digitized at a rate of 3.13 kHz. Off-line
data analysis was performed using Igor software (WaveMetrix). The
paired-pulse ratio was calculatedas the ratio of second peak amplitude
divided by the first peak amplitude.
Amperometric DA recording in vivo
Amperometric DA recording in the mPFC in vivo was performed fol-
lowing the protocols described in previous studies33,36. Briefly, mice
were anesthetized with urethane (1.5 g/kg, i.p.) and secured on a ste-
reotaxic instrument (Narishige, Tokyo, Japan). Body temperature was
maintained at 37°C using a heating pad (KEL-2000, Nanjing, China). A
recording carbon fiber electrode with a diameter of 7 µmwas
implanted in the mPFC at the following coordinates (in mm): AP + 2.8,
ML ± 0.5, DV –1.7. An Ag/AgCl reference electrode was placed in the
contralateral cortex. A bipolar stimulating electrode was implanted in
Article https://doi.org/10.1038/s41467-024-54604-4
Nature Communications | (2024) 15:10571 18
Content courtesy of Springer Nature, terms of use apply. Rights reserved

the medial forebrain bundle (AP –2.1, ML ± 1.1, DV –4.5). Electrical sti-
mulation was delivered as a train of biphasic square-wave pulses
(0.6 mA, 1 ms duration) using an isolator (A395, WPI, USA). The carbon
fiber electrode was clamped at a potential of 780 mV using an EPC10/2
amplifier controlled by Pulse software (HEKA Electronic, Germany).
The amperometric signal (I
amp
) was low-pass-filtered at 50 Hz and
digitized at a rate of 3.13 kHz. Off-line data analysis was performed
using Igor software (WaveMetrix).
RNA extraction and qRT-PCR
Total RNA was extracted from peripheral blood using the PAXgene
Blood RNA Kit (BD Biosciences, USA) following the manufacturer’s
instructions.The extracted RNAwas then reverse transcribed using the
Reverse Transcriptase M-MLV kit (TaKaRa, Japan). Quantitative reverse
transcription PCR (qRT-PCR) was performed using the SYBR Premix Ex
Taq II kit (TaKaRa, Japan) on a Bio-Rad CFX96 detection instrument
(Bio-Rad, USA). The primer sequ ences for SYT11 used in this study were
AGCTTTGACCGCTTCTCTCG (forward) and CCTCTGCTGATG-
CACTTCTGG (reverse).
RNA-sequencing data analysis
Total RNA was extracted from the mPFC of adult (3 months old) male
mice using Trizol reagent (Invitrogen, USA). After quality control of the
RNA samples using the NanoDrop 2000 and Agient 2100 (Agilent
Technologies, USA), the quantified RNA samples were used for library
preparation. Sequencing was carried out on an Illumina NovaSeq 6000
platform (Novogene Bioinformatics Institute, China) using the PE150
mode.TheRNA-Seqdataweremappedtothewholemousegenome
sequence. Differential expression analysis was performed to compare
control vs Syt11 cKO or control virus vs chemogenetic manipulated
mice. Genes with a fold change >1.5 and a P-value < 0.05 were con-
sidered as differentially expressed genes (DEGs) and visualized using a
volcano plot. Gene ontology (GO) analysis of the DEGs was performed
using the cluster Profiler R package, with default parameters for the
categories of Cellular Component and Biological Process of GO terms.
Go terms with a P-value < 0.05 were considered as enriched GO terms.
