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CNS Neuroscience & Therapeutics, 2025; 31:e70227
https://doi.org/10.1111/cns.70227
CNS Neuroscience & Therapeutics
ORIGINAL ARTICLE OPEN ACCESS
Modulating Anxiety- Like Behaviors in Neuropathic Pain:
Role of Anterior Cingulate Cortex Astrocytes Activation
QingqingZhou1 | QiZhong1 | ZhuangLiu2,3,4 | ZiyueZhao2,3,4 | JieWang2 | ZongzeZhang1
1Department of Anesthesiology, Zhongnan Hospital, Wuhan University, Wuhan, China | 2Department of Neurology, Song jiang Research Institute,
Shanghai Key Laborator y of Emotions and Affective Disorders, Songjiang Hospital Affiliated to Shanghai Jiao Tong University School of Medicine,
Shanghai, China | 3Key Laboratory of Magnetic Resonance in Biological Systems, State Key Laboratory of Magnetic Resonance and Atomic and Molecular
Physics, National Center for Magnetic Resonance in Wuhan, Innovation Academy for Precision Measurement Science and Technology, Chinese Academy of
Sciences, Wuhan, China | 4University of Chinese Academy of Sciences, Beijing, China
Correspondence: Jie Wang (jie.wang@shsmu.edu.cn) | Zongze Zhang (zhangzz@whu.edu.cn)
Received: 27 August 2024 | Revised: 19 December 2024 | Accepted: 8 January 2025
Funding: This work was supported by National Natural Science Foundation of China, 82201350. Hubei Provincial Health Commission joint fund project,
PTXM2023011.
Keywords: anterior cingulate cortex (ACC)| astrocyte| chemogenetic approach| excitatory neurons| spared nerve injury
ABSTR ACT
Aims: The comorbidity of anxiety- like symptoms in neuropathic pain (NP) is a significant yet often overlooked health concern.
Anxiety sufferers may have a lower tolerance for pain, but which is difficult to treat. Accumulating evidence suggests a strong
link between astrocytes and the manifestation of NP with concurrent anxiety- like behaviors. And the anterior cingulate cortex
(ACC) has emerged as a key player in pain modulation and related emotional processing. However, the complex mechanisms that
astrocytes in ACC influence anxiety behavior in mouse models of NP remain largely unexplored.
Methods: Utilizing the traditional spared nerve injury (SNI) surgical model, we employed chemogenetic approaches, immuno-
fluorescence, and western blot to investigate the functional significance and interactive dynamics between ACC astrocytes and
excitator y neurons.
Results: Our results revealed that SNI surgery induces NP and delayed anxiety- like behaviors, accompanied by increased as-
trocyte activity in the ACC. Chemogenetic manipulation demonstrated that inhibiting astrocytes alleviates anxiety symptoms,
while activating them exacerbates anxiety- like behaviors, affecting local excitatory neurons and synapse density. Direct manip-
ulation of ACC excitatory neurons also significantly impacted anxiety- like behaviors.
Conclusion: Our results highlight the pivotal role of ACC astrocytes in modulating anxiety- like behavior, suggesting a novel
therapeutic strategy for anxiety associated with NP by targeting astrocyte function.
1 | Introduction
Neuropathic pain (NP), which is caused by damage or disease
of the somatosensory system, has been demonstrated to be
highly comorbid with various psychiatry conditions, such as
anxiety, depression, and posttraumatic stress disorder (PTSD)
[1–4]. Among these comorbidities, anxiety plays a significant
role in intensifying pain perception and reducing pain toler-
ance, rendering it intractable and hard to treat. Despite the high
comorbidity of anxiety among NP patients, more than half of
comorbid anxious patients could not benefit from effective treat-
ments, which highlights the urgent need for better therapeutic
This is a n open access ar ticle under the terms of t he Creative Commons Attr ibution License, which p ermits use, dis tribution and repro duction in any medium, p rovided the orig inal work is
properly cited.
© 2025 T he Author(s). CNS Neuroscience & Therapeutics published by John Wiley & S ons Ltd.
Qingqin g Zhou and Qi Zhong contr ibuted equally t o this study.
2 of 15 CNS Neuroscience & Therapeutics, 2025
strategies [5–7]. Currently recommended first- line anti- pain
(opioid analgesic) treatments for NP have obvious side effects
that prolongs the duration of hyperalgesia and induce anxiety
disorder [7–9]. This problematic feature of the current NP ther-
apeutic strategies urges us to advance our understanding of the
NP- mediated anxiety mechanism.
Many brain regions, such as the anterior cingulate cortex (ACC),
medial prefrontal cortex (mPFC), central amygdala (Ce), insu-
lar cortex (IC), and ventrolateral periaqueductal gray (vlPAG),
contribute to the central mechanisms of NP [1, 10]. Among
these regions, ACC is particularly notable for its role in anxiety
regulation. For example, elevated activity was observed in this
region during a mammal model with experimental anxiety, de-
pression, and social deficit [11]. Furthermore, functional MRI
and experimental evidence also verified that ACC exhibited
hyperactivity in SNI mice with anxiety- like behaviors [12, 13].
Optogenetic activation or inhibition of ACC pyramidal neurons
could respectively induce or ameliorate anxiety- like actions
[8, 14, 15]. Stimulation and transcranial magnetic stimulation of
ACC could serve as effective treatments of the affective compo-
nent of chronic pain [16]. Furthermore, one research proposed
that the presynaptic LTP in ACC neurons could be a key cellular
mechanism for anxiety behavior caused by chronic pain [17].
Meanwhile, a recent study suggests a functional involvement
of ACC PVINs in mediating the anxiety induced by maternal
separation in mice [18]. These pieces of evidence suggest that
the ACC plays a vital role in NP comorbid anxiety, serving as a
critical target for understanding and potentially mitigating the
emotional disturbances associated with NP.
