1-s2.0-S2589004226010047-main-html.html

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Neuronal Igfbp2 deficiency in the prefrontal cortex impairs cognition through synaptic

dysfunction in male mice

Weiming Zhao, Yanan Gao, Yichan Wang, Ke Peng, Shaoyong Song, Yufan Yang,

Baojian Zhao, Xisheng Shan, Li Deng, Ruixia Weng, Hong Liu, Xiaowen Meng,

Huayue Liu, Fuhai Ji
PII:

S2589-0042(26)01004-7

DOI:

https://doi.org/10.1016/j.isci.2026.115629

Reference:

ISCI 115629

To appear in:

iScience

Received Date: 25 October 2025
Revised Date: 2 February 2026
Accepted Date: 2 April 2026

Please cite this article as: Zhao, W., Gao, Y., Wang, Y., Peng, K., Song, S., Yang, Y., Zhao, B., Shan,

X., Deng, L., Weng, R., Liu, H., Meng, X., Liu, H., Ji, F., Neuronal Igfbp2 deficiency in the prefrontal

cortex impairs cognition through synaptic dysfunction in male mice,

iScience

(2026), doi:

https://

doi.org/10.1016/j.isci.2026.115629

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Neuronal Igfbp2 deficiency in the prefrontal cortex impairs cognition through

synaptic dysfunction in male mice

Weiming Zhao

1,2,7

, Yanan Gao

1,2,7

, Yichan Wang

1,2,7

, Ke Peng

1,2

, Shaoyong Song

1,2

Yufan Yang

1,2

, Baojian Zhao

, Xisheng Shan

1,2

, Li Deng

1,2

, Ruixia Weng

, Hong Liu

Xiaowen Meng

1,2

, Huayue Liu

1,2,3

, Fuhai Ji

1,2,8

Department of Anesthesiology, First Affiliated Hospital of Soochow University,

Suzhou, Jiangsu, China

Institute of Anesthesiology, Soochow University, Suzhou, Jiangsu, China

Ambulatory Surgery Center, First Affiliated Hospital of Soochow University, Suzhou,

Jiangsu, China

Department of Anesthesiology, Nanjing Stomatological Hospital, Affiliated Hospital

of Medical School, Nanjing University, Nanjing, Jiangsu, China

Department of Anesthesiology and Pain Medicine, University of California Davis

Health System, Sacramento, CA 95817, USA

Department of Gastroenterology, First Affiliated Hospital of Soochow University,

Suzhou, Jiangsu, China

These authors contributed equally

Lead contact

*Correspondence: jifuhaisuda@163.com (F-H.J.); docliu.hy@163.com (H-Y.L.);

lingximxw@163.com (X-W.M.).

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SUMMARY

Insulin-like growth factor binding protein 2 (Igfbp2) is known to participate in brain

development and synaptic regulation, but its specific contribution to prefrontal cortex

(PFC)-dependent cognitive function remains unclear. Here, we demonstrate that

neuronal Igfbp2 in the PFC plays a critical role in maintaining cognitive performance

and synaptic integrity. Neuron-specific Igfbp2 knockdown impaired recognition and

spatial memory without altering emotional or locomotor behaviors. Mechanistically,

loss of Igfbp2 reduced dendritic spine density, downregulated synapsin-1 and PSD-95

expression, and weakened excitatory synaptic transmission. Remarkably, local infusion

of recombinant Igfbp2 protein restored synaptic architecture and rescued cognitive

deficits. Together, these findings identify neuronal Igfbp2 as an essential regulator of

PFC-dependent cognition and highlight its potential as a therapeutic target for cognitive

dysfunction.

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Introduction

The insulin-like growth factor binding protein (Igfbp) family regulates cell growth,

differentiation, and metabolism by modulating the bioavailability and activity of

insulin-like growth factors.

1-3

Among these proteins, Igfbp2 is the most abundant in

cerebrospinal fluid and exhibits high expression levels in the central nervous system

throughout development, from the embryonic period into adulthood.

Igfbp2 is

primarily synthesized by neurons and astrocytes, functioning through autocrine and/or

paracrine pathways to regulate neuronal excitability, synaptic transmission, as well as

central nervous system injury and repair.

Recent evidence indicates that Igfbp2 in

hippocampal neurons is critical for early cognitive development through the regulation

of neuronal plasticity.

Our previous study demonstrated that Igfbp2 in glutamatergic

neurons within paraventricular thalamus afferents to the central amygdala mediates

neonatal anesthesia-induced fear memory deficits.

In addition, Igfbp2 exerts

neuroprotective effects by reducing Aβ-induced tau phosphorylation and neuronal

death in Alzheimer’s disease.

Another study also demonstrated that exogenous

supplementation of recombinant Igfbp2 protein can alleviate posttraumatic stress

disorder by facilitating brain plasticity.