Protein preparation and western blotting
Western blotting was performed following previously established
protocols27,30 to assess protein expression. Mice wereanesthetized and
perfused with ice-cold sectioning aCSF to obtain brain slices (300 μm
thick). The midbrain was meticulously dissected from the brain slices
under a dissecting microscope, followed by homogenization in ice-
cold buffer composed of 20 mM HEPES at pH 7.4, 100 mM KCl, 2 mM
EDTA, 1%NP40, 1 mM PMSF, and2% protease inhibitor cocktail (P8340,
Sigma). The homogenates were then centrifuged at 16,000 g for 15 min
at 4 °C, and the supernatants were collected and denatured in sam-
pling buffer. Proteins were separated by electrophoresis and trans-
ferred onto nitrocellulose filter membranes. The membranes were
blocked by incubating in a phosphate-balanced saline (PBS) solution
containing 0.1% Tween-20 (v/v) and 5% non-fat dried milk
(w/v) for 1 h. After washing with PBST (PBS containing 0.1% Tween-20),
the blots were incubated overnight at 4°C with primary antibodies
diluted in PBST containing 2% bovine serum albumin (BSA). The pri-
mary antibodies used in the present study were rabbit anti-tyrosine
hydroxylase (TH) (AB152, Millipore, 1:1000), rabbit anti-Syt11 (270003,
Synaptic Systems, 1:1000), and rabbit anti-GAPDH (ab9485, Abcam,
1:1000). Subsequently, the membranes were washed with PBST con-
taining 0.05% Tween-20 and incubated with HRP-conjugated fluores-
cence-labelled secondary antibodies at room temperature for 1 h. The
secondary antibodies employed were goat anti-rabbit IgG (149393,
Jackson Immuno Research 1:3000) and goat anti-mouse IgG (148774,
Jackson Immuno Research, 1:3000). The blots were visualized using
the Clarity Western ECL substrate, scanned with the Clinx chemical
capture system (Clinx Science Instruments Co., Ltd), and quantified
with ImageJ (National Institutes of Health).
Immunofluorescence
The mice were anesthetized with avertin and perfused with 0.9% saline
followed by 4% paraformaldehyde (PFA, Sigma) in PBS 3 weeks or
6 weeks (virus injection at P0) post-virus injection. The brain was
swiftly removed and post-fixed in 4% PFA for 24 h at 4 °C. Following
dehydration in 10%, 20%, and 30% sucrose at 4 °C for 3–4 days, a series
of coronal sections (30 μm thick) were sliced using a cryostat micro-
tome system (MEV, SLEE, Germany). The sections were rinsed three
times with PBS and then permeabilized with 0.3% Triton X-100 in PBS
containing 2% BSA for 8 min at room temperature. After a 1-hour
blocking step with 2% BSA in PBS, the sections were incubated with the
primary antibodies overnight at 4 °C. The primary antibodies used
were rabbit anti-TH (213102; 1:1000), mouse anti-TH (213211; 1:1000),
and rabbit anti-Syt11 (270003; 1:500) from Synaptic Systems. After five
washes with blocking solution, the samples were incubated with the
secondary antibodies at room temperature for 2 h. The secondary
antibodies employed were donkey anti-rabbit Alexa488 (2289872;
1:1000), donkey anti-mouse Alexa488 (2229195; 1:1000), donkey anti-
rabbit Alexa594 (2266563; 1:1000), donkey anti-mouse Alexa594
(2234977; 1:1000), and donkey anti-rabbit Alexa647 (LC-307589;
1:1000) from Invitrogen. Following five washes in blocking solution
and one wash in PBS, the sections were stained with DAPI and mounted
on microslides with anti-fade mounting medium. Fluorescence images
were captured on a Leica TCS SP8 STED inverted confocal microscope
(Leica, Germany). The images were analyzed using ImageJ software
(National Institutes of Health, Bethesda, MD) and packaged with
Adobe Photoshop (Adobe Systems Inc.).
TIRF imaging
TIRF imaging was performed on an inverted microscope equipped
with a 100× TIRF objective lens (Nikon ECLIPSE Ti-U; numerical aper-
ture 1.45). Images were captured using an Andor EMCCD camera with
NIS-Elements BR software, with an exposure time of 200 ms. Surface
D2R fluorescence intensity values were calculated and analyzed using
ImageJ software. Both the transfected and non-transfected cells were
calculated and the fluorescence intensity of transfected cells were
normalized by the non-transfected cells.