Several former studies of NP were mainly focused on the vari-
ations of neurons or microglia, often paying insufficient atten-
tion to the function of astrocytes [19, 20]. Previous findings
suggested that NP- mediated development of negative emotions
was triggered and maintained by changes in neuronal plasticity
and neuronal firing activity [21, 22]. However, astrocytes are
the most abundant glial cell type in the central nervous sys-
tem (CNS), historically thought to primarily provide necessary
support for neurons [23, 24]. Emerging evidence indicates that
astrocytes in the CNS are involved in pain processing and emo-
tional regulation. For instance, astrocyte ablation is sufficient
to induce negative emotions and impair cognitive flexibility
[25]. Reactivation of astrocytes in ACC contributes to allergic
inf lammation- induced anxiety- like behavior [26]. Furthermore,
reduced level of GFAP in the hippocampus could be detected
in anxious rats which ongoing PTSD [27]. By using astrocyte-
specific manipulation approaches in the central amygdala of
mice and rats, researchers highlights astrocytes involve in mod-
ulation of emotional states under normal and chronic pain con-
ditions [28]. These prior studies suggested that astrocyte may
play a significant role in the development of anxiety behavior.
However, studies on the modulating effects of astrocytes on anx-
iety have focused on other brain region and diseases. Besides,
there are many studies reports that astrocytes should play a
significant role in the initiation and maintenance of chronic
pain through the release of key pro- inf lammatory cytokines
[29, 30]. Some researches also find that low intensity transcra-
nial direct current stimulation (tDCS), allodynia- like behav-
iors of mice that performed partial sciatic nerve ligation (PSL)
surgery can be reversed [31]. However, previous evidence in
terms of ACC astrocytes mainly focused on remyelination and
cognitive deficits [32], inflammatory pain [33], and lactic acid
metabolism changes, followed by pain- related aversive learning
and decision- making performance [34, 35]. Ambiguous results
still exist regarding the putative mechanisms of how ACC astro-
cytes influence SNI mice anxiety behavior. This gap in research
points to an underexplored area in understanding the role of as-
trocytes in NP and its comorbid emotional disorders.
To determine whether astrocy tes in ACC may contribute to emo-
tional disorders, we used the spared nerve injury (SNI) model,
which is one of the most well- validated NP models [36, 37]. By
employing the designer receptor exclusively activated by de-
signer drugs (DREADDs) approach, along with behavioral tests
and immunohistochemical methods, our results highlight the
critical involvement of astrocytes and astrocyte- mediated acti-
vation of excitatory neurons in the ACC in NP comorbid anxi-
ety, offering new avenues for therapeutic intervention targeting
astrocyte function. The integration of these findings into the
broader context of NP research underscores the necessity of a
more comprehensive approach that includes the role of astro-
cytes in both pain and emotional regulation.
2 | Materials and Methods
2.1 | Animals
Experiments were performed using nine- week- old male C57BL/6J
mice (25–28 g) obtained from L iaoning Changsheng Biotechnology
Co., Benxi, Liaoning, China. The mice were group- housed with
a maximum of five animals per cage and maintained under a
controlled environment with a temperature of 23°C ± 1°C and a
12/12- h light–dark cycle. All animals were randomly divided into
different experimental groups to ensure unbiased results, and the
whole experimental procedures were collected and illustrated in
Figure 1. The experiments were conducted in accordance with
the Animal Ethics Committee of Wuhan University's Zhongnan
Hospital (ZN2023265) and adhered to the National Institutes of
Health Guidelines for the Care and Use of Laboratory Animals.
2.2 | Drugs
There were several viruses involved in the current study, such
as rAAV- GfaAB C1D- hM3D (Gq)- P2A- mCherry (10 × 1012 V.G./
mL), rA AV- GfaA BC1D- hM4D(Gi)- P2A- mCherry (10 × 1012 V.G./
mL), rAAV- Gfa ABC1D- mCherry (10 × 1012 V.G./mL), rAAV-
CaMK IIα- hM3D(Gq)- EGFP (10 × 1012 V.G./mL), rAAV- Ca MK
IIα- hM4D(Gi)- EGFP (10 × 1012 V.G./ml), rAAV- CaM KIIα- EGFP
(10 × 1012 V.G./mL), which were purchased from Shenzhen
Brain Case Limited Company. Clozapine- N- oxide (CNO) (Tocris,
4936) were dissolved in dimethyl sulfoxide (DMSO) and subse-
quently diluted to their final concentrations (1 mg/kg) in sterile
0.9% saline.
2.3 | Surgical Procedures
Surgical procedures were performed under ketamine an-
esthesia (dissolved in 1% pentobarbitone, intraperitoneal
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injection, 40 mg/kg, Jiangsu Hengrui Pharmaceuticals Co.
Ltd., Lianyungang, China). It's worth mention that previous
researches reported the rapid antidepressant effects only oc-
curred in low dose ketamine [38, 39]. Studies suggest that only
subanesthetic ketamine elicits antidepressant and anxiolytic
effect [40]. However, in our experiments, we injected high dose
ketamine, which only acted as an acute analgesic and the in-
jected dose of each group was the same to counteract its other
effect. Furthermore, the anxiety- related effects of ketamine are
scarce and controversial, our open field test (OFT) and elevated
plus maze (EPM) tests were performed at least one- week after
injection of ketamine, the behavioral evalution could be almost
unaffected. During the surgical procedures, mice were placed
on a heating pad to maintain body temperature, which is cru-
cial for ensuring physiological stability and facilitating recov-
ery from anesthesia. Postsurgery, the mice were returned to
their original cages and carefully monitored to ensure proper
recovery.
2.3.1 | Neuropathic Pain Surgery
SNI surgery was conducted following the established protocol
from a previous study [41]. Briefly, the fur on the left lateral
thigh of the mice was shaved after administering general an-
esthesia, and a minor incision was made through the skin. The
femoris muscle was carefully dissected to expose the sciatic
nerve along with its three peripheral branches: the common
peroneal nerve (CPN), the tibial nerve (TN), and the sural
nerve. The CPN and TN were ligated using a 5–0 nylon su-
ture and then cut 2 mm distal to the ligation to prevent nerve
regeneration. It's important to protect the sural nerve to avoid
causing paralysis. After completing the nerve ligation and sec-
tioning, the incision was sutured using a 5–0 nylon suture.