Collectively, these findings underscore the

broad and pivotal role of Igfbp2 in regulating cognitive processes across multiple brain

regions and pathological conditions.

The prefrontal cortex (PFC) plays a pivotal role in cognition and higher-level executive

functions. Accumulating evidence indicates that neural network disturbances within the

PFC are major contributors to cognitive dysfunction.

10, 11

Ageing-related structural and

functional changes in the PFC, including reduced connectivity and volumetric loss,

frequently precede the broader cognitive impairments

observed in dementia

syndromes.

PFC dysfunction is also a prominent feature of neurodegenerative

diseases such as Alzheimer's disease, where it contributes to early cognitive decline

characterized by deficits in executive function and memory retrieval.

However,

whether Igfbp2 in the PFC directly regulates cognitive function remains largely

unknown.

In this study, we investigated the role of neuronal Igfbp2 in the PFC in mediating

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cognitive function. Using adeno-associated virus (AAV)-mediated gene knockdown,

we found that neuron-specific depletion of Igfbp2 in the PFC resulted in cognitive

dysfunction in mice. Mechanistically, Igfbp2 knockdown decreased dendritic spine

density and impaired excitatory synaptic transmission within the PFC. Importantly,

local supplementation with exogenous recombinant Igfbp2 protein rescued the

cognitive deficits induced by Igfbp2 knockdown through restoration of dendritic spine

density and excitatory synaptic transmission. Therefore, our results reveal that Igfbp2

in PFC neurons is essential for cognitive function and highlight its potential as a

therapeutic target for cognitive impairment.

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Results

Igfbp2 is predominantly expressed in PFC neurons

Although Igfbp2 in hippocampal pyramidal neurons has been shown to be essential for

early cognitive development,

its role in cognitive regulation within the PFC remains

poorly understood. To address this question, we first examined the cellular distribution

of Igfbp2 in the PFC. Brain tissue sections were prepared and subjected to double

immunofluorescence staining for Igfbp2 (Santa Cruz, sc-515134) in combination with

NeuN (neuronal marker), Sox9 (astrocytic marker), or IBA1 (microglial marker). Our

results showed that Igfbp2 was predominantly colocalized with NeuN, while exhibiting

sparse colocalization with Sox9 or IBA1 in the PFC (Figure 1A-D). To further validate

antibody specificity and assess staining reproducibility, we performed co-

immunostaining using a second independent anti-Igfbp2 antibody (R&D Systems,

AB5535) together with the same neuronal, astrocytic, and microglial markers.

Consistent with our initial observation, Igfbp2 signal was predominantly detected in

NeuN-positive neurons, with minimal colocalization with Sox9 or IBA1 (Figure S1).

Together, the concordant results obtained using two independent Igfbp2 antibodies

indicate that Igfbp2 is primarily localized to neurons in the PFC. These findings indicate

that Igfbp2 is primarily localized to neurons in the PFC, providing a cellular basis for

investigating its potential role in cognitive function.

Neuron-specific Igfbp2 deficiency in the PFC induces cognitive dysfunction.

To examine the functional consequences of Igfbp2 deficiency in PFC neurons, we

achieved neuron-specific Igfbp2 knockdown by bilaterally injecting AAV-hSyn-shRNA

(Igfbp2)-EGFP (AAV-shRNA) into the PFC of wild-type mice, with AAV-hSyn-shRNA

(Scramble)-EGFP (AAV-Scramble) serving as a control. Mice then underwent a

comprehensive battery of behavioral assays (Figure 2A). All behavioral experiments

were performed in adult mice.

At 3 weeks post-injection, PFC tissue was collected 24

h after completion of the behavioral tests to assess knockdown efficiency. Viral

infection in the PFC was confirmed by EGFP fluorescence (Figure 2B), and Igfbp2

knockdown efficiency was quantified at the protein level by Western blot analysis

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(Figure 2C). In addition, Igfbp2 knockdown was further validated at the mRNA level

by quantitative RT-PCR and at the cellular level by Igfbp2 immunofluorescence co-

staining with viral EGFP (Figure S2).

In the novel object recognition test, AAV-shRNA group spent significantly less time

investigating and made fewer approaches of the novel object compared with AAV-

Scramble group (Figure 2D-G). Similarly, in the Y-maze test, the AAV-shRNA group

exhibited significantly reduced time in and fewer entries into the novel arm compared

to the AAV-Scramble group (Figure 2H-K). These results indicated that neuron-specific

knockdown of Igfbp2 in the PFC impairs cognitive performance.

In contrast, both the forced swim test and tail suspension test revealed no significant

differences in immobility time between the AAV-shRNA and AAV-Scramble groups

(Figure S3), suggesting that Igfbp2 deficiency in PFC neurons did not induce

depression-like behavior. Similarly, the elevated plus maze test revealed no significant

differences in open-arm time between groups, and the open field test showed

comparable time spent in the central zone as