Behavioral tests
The procedures for behavioral tests were conducted following the
methodsdescribedinapreviousstudy
36. Mice were transported in
their home cage to the testing room 1 h before the tests for environ-
mental habituation. The behaviorsof the animals were recorded using
an overhead cameraand the Anymaze tracking system (Stoelting Inc.),
unless otherwise specified. Pharmacological, chemogenetic, and
optogenetic manipulations were applied as described below. Dim light
( ~ 20 lux) was used in the tes ting room to minimize anxiety in the m ice.
Social and social novelty test. This assay was performed using a
Plexiglas rectangular box (40 × 20 × 22 cm) consisting of three inter-
connected chambers of equal size. For habituation, the test mouse was
placed in the central chamber and allowed to freely explore all three
chambers for 10 min. Mice that showed a preference for a specificside
chamber were excluded from the test. After habituation, the test
mouse was placed in the center chamber with both gates to the side
chambers closed. An age- and gender-matched stranger mouse (M1)
was introduced ina mesh cage in one side chamber,while a fake mouse
(F) was placed in a similar mesh cage in the other side chamber. The
test mouse was allowed to freely explore for 10 min after the opening
of both gates. The interaction time was measured as the sum of all time
intervals the test mouse spent sniffing and approaching the M1 or F
Article https://doi.org/10.1038/s41467-024-54604-4
Nature Communications | (2024) 15:10571 19
Content courtesy of Springer Nature, terms of use apply. Rights reserved

mouse.Thesocialpreferenceindexwascalculatedastheratioof
sniffing time with M1 versus M1 + F, as previously described78.
In the social novelty test, the same test mouse was placed in the
center chamber with both gates to the side chambers closed. The
former stranger mouse (M1, familiar mouse in novelty test) remained
unchanged, but the fake mouse was replaced by another age- and sex-
matched stranger mouse (M2). The test mouse was allowed to freely
explore and was monitored for an additional 10min. The interaction
times that the test mouse spent sniffing and approaching the M1 or M2
mouse were analyzed. The arena was thoroughly cleaned with 75%
ethanol after each trial, and the mouse was returned to its own
home cage.
Social approach test. This test was performed in an open field
apparatus (40 × 40 × 40 cm) with a cylindrical mesh cage fixed in the
center area. An age- and gender-matched stranger mouse was placed
inside the central cage, while the test mouse was placed in the appa-
ratus and allowed to freely explore its surroundings for 30min. The
movement and activity of the mice were monitored using a video
tracking system and analyzed with Anymaze software (Stoelting Inc.).
The time each mousesniffingthe cylindrical mesh cage or the stranger
mice was measured. The apparatus was cleaned with 75% ethanol after
each test, and the mouse was returned to its home cage.
Home-cage social test. The test mouse was individually housed and
fed for 5 days without changing the padding before the experiment. An
age- and sex-matched intruder mouse was introduced into the resi-
dent’s home cage and allowed to freely explore for 30min. The time
and frequency of contact between the resident and the intruder were
monitored using an overhead camera and analyzed with Anymaze
software (Stoelting Inc.).
Prepulse inhibition (PPI) test. The PPI test was conducted in a stan-
dard sound-attenuated cabinet (Zhongshi technology Inc., Beijing,
China). Prior to the test, the device was calibrated and standardized for
each mouse. The test mouse was acclimatized to a Plexiglas cylinder
with a background noise of ~65 dB (white noise) for 5 min. Subse-
quently, the mouse was exposed to six blocks of seven trial types
presented in a pseudorandom order,with an averageinter-trialinterval
of 15 s. The seven trial types included: trial 1, basal startle responses
(40-ms 120 dB startle-only pulse, 5 pulses with a 15-s inter-pulse
interval); trials 2–6, prepulse tests (three paired-pulse stimuli applied
in a random order, with each consisting of a 20-ms prepulse of 72, 76,
or 80 dB acoustic stimulus followed by a 120 dB startle stimulus
100 ms after prepulse); and trial 7, 120 dB startle-only, 5 pulses with a
15-s interval. The startle response was measured as the maximum
response within the 65-ms sampling window following each set of
stimuli. The averaged startle response was used to calculate the per-
centage inhibition (PPI %) of each type of stimulus, representing the
percentage reduction in startle response compared with the startle
stimulus.