The sham- operated mice were undergone the same surgical
procedures, but the sciatic nerve was only exposed without
ligation.
2.3.2 | Virus Injection
Mice were anesthetized and their heads were fixed in a stereotac-
tic frame (RWD Life Science, Shenzhen, China). A small incision
was made to expose the skull. The virus (150 nL/dosage) was bilat-
erally injected into ACC region (AP, +1.0 mm, ML, +0.3 mm, DV,
−1.8 mm) at a rate of 100 nL/min using a glass pipette [42, 43]. Then
the micro- syringe was left in place for at least 10 min to prevent
virus backflowing during the retraction. Following the injection,
mice were placed on the heating pad for recovery, and then put
them back in their original cages. At least 3 weeks later, mice were
intraperitoneally administrated with 0.5 mg/kg CNO for pharma-
cogenetics studies before the undergoing behavioral experiments.
2.4 | Animal Behavioral Assessments
The animal behavioral testing was performed under the same
conditions to avoid any experimental bias. Before testing, all an-
imals were habituated in the testing room with dim light (~20 lx)
at least 1 h, and the software of ANY- maze (Stoelting Inc., Kiel,
WI, USA) was used to analyze their behaviors.
2.4.1 | Mechanical Allodynia Test
As previously described, the mechanical allodynia of the NP
models was tested using a series of von Frey filaments (ranging
from 0.00 8 g to 2.0 g) [10]. Mice were individually placed in acrylic
compartments (7 × 9 × 7 c m3) with wire mesh pads at the bottom
for at least 1 h of habituation until they calmed down. Pressure
was then applied to the plantar surface of the left hind paw using
the filaments. A positive response was defined as brisk paw with-
drawal, f linching, or licking, indicating pain perception. Each
test began with a 0.16 g filament; if a positive response occurred, a
lower pressure filament was used next, and if a negative response
occurred, a higher- pressure filament was used. Each filament
FIGUR E | Flowchart of the experimental design.
4 of 15 CNS Neuroscience & Therapeutics, 2025
was applied five times per mouse. The up- down method of Dixon
was utilized to analyze the mechanical allodynia, providing a ro-
bust measure of pain sensitivity in the mice.
2.4.2 | Open Field Test
Before every testi ng, the apparatus (40 × 40 × 70 cm3) was
cleaned with 75% ethanol. Mice were then placed in the appa-
ratus to explore freely for 10 min. The time spent and the route
taken in the center zone (20 × 20 cm2) were recorded and ana-
lyzed using ANY- maze software.
2.4.3 | Elevated Plus Maze
The EPM apparatus consisted of two mutually perpendicular
arms (74 × 5 cm2) raised 50 cm above the floor, with one closed
arm having 14- cm- high walls and one open arm without walls.
Before testing, each mouse was placed in the center zone facing
an open arm and allowed to explore freely for 10 min. The time
spent and the path taken in the open arm were recorded and an-
alyzed using ANY- maze software. After each test, the apparatus
was thoroughly cleaned with 75% ethanol to eliminate any resid-
ual scents and ensure consistent conditions for subsequent tests.
2.5 | Immunochemistry
To conduct immunohistochemical analysis on mouse brain tissue,
mice were first subjected to deep anesthesia using 2% to 4% isoflu-
rane before being transcardially perfused with phosphate- buffered
saline (PBS) to clear the blood from the vasculature. This was fol-
lowed by perfusion with 4% paraformaldehyde (Merck, Darmstadt,
Germany) to fix the tissues. Subsequently, the mice were decapi-
tated, and their brains were carefully excised and immersed in 4%
paraformaldehyde for 48 h to ensure thorough fixation. Following
fixation, the brains underwent dehydration through immersion
in 30% sucrose solution until they sank, indicating adequate de-
hydration. Then the dehydrated brains were frozen and sectioned
into 40 µm- thick coronal slices using a Thermo Fisher CRYOSTAR
NX50 cryostat microtome (Thermo Fisher Scientific).
For immunostaining, the brain slices were washed three times
for 10 min each in PBS to remove any residual fixative. Then,
they were blocked for 2 h in solution containing 10% fetal bo-
vine serum (FBS) with 0.3% Triton X- 100 in PBS to reduce non-
specific binding. The slices were incubated overnight at 4°C
with primary antibodies against GFAP (goat- anti- GFAP, 1:500
dilution, ab53554, Abcam), NeuN (anti- NeuN, 1:500 dilution,
ab177487, Abcam), Iba- 1 (anti- Iba- 1, 1:500 dilution, WX331590,
ABclonal), CamKII (goat- anti- CamKII, 1:500 dilution, ab87597,
Abcam), or c- Fos (rabbit- anti- c- Fos, 1:1000 dilution, 2250S, Cell
Signaling Technology; rat- anti- c- fos, 1:1000 dilution, 226,017,
SYSY). After washing, the slices were incubated with species-
specific fluorescently labeled secondary antibodies (Alexa Fluor
488 Donkey Anti- Goat IgG (H + L), 705–545- 003; Alexa Fluor
488 Donkey Anti- Rabbit IgG (H + L), 711–545- 152; Cy3 Donkey
Anti- Goat IgG (H + L), 705–165- 003; Cy3 Donkey Anti- Rabbit
IgG (H + L), 711–165- 152; Goat anti- Rabbit Alexa Fluor 647,
111–607- 008; Jackson ImmunoResearch; Alexa Fluor 488 Goat
Anti- Rat IgG (H + L), A11006, Thermo Fisher Scientific) for
2 h at room temperature. Finally, all slices were counterstained
with 4′, 6- diamidino- 2- phenylindole (DAPI, C1002, Beyotime
Biotechnology) for 10 min. The immunofluorescent images
were captured using confocal microscope (Leica, TCS SP8,
Buffalo Grove, IL) or a virtual microscopy slide- scanning system
(Olympus, VS. 120, Tokyo, Japan) for image acquisition. The im-
ages were further analyzed and processed using ImageJ software
(National Institutes of Health, USA) to quantify and visualize the
expression of the targeted proteins within the ACC region.
2.6 | Western Blot
After various treatments, under the general anesthesia with iso-
flurane (2%–4%), mice brains were collected on an acrylic plate
with ice in the bottom. Then, we harvested the ACC tissues and
stored at −80°C. For protein extraction, ACC tissue is homoge-
nized in RIPA buffer containing protease inhibitors (beyotime,
P0013B), followed by centrifugation at 12,000 rpm for 15 min at
4°C to collect the supernatant for protein concentration measure-
ment using a BCA assay (beyotime, P0012). Equal amounts of
protein are then loaded onto SDS- PAGE gels along with molec-
ular weight markers (thermofisher, 26,617) and run at constant
voltage. Proteins are transferred from the gel to a PVDF mem-
brane. Then, membranes are blocked in 5% BSA (roche, G5001)
in TBST for 2 h at room temperature, followed by overnight incu-
bation at 4°C with primary antibodies against PSD95 (Abclonal,
A0131) and GAPDH (Cell Signaling, 2118). After washing with
TBST, membranes are incubated with HRP- conjugated second-
ary antibodies (bioss, bs- 0295G) for 1 h at room temperature.
Chemiluminescent substrate is applied to the membrane, which
is then analyzed using a digital imaging system (Tanon- 4800).
Finally, band intensities are quantified using image J software.
2.7 | Statistics
All data were processed in GraphPad Prism v9.0 software, ex-
pressing as the mean ± standard error of mean (SEM). We used
an unpaired t- test for comparison between two groups, one- way
analysis of variance (ANOVA) followed by Tukey's post hoc test
for comparison among ≥ 3 groups, two- way ANOVA followed
by unpaid t test for the comparisons of mechanical pain thresh-
old between two groups, with statistical significance assessed as
*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
3 | Results
3.1 | SNI Induced Time- Dependent Anxiety- Like
Behaviors and Correspondingly Overexcited ACC
Astrocytes
The SNI- induced NP model is a widely accepted chronic pain
model that also presents with comorbid anxiety- like symptoms.
Following SNI surgery, SNI mice exhibited a significant reduction
in pain threshold compared to sham- operated mice, and this re-
duction persisted for 6 weeks (Figure2A). To further investigate
the anxiety- like behavior in SNI mice, we conducted an OFT and
found that, SNI mice at 6 weeks' postsurgery spent significantly
5 of 15
less time in the center zone compared to their behavior at one-
week postsurgery (Figure 2B). Similarly, in the EPM test, SNI
mice spent a shorter duration in the open arms at 6 weeks post-
surgery compared to one- week postsurgery (Figure2C).
To determine whether astrocytes in the ACC were active in
SNI mice with comorbid anxiety- like behavior at 6 weeks, the
ACC slices were immunostained for glial fibrillary acidic pro-
tein (GFAP), a marker of astrocytes. Results of immunostain-
ing revealed a significant increase in GFAP expression in ACC
of SNI mice at 6 weeks compared to one- week postsurgery
(Figure 2D,E). These findings suggested a temporal sensitivity
in the development of anxiety- like behavior following SNI sur-
gery, with a corresponding increase in astrocyte activity in ACC.
3.2 | Inhibited ACC Astrocytes Ameliorate
Anxiety- Like Behaviors in SNI Mice
To investigate the relationship of astrocyte activity and the anxiety-
like behaviors in SNI mice, the astrocytes in ACC were manipu-
lated and their function were further evaluated. SNI surgery was
performed concurrently with the virus injection (GfaABC1D-
hM4D- mCherry), and the paw withdrawal threshold was evalu-
ated on days −1, 5, 7, and 9 to screen for mice with mechanical
allodynia (FigureS1A–C). Six weeks postsurgery, selected mice
were injected intraperitoneally with 5 mg/kg CNO and subjected
to behavioral tests 2 h later (Figure 3A). Our findings revealed
that, following the chemogenetic inhibition of astrocytes, the
mice in hM4D- mCherry group spent significantly more time in
the center zone of the OFT and in the open arm of the EPM test
compared to mCherry control mice, while the total distances trav-
eled showed no significant difference (Figure3B,C). These results
suggested that chemogenetic inhibition of ACC astrocytes could
alleviate anxiety- like behavior in SNI mice.
Six weeks postinjection, red fluorescence (~mCherry tag) was
observed to be confined to the ACC region, confirming the virus
infection (Figure 3A). The immunohistochemical staining re-
vealed that most mCherry signals (about 90%) co- localized with
GFAP (Figure3H), rather than NeuN or Iba- 1 (FigureS3A–D),
indicating that the GfaABC1D- hM4D- mCherry virus specifi-
cally infected ACC astrocytes (Figure3D–3F).
It is noteworthy that a study found that clozapine- N- oxide (CNO)
could rapidly convert to clozapine invivo, which could alter the
mice behaviors [44]. To eliminate the potential confounding ef-
fects of CNO, a group of control animals were also injected with
the GfaABC1D- mCherry virus and compared. In comparison to
the mCherry control group, CNO treatment could significantly
inhibit ACC astrocytes in the hM4D- mCherry group, demon-
strating the virus's intended effect (Figure3G,I).
3.3 | Activated ACC Astrocytes Aggravate
Anxiety- Like Behavior and Regulate Excitatory
Neurons
Three weeks after virus injection (GfaABC1D- hM3D), the
treated mice were intraperitoneally injected with CNO,
FIGUR E | SNI mice manifested time- dependent anxiety- like behavior and correspondingly overexcited ACC astrocytes. (A) Time course of
SNI- induced the mechanical pain threshold, (n = 8 mice per group); Two- way repeated- measures ANOVA follow by unpaired t test; (B and C) The
representative tracking plot, time in center zone (left) and total travel distance of for OF T (right) (B, n = 8 mice per group) and time in open arm (left)
and total travel distance (right) for EPM test (C, n = 8 mice per group); Two- way repeated- measures ANOVA followed by post hoc Sidak's test; (D and
E) Immunohistochemical staining and statistical data analysis of GFAP in the ACC after surgery (n = 10 brain slices from 5 mice); unpaired t test;
Scale bar:100 µm. Ns p > 0.05, *p < 0. 05, **p < 0.01, ***p < 0.001, ****p < 0.0001 sham versus SNI group.