Locomotion and grooming.Eachmousewasplacedinthecenterarea
of an open field apparatus (40 × 40 × 40cm) and allowed to freely
explore its surroundings. The movement and activity of the mice were
tracked and analyzed using an overhead camera and the Anymaze
tracking system (Stoelting Inc.). Grooming behaviors, including face-
wiping, scratching/rubbing of the head and ears, or whole-body
grooming, were quantified over a 15-min period. The average speed,
maximum speed, and total travel distance in 15 min were measured to
assess locomotor activity. The apparatuswas cleaned with 75% ethanol
after each test.
Marble burying. Before the test, the home-cage padding was changed
to corncob for at least 4 days. The testing mouse was then placed in a
new home cage with fresh corncob padding (5 cm in depth) with 18
clean marbles prearranged in a 3 by 6 grid. The mouse was allowed to
bury the marbles for 15 min, and the number of buried marbles was
counted immediately after the test. Only marbles that were at least 2/3
covered with corncob padding were considered as buried.
Food-induced T-maze. The mice were food-deprived for 24 h before
the test. The T-maze consisted of a start arm and two identical goal
arms (30 × 10 × 20 cm for each arm). A 2-g pellet of regular chow was
placedinthecornerofoneofthegoalarms.Thetestmousewas
habituated in the start arm for 90 s. After opening the gate in the start
arm, the mouse was allowed to freely explore the T-maze. When the
test mouse entered the goal arm and found the food, the gate in the
goal arm was closed. The mouse was returned to its home cage after
consuming the food, and 10 min later, the trial was repeated but
without any food inthe goal arm. Thetime taken by themouse to reach
the goal arm was recorded to assess short-term memory. To prevent
discrimination of the goal arm based on odor, the entire apparatus was
thoroughly cleaned with 75% ethanol before and after each trial.
Y-maze. The test mouse was transferred into the central area of a
Y-maze apparatus consisting of three dark gray arms (L: 30cm,
W: 8 cm, H: 15 cm for each arm). The mouse was allowed to freely
explore all three arms for 10 min, and its movements were monitored
using an overhead camera and the Anymaze tracking system (Stoelting
Inc.). The number of entries into the arms and the number of alter-
nations were quantified. The ratio of correct alternations to the total
number of new arm entries was used to determine short-term memory.
Spontaneous alternation was calculated as: SPA% = (number of alter-
nations / [total number of arm entries –2]) × 100.
Behavioral pharmacology
Drugs were administered through a bilateral stainless-steel inner can-
nula (RWD, China) connected to a dummy micro-tube, controlled
by a micro-syringe pump. The D2 agonist quinpirole(QP, 1 µg/µl, 0.2 µl/
side), D2 antagonist haloperidol (Halo, 50 µM, 0.2 µl/side), or saline
were locally infused at a rate of 100 nl/min. All drugs were prepared
in buffered saline. The internal cannula was withdrawn 2min after
infusion, and social testing was conducted 10 min after drug
administration.
Optogenetic manipulation
Behavioral testing was performed at least 3 weeks after virus injection
to ensure the expression of ChR2 in vivo. Optical fiber implants were
connected to a patch cable using a ceramic sleeve (RWD, China), which
was further connected to a commutator (Newdoon Technology Co.,
Ltd, China) via an FC/PC adapter to allow unrestricted movement of
the test mouse. Bursts of 473-nm light (5-ms, 8 pulses at 30Hz) were
delivered once every 5 s at an output power of 10 mW, controlled by an
Intelligent Light System (Newdoon Technology Co., Ltd, China).