6 of 15 CNS Neuroscience & Therapeutics, 2025
followed by a series of behavioral tests (Figure4A). The naïve
mice did not develop any anxiety- like symptoms, indicating
that the combination of GfaABC1D- hM3D and CNO alone
did not induce any anxiety- like behaviors (Figure S2A,B).
Furthermore, previous measurements indicated that mice did
not exhibit anxiety- like behaviors 1 week after SNI surgery,
thus we try to proceeded with the animal behavior tests on the
mice one- week post- SNI to investigate the roles of astrocytes
in SNI. The mice were intraperitoneally injected with CNO,
followed by OFT and EPM tests. These tests showed reduced
FIGUR E | Inhibited ACC astrocytes ameliorate anxiety- like behavior in SNI mice. (A) Timeline of the experimental procedures and a represen-
tative image illustrating gfaABC1D- mCherry expression in the ACC; (B and C) The representative tracking plot, time in center zone (left) and total
travel distance (right) for OFT (B, n = 11 mice per group) and time in open (left) and total distance (right) for EPM test (C, n = 11 mice per group); un-
paired t test; (D–F) Immunohistochemical verification of hM4D expression in ACC astrocytes. The marker of astrocyte (D, GFAP, green), neuron (E,
NeuN, green), microglial cell (F, Iba1, green) co- immunostains with the GfaABC1D- hM4D- mCherry virus (red). Scale bar: 100 µm; (H) Histogram
presenting the percentage of GFAP- positive cells among hM4D- positive cells. (n = 6 brain slice from 3 mice); (G and I) Immunohistochemical ver-
ification of GfaABC1D - hM4D inhibition of astrocytes. (n = 15 brain slices from 5 mice); unpaired t test; Scale bar:100 µm. Ns p > 0.05, *p < 0.05,
**p < 0.01, **p < 0.01, ****p < 0.0 001 SNI 6 week s + GfaA BC1D- mCherry versus SNI 6 week s + GfaAB C1D- hM4D- mCher ry group.
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FIGUR E | Activated ACC astrocytes aggravate anxiety- like behavior and regulate excitatory neurons. (A) Timeline of the experimental proce-
dures; (B and C) T he representative tracki ng plot, time in c enter zone (left) and tota l travel distance (right) for OFT (B, n = 11 mice per group) and time
in open arm (left) and total distance (right) for EPM test (C, n = 11 mice per group); unpaired t test; (D and F) Immunohistochemical verification of
GfaABC1D- hM3D activation of astrocytes (n = 15 brain slices from 5 mice); unpaired t test; Scale bar:100 µm; (E and G) Immunohistochemical stain-
ing and stat istical data of c- fos positive cel ls in the ACC (n = 15 brain slices f rom 5 mice); unpaired t test; Sc ale bar:100 µm; (H, I) Im munohistochemical
staining and statistical data of c- fos positive cells in individual layers in ACC (n = 15 brain slices from 5 mice); Two- way repeated- measures ANOVA
follow by unpaired t test; Scale bar:100 µm (J) Histogram presenting the percentage of CamKII- positive cells among c- fos- positive cells (n = 6 brai n
slice from 3 mice); (K) c- fos colocalized with CamKII after GfaABC1D- hM3D activation of astrocytes. Scale bar:100 µm. Ns p > 0.05, *p < 0.05,
**p < 0.01, **p < 0.01, ****p < 0.0 001 SNI 1 week + Gf aABC1 D- mCherry ver sus SNI 1 week + Gfa ABC1D- hM3D- mCherry group.
8 of 15 CNS Neuroscience & Therapeutics, 2025
time spent in the center zone and open arm zone, respectively,
without affecting the total travel distance (Figure 4B,C).
As expected, mice in the hM3D- mCherry group developed
anxiety- like behaviors as early as one- week post- SNI. After
completing the behavioral experiments, the mice were per-
fused 2 h after CNO treatment, and their brains were collected
for staining. The GFAP density increased in hM3D- mCherry
mice, demonstrating that the virus effectively activated astro-
cytes (Figure4D,F).
Previous studies have shown that the ACC excitatory neurons
are overexcited in mice with anxiety- like behaviors, and astro-
cytes are closely connected to neurons [45, 46]. To determine
whether manipulating ACC astrocytes affected neurons, we
activated astrocytes in hM3D- mCherry mice and observed a
significant enhancement in ACC c- Fos expression, indicat-
ing that astrocytes influence the local neurons (Figure4E,G).
Besides, it is generally accepted that pyramidal cells in layer
5 project to the amygdala and periaqueductal gray, the former
is a structure that has a key role in processing fear and anx-
iety, and the latter is involved in the descending modulation
of spinal sensory transmission [11]. We investigated the tis-
sue slices that attained from mice brains after activation of
ACC astrocytes to count the number of c- Fos positive cells. We
found a significant increase in c- Fos expression in the layer 5
compared with other layers in ACC (Figure 3H,I). And this
result demonstrated that the function of ACC astrocytes in
regulation of anxiety- like behavior under the NP conditions
should be through the regulation of layer 5 neurons. We then
co- stained layer 5 c- Fos activated by astrocytes in hM3D-
mCherry mice with CaMKII, a marker of excitatory neurons,
to detect the effects on local excitatory neurons. Most c- Fos
signals co- localized with CaMKII (approximately 81.5%), sug-
gesting that the majority of neurons activated by astrocytes
were excitatory (Figure4J,K). Overall, these results indicated
that ACC astrocytes should play an important role in regulat-
ing the anxiety- like behavior by modulating the L5 excitatory
neurons.
3.4 | Regulation of Anxiety- Like Behaviors in SNI
Mice by Manipulating ACC Excitatory Neurons
As showed above, ACC excitatory neurons were modulated
by the local astrocytes. However, we needed to determine
whether direct regulating the excitatory neurons would have
the same effect on the anxiety- like behaviors. To verify this,
we injected CaMKII- hM4D- EGFP, CaMKII- hM3D- EGFP, or
CaMKII- EGFP viruses into the ACC of mice and waited at
least 3 weeks for viral transduction (Figure5A,B). Within the
ACC, CaMKII- hM4D- EGFP expression was limited to NeuN,
with small penetrance of GFAP or Iba1 (Figure S4A–D). Six
weeks after SNI surgery, all mice were intraperitoneally in-
jected with CNO. As expected, The CaMKII- hM4D- EGFP
group mice spent more time in the center zone of the OFT test
and the open arm of the EPM test, while the total distances
they traveled were similar to the control group (Figure5C,D).
Conversely, after activating ACC excitatory neurons with
CNO, the CaMKII- hM3D- EGFP group mice spent less time in
the center zone and open arm compared to the animals in the
CaMKII- EGFP group (Figure5E,F).
To further demonstrate that the CNO specifically inhibited or
activated CaMKII- hM4D- mCherry or CaMKII- hM3D- mCherry
respectively infected neurons, we examined the expression of c-
Fos in the ACC region. Following the activation of the CaMKII-
hM4D- EGFP receptor by CNO, c- Fos expression was scarcely
detectable in the ACC (Figure5G,I). In contrast, activation of
ACC excitatory neurons led to a marked increase in c- Fos ex-
pression (Figure5H,J). These results indicated that modulating
ACC excitatory neurons, much like the inhibition or activation
of ACC astrocytes, significantly influences the anxiety- like
behaviors in SNI mice. In general, our results suggested that
both astrocytes and excitatory neurons in the ACC played cru-
cial roles in regulation of anxiety- like behaviors under the NP
conditions.
3.5 | Activating Astrocytes Combined With
Excitatory Neurons Inhibition in ACC Did Not
Aggravate Anxiety- Like Behavior
To further investigate the combined effect of astrocytes
and excitatory neurons in ACC, we developed two addi-
tional experimental groups: GfaABC1D- hM3D- mCherry +
CaMKII- hM4D- EGFP, and GfaABC1D- hM3D- mCherry +
CaMKII- EGFP (Figure6A). The viruses GfaABC1D- hM3D-
mCherry, CaMKII- hM4D- EGFP, and CaMKII- EGFP were all
restricted to the ACC region (Figure 6B). Three weeks after
virus injection, we conducted the behavioral tests. CNO led to
decreased time spent in the center zone and open arm in the
OFT and EPM tests, respectively, for mice in the GfaABC1D-
hM3D- mCherry + CaMKII- EGFP group compared to the
GfaABC1D- hM3D- mCherry + CaMKII- hM4D- EGFP group
(Figure 6C,D). To further investigate the combined effect of
astrocytes and excitatory neurons, we examined the level of
c- Fos in ACC. The GfaABC1D- hM3D- mCherry + CaMKII-
hM4D- EGFP group showed a significant increase in c- Fos
expression compared to the GfaABC1D- hM3D- mCherry +
CaMKII- EGFP group (Figure6E,F). These results suggested
that the activation of ACC astrocytes may regulate local ex-
citatory neurons, contributing to the anxiety- like behaviors in
SNI mice.
3.6 | Regulation of Synapse Density by
Manipulating ACC Astrocytes
In recent years, it has become clear that astrocytes play an
important role in neuronal activity and plasticity [47]. To in-
vestigate whether synapse plasticity is involved in pain and con-
sequent anxiety relief after the modulation of ACC astrocytes,
we used GfaABC1D- hM4D- mCherry or GfaABC1D- hM3D-
mCherry to inhibit or promote the ACC astrocyte activity, re-
spectively, and then examined the effects of modulating ACC
astrocyte activity on PSD 95 expression after behavior tests
(Figure 7A). Immunofluorescence analysis revealed a lower
density of the postsynaptic marker PSD95 in GfaABC1D- hM4D-
mCherry mice in ACC L5 (Figure7C,D). Conversely, activation
of ACC astrocytes (GfaABC1D- hM3D- mCherry) resulted in
higher PSD 95 expression compared to the GfaABC1D- mCherry
group (Figure7E,F). Western blot of ACC homogenates also dis-
played a decreased expression of postsynaptic density protein
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FIGUR E | Chemogenetic manipulation ACC excitat ory neurons can regu late anxiety- li ke behavior in SNI mice. (A and B) Timeli ne of the exper-
imental procedure; (C and E) The representative tracking plot, time in center zone (left) and total travel distance (right) for OFT after CamK II- hM4D
activation of astrocytes (C, n = 11 mice per group) or after CamKII- hM3D activ ation of astrocytes (E , n = 10 mice per group); unpaired t test; (D and F)
The representat ive tracking plot, ti me in center zone (left) and total travel d istance (right) for EPM test af ter CamKII- hM4D activation of astroc ytes (D,
n = 11 mice per group) or after CamKII- hM3D activation of astrocytes (F, n = 10 mice per group); unpaired t test; (G–J) Immunohistochemical stain-
ing and statistical analysis of c- fos positive cells in the ACC (n = 15 brain slices from 5 mice); unpaired t test; Scale bar:100 µm. Ns p > 0.05, *p < 0.05,
**p < 0.01, ** p < 0.01, ****p < 0.00 01 SNI 6 weeks + CamK II- mCherry versus SN I 6 weeks + C amKI I- h M4D- mC herry group; SNI 1 w eek + CamK II-
mCherry versus SNI 1 week + CamKII- hM3D- mCherry group.
10 of 15 CNS Neuroscience & Therapeutics, 2025
(PSD95) in GfaABC1D- hM4D- mCherry mice and an increased
expression in GfaABC1D- hM3D- mCherry mice (Figure7G,H).
In general, these results demonstrated that ACC astrocytes may
induce increase synapse- related proteins and modulate anxiety-
like behaviors.