Chemogenetic manipulation
The TH-Cre virus and Cre-dependent chemogenetic activation virus
(DIO-hM3Dq) were co-injected into the VTA of P0 C57 mice or mice
aged 3 weeks. The ligand clozapine-N-oxide (CNO) from Sigma-Aldrich
(St. Louis, MO) was dissolved in saline. For electrophysiological and
electrochemical slice recordings, CNO (5 μM) was delivered using a
gravity-fed perfusion system (MPS-2, Yibo Inc., Wuhan, China). Acute
chemogenetic activation in vivo was applied three weeks after virus
injection, and behavioral tests were conducted 20 min after CNO
administration (0.5 mg/kg, i.p.). To assess the long-lasting effect of
repetitive chemogenetic activation in vivo, systemic treatment with
CNO (0.5 mg/kg, i.p. injection) was administered every second day
between P7 and P14, and behavioral tests were conducted 4–6weeks
after the CNO treatment or during adulthood as indicated.
Article https://doi.org/10.1038/s41467-024-54604-4
Nature Communications | (2024) 15:10571 20
Content courtesy of Springer Nature, terms of use apply. Rights reserved

Statistics and reproducibility
All experiments were performed with side-by-side controls and in a
random order, and were replicated at least three times. Sample sizes
were not predetermined using statistical methods, but were consistent
with those reported in similar studies. No samples or animals that
yielded successful measurements were excluded from the analysis. To
preprocess the three RNA-Sequencing datasets of humanbrain tissues
(LIBD, CMC, and HBCC), we normalized raw counts to counts per
million (CPM) by dividing each count with the total count of the cor-
responding samples in each dataset, and then multiplying by one
million to account for differences in sequencing depth across samples.
To mitigate the effects of low expression values, we performed a log2
transformation of the CPM values by calculating the base-2 logarithm
of each value. All statistical tests were performed using GraphPad
Prism version 9.0 (GraphPad Software Inc.), R version 4.1 (https://cran.
r-project.org/), or SPSS version 18.0. The Shapiro-Wilk test was used to
test the normality of data, and Lenene’s test was used to assess the
equality of variance. Statistical comparisons were conducted using the
Wilcoxon–Mann-Whitney non-parametric test, Pearson correlation
analysis, paired Student’s t-test, one-way ANOVA, or two-way ANOVA
(followed by Bonferroni’sorTukey’s multiple comparisons were used
to make comparisons) as indicated. In all between-group comparisons,
the type I error rate was set at 0.05 (α= 0.05, two-tailed). Significant
differences were accepted at P< 0.05. Data are shown as box-and-
whisker plots or mean ± s.e.m. as indicated, and the numbers of cells,
slices, mice, or human samples analyzed are presented in the figures.
Reporting summary
Further information on research design is available in the Nature
Portfolio Reporting Summary linked to this article.
Data availability
All data presented in this study are either included in this article and its
Supplemental Information or are available upon request to the cor-
responding author. The RNA-Seq data generated in this study have
beendepositedinSequenceReadArchive(SRA)underaccessioncode
PRJNA1162940. Source data are provided with this paper.
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Acknowledgements
We thank Drs. Han Xu (Zhejiang Univerisity, China) for discussion and
comments, Minmin Luo (National Institute of Biological Sciences, China)
for DAT-Cre mice, Xianghui Zhao (Fourth Military Medical University,
China) for discussion and prepulse inhibition test, Ying Hao (Core Facil-
ities Sharing Platform, Xi’an Jiaotong University, China) for assistance with
confocal imaging, Enle Wang (High School Affiliated to Xi’an Jiaotong
University, China) for art work, and Iain C. Bruce (Peking University, China)
for reading the manuscript. This work was supported by the National
Natural Science Foundation of China (32171233 to C.W., 81901308 to H.X.,
81974203 to X.K., 82222031 to F.G., 32300819 to Y.C., and 32000704 to
Q.S.), the Natural Science Foundation of Shaanxi Province of China (2023-
ZDLSF-23, 2021TD-37, and 2019JC-07 to C.W.; 2020JQ-029 to Q.S.; 2023-
JC-QN-0236 to N.D.; 2024JC-YBMS-146 to H.X.; JC-YBQN-0172 to
C.Z.; and 2024RS-CXTD-80 to F.G.), the Sichuan Science and Technology
Program (2022YFS0615 and 2024ZYD0077 to X.K.), the China Post-
doctoral Science Foundation (2018M640972 to Q.S.; 2022M712543 to
N.D.; 2024M752559, 2024T170724, and GZC20232111 to Y.C.), and the
Shaanxi Postdoc Funding (2023BSHTBZZ15 to H.X., 2023BSHYDZZ39 to
Y.C., and 2023BSHEDZZ67 to C.Z.). Figure 2r and Supplemental Fig. 19
are created at BioRender.com with the publication license.