4 | Discussion
4.1 | Main Findings
In this study, we addressed the knowledge gap concerning the
contribution of ACC astrocytes to comorbid anxiety- like behav-
ior in the SNI model. During the course of post- SNI, we found
no significant differences in mice anxiety- state and GFAP den-
sity in 1 week after surgery procedure while they manifest ob-
vious decrease pain threshold. Mice would develop in anxiety
and consequently increase the GFAP density when experiencing
long- term NP. Using immunohistochemical staining and west-
ern blot following chemogenetic manipulation of ACC astro-
cytes, we have demonstrated that activation of astrocytes in ACC
could activate local excitatory neurons and increased synapse
density. Additionally, our experiments revealed that manipulat-
ing the local excitator y neurons resulted in the same anxiety- like
behavior observed when manipulating astrocytes. These find-
ings indicated a significant interplay between astrocytes and
excitatory neurons in ACC, emphasizing that ACC astrocytes
played a crucial role in the activation of excitatory neurons, and
consequently, the development of anxiety- like behavior in SNI
mice. This study highlights the importance of targeting astro-
cyte activity in the ACC as a potential therapeutic strategy for
managing anxiety in chronic pain conditions, providing a deeper
understanding of the cellular mechanisms underlying these
behaviors.
FIGUR E | Activating astrocytes combined with excitator y neurons inhibition in ACC did not aggravate anxiety- like behavior. (A) Timeline of
the experimental procedures; (B) Representative image illustrating gfaABC1D- mCherry and CamKII- EGFP expression in the ACC; (C and D) The
representative tracking plot, time in center zone (left) and total travel distance (right) for OFT (C, n = 8 mice per group) and time in the open arm
(left) and total travel distance (right) for EPM test (D, n = 8 mice per group); (E and F) Immunohistochemical staining and statistical analysis of c- fos
positive cells in ACC (n = 6 brain slices from 3 mice). Scale bar: 100 µ m. Ns p > 0.05, *p < 0.0 5, ** p < 0.01, * *p < 0.01, ****p < 0.00 01 Gf aABC1 D- hM3D-
mCherry+CamKII- mCherry versus GfaABC1D- hM3D- mCherry+CamKII- hM4D- mCherry group.
11 of 15
4.2 | Anxiety Like Behaviors and Neuropathic Pain
Animal Models
Among the NP animal models, SNI model has been extensively
characterized, closely mimicking clinical NP symptoms [21].
Clinical and preclinical studies have demonstrated a strong
association between NP and psychiatric conditions such as
anxiety [48]. However, the published data regarding SNI's as-
sociation with anxiety- like behavior are contradictory. Some
studies report no association between SNI and anxiety- like
FIGUR E | ACC astrocytes induced synapse- related proteins. (A, B) Timeline of the experiment. (C–F) Representative fields (C, E) and rela-
tive quantification (D, F) of the PSD95 in the ACC L5 region. (n = 15 brain slices from 5 mice); unpaired t test; Scale bar: 50 µm. (G, H) Western blot
analysis (G) and quantification (H) of in the whole ACC. (n = 3mice); Two- way repeated- measures ANOVA followed by unpaired t test. Ns p > 0.05,
*p < 0.05, **p < 0.01, **p < 0.01, ****p < 0.00 01 SNI 6 weeks + GfaA BC1D- mCherry versus SNI 6 week s + GfaAB C1D- hM4D- mCherry group, SNI
1 weeks + Gfa ABC1D - mCher ry versus SNI 1 we eks + GfaA BC1D- hM3D- mC herry group.
12 of 15 CNS Neuroscience & Therapeutics, 2025
actions [49], while others show a significant association be-
tween SNI and psychiatric disorders, including anxiety be-
havior [50, 51]. Our results suggested that mice developed
psychiatric disorders in the later stages (at least 6 weeks post-
surgery) and that this development was in a temporal fash-
ion. Other studies on chronic pain comorbid with psychiatric
disorders also show that experimental animals do not exhibit
anxiety behavior 1 week after persistent pain stimuli. For in-
stance, some studies reported that SNI mice displayed anxiety
behavior after 2 weeks [8, 52], while others observed it after
more than 4 weeks [53, 54]. This variability may result from
different experimental conditions, such as mouse feeding, en-
vironment, and individual differences, but all studies agree
that emotional disorders do not develop until the later stages
postsurgery. Interestingly, we found that acute activation of
astrocytes in naïve mice could not induce anxiety behavior.
However, when activation occurred during the advanced
stage of SNI, while anxiety behavior was not developing, mice
manifested obvious anxiety disorders. Oher study also finds
that in naïve mice without NP, acute ACC activation is not suf-
ficient to trigger depressive- like effects [3]. This suggests that
the effect of astrocytes in the ACC on anxiety- like behavior
may be context- dependent. Anxiety- like behavior may be ini-
tially triggered by SNI surgery and subsequently promoted by
the activation of astrocytes. These findings highlight the im-
portance of timing and context in the development of anxiety-
like behaviors in NP models and underscore the crucial role of
ACC astrocytes in modulating these behaviors.
4.3 | Astrocyte Function for the NP- Mediated
Anxiety Animal Model
Historically, astrocytes were considered primarily as elements
that maintain homeostasis and provide support for brain func-
tion. However, emerging evidence establishes that astrocytes
act as active modulatory cells in synaptic function, neural cir-
cuits, and consequently on brain function and animal behavior
[55]. Multiple studies over the past decade have reported as-
trocyte dysfunction in the brains of rodent models of chronic
pain- mediated anxiety. For instance, genetic or pharmacologi-
cal inhibition of astrocytes in the ventral hippocampus (vHPC)
compelety attenuated anxiodepressive- like behaviors in chronic
pain mouse model [56].;Beside, GFAP in the central nucleus
of amygdala (CeA) were demonstrated to be increased at the
chronic (4 weeks post- SNL), while manipulating anxiety like be-
havior, but not acute (1 week post- SNL), stage of NP [57]. Our
research found that, in the context of NP and anxiety, astrocytes
in the ACC play a pivotal role. Our results suggested that the
activation of ACC astrocytes can exacerbate anxiety- like behav-
iors, whereas their inhibition can ameliorate these behaviors.
This modulation is mediated through the interaction between
astrocytes and local excitatory neurons, highlighting the impor-
tance of astrocytic activity in regulating emotional responses
associated with NP. Additionally, astrocyte activation is linked
to other diseases, such as Alzheimer's disease [58], Parkinson's
disease [59], Huntington's disease [59]. The role of astrocytes in
comorbid anxiety behavior in these diseases warrants further in-
vestigation. Understanding the broader implications of astrocyte
function could provide new insights into the treatment of anxiety
across various neurological conditions.
4.4 | Communications of Astrocytes and Neurons
for the NP and Anxiety Animal Model
Accumulating evidence on astrocytes has established their
role in bidirectional communication with neurons. Astrocytes
respond to neurotransmitters and, in turn, release neuroac-
tive substances that influence neuronal and synaptic activ-
ity [60, 61]. To further determine the relationship between
astrocytes and neurons, we utilized SNI- induced anxiety
behavior mice and performed experiments on the interplay
between astrocytes and excitatory neurons in the ACC in
modulating anxiety- like behavior. We found that activated as-
trocytes alone upregulated the c- Fos level, whereas inhibited
CaMKII neurons alone decreased it. However, the activation
of astrocytes combined with the inhibition of neurons led to
attenuated expression of c- Fos and improved behaviors. This
indicates that astrocytes may regulate local excitatory neu-
rons by releasing neuroactive substances or through other
molecular mechanisms in the development of anxiety- like
symptoms in the SNI model. By demonstrating the combined
effects of manipulating both astrocytes and excitatory neu-
rons, we provide evidence that these cell types work together
to regulate anxiety- like behaviors. Recent rodent research has
demonstrated that CaMKII neurons are well correlated with
anxiety behavior. For instance, adolescent cocaine exposure
(ACE) mice exhibited anxiety- like behavior accompanied by
the activation of CaMKII- positive neurons in the claustrum.
By suppressing CaMKII activity, the anxiety behavior of ACE
mice was reversed [62]. Similarly, in mice with plantar injec-
tion of complete Freund's adjuvant (CFA), activation of the
dorsal raphe nucleus (DRN) GABAergic projections to ACC
CaMKII neurons reversed comorbid anxiety behavior [63].
Additionally, administering a DREADD agonist (CNO) to
selectively inhibit CaMKII neurons in the basolateral amyg-
dala (BLA) significantly alleviated anxiety behavior induced
by paclitaxel [64]. However, a recent study demonstrated that
chemogenetic activation of CaMKII neurons in the ACC could
robustly improve LPS- induced behavioral deficits [65]. This
contrast may be due to the different animal models used to in-
duce anxiety behavior. This insight could inform future ther-
apeutic strategies targeting both astrocytes and neurons to
more effectively manage anxiety in chronic pain conditions.
4.5 | Astrocyte- Mediated Synapse Plasticity in NP
and Anxiety Mouse Model
In general, Gq pathway activation of astrocytes is always consid-
ered depending on metabotropic glutamate receptor (mGluR)-
mediated intracellular calcium inf lux and tending to produce
artificial phenotypes or mechanistic profiles[47]. Recent studies
found that astroc ytes also contributed to s ynaptic plasticity events
and consequently accelerated chronic pain and anxiety- like
states [11, 47]. Astroglial processes enwrap most synaptic struc-
tures, forming the tripartite synapse [66]. Astrocytes become re-
active in response to injury or disease in the nervous system, and
reactive astrocytes were demonstrated to upregulate some genes
responsible for the induction of synapse formation, which in-
cluded unwanted synapses that lead to NP [67]. Initial studies on
astrocyte- mediated synapses plasticity in NP model focused on
the spinal cord level, suggesting that reactive astrocytes release
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complex array of substances, such as gliotransmitters, chemok-
ines, cytokines, and synaptogenic molecules to induce functional
and structural synaptic plasticity [68]. Multiple lines of following
evidences suggested maladaptive plastic changes in the “pain
matrix” cortical regions, such as ACC, mPFC and primary so-
matosensory cortex also existed [68]. Besides, chronic restraint
stress model with obvious anxiety- like behavior displayed lasting
dendritic hypertrophy, such as increased size of dendritic spine
heads and the number of mature, mushroom- shaped spines in
basolateral amygdala (BLA) [69]. Similarly, these dendritic hy-
pertrophy also existed in dorsomedial prefrontal cortex (dmPFC)
and hippocampus after chronic stress [70]. On the other hand,
LTP and other types of functional synaptic changes have been
particularly well studied in the ACC, with reports on both pre-
synaptic and postsynaptic contributions and increased AMPA
receptor insertion [71]. By examining the synapse- related pro-
teins, such as PSD95, our results demonstrated that activation
of astrocytes led to enhanced synaptic plasticity, which may in-
crease inappropriate neural connections and result in aggrava-
tive anxiety- like behavior in NP model.
5 | Conclusions
In summar y, our research emphasized the pivotal role of ACC
astrocytes in modulating NP comorbid with anxiety behaviors.
We found that the inhibition of ACC astrocytes could improve
anxiety symptoms, while activation of astrocytes could induce
these disorders in the advanced stage of SNI surgery. The mod-
ulatory effect of astrocytes on anxiety may occur by inf luencing
the synapse plastic, which in turn affects the activity of excit-
atory neurons. This work highlights the importance of astrocyte
dysfunction in the development of anxiety disorders associated
with NP and underscores their potential as a target for antide-
pressant treatment. By targeting astrocytes, the current work
may develop a more effective therapeutic strategy for managing
anxiety in chronic pain conditions.
Author Contributions
Qingqing Zhou: Conception and design, collection and assembly of
data, data analysis and interpretation, and manuscript writing; Qi
Zhong: Conception and design, data analysis, manuscript writing, and
financial support; Zhuang Liu and Ziyue Zhao: Conception and design,
collection and assembly of data, and data analysis; Jie Wang: conception
and design, manuscript writing, and supervision; Zongze Zhang: man-
uscript writing, superv ision, and financial support.
Conflicts of Interest
The authors declare no conflicts of interest.
Data Availability Statement
The data that support the findings of this study are available from the
corresponding author upon reasonable request.
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Supporting Information
Additional supporting information can be found online in the
Supporting Information section.