Author contributions
C.W., X.K., H.X. and F.G. conceived the study and designed the experi-
ments with the help of S.Z. and Q.Q. Y.C., Y.G., B.W., A.W., N.D., Y. J., X.L.,
L.Z., F.Z., T.T., Z.J., F.M., Y.Z., J.Y., Y.Y., H. Wang, H. Wu, H.L., C.Z., X.D.,
J.H., X.W., S.H., A.Z., Z.L., X.C., Y.Q., and Q.S. performed the experiments
and analyses. C.W., X.K., H.X., F.G., and Y.C. wrote the manuscript. All
authors reviewed the manuscript and approved its submission.
Competing interests
The authors declare no competing interests.
Additional information
Supplementary information The online version contains
supplementary material available at
https://doi.org/10.1038/s41467-024-54604-4.
Correspondence and requests for materials should be addressed to
Fanglin Guan, Huadong Xu, Xinjiang Kang or Changhe Wang.
Peer review information Nature Communications thanks the anon-
ymous reviewers for their contribution to the peer review of this work. A
peer review file is available.
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© The Author(s) 2024
1
Department of Neurology, the Second Affiliated Hospital, Neuroscience Research Center, Key Laboratory of Biomedical Information Engineering of Ministry
of Education, School of Life Science and Technology, Xi’an Jiaotong University, Xi’an 710049, China.
2
Department of Neurosurgery, the Affiliated Hospital of
Southwest Medical University, Luzhou Sichuan 646000, China.
3
Key Laboratory of Medical Electrophysiology, Ministry of Education of China, Collaborative
Innovation Center for Prevention and Treatment of Cardiovascular Disease, and the Institute of Cardiovascular Research, Southwest Medical University,
Luzhou 646000, China.
4
College of Life Sciences, Liaocheng University, Liaocheng 252059, China.
5
Key Laboratory of National Health Commission for
Forensic Sciences, College of Medicine & Forensics, Xi’an Jiaotong University, Xi’an 710061, China.
6
Center for Translational Medicine, the First Affiliated
Hospital of Xi’an Jiaotong University, Xi’an 710061, China.
7
Oujiang Laboratory (Zhejiang Lab for Regenerative Medicine, Vision and Brain Health), Institute of
Aging, Key Laboratory of Alzheimer’s Disease of Zhejiang Province, Zhejiang Provincial Clinical Research Center for Mental Disorders, The Affiliated Wenzhou
Kangning Hospital, Wenzhou Medical University, Wenzhou 325035, China.
8
Department of Neurology, the First Affiliated Hospitalof Xi’an JiaotongUniversity,
Xi’an 710061, China.
9
Department of Psychology, Chengwu People’s Hospital, Heze 274200, China.
10
These authors contributed equally: Yang Chen, Yuhao
Gu, Bianbian Wang, Anqi Wei. e-mail: fanglingguan@163.com;hdxu@pku.edu.cn;kxj335@163.com;changhewang@xjtu.edu.cn
Article https://doi.org/10.1038/s41467-024-54604-4
Nature Communications | (2024) 15:10571 23
Content courtesy of Springer Nature, terms of use apply. Rights reserved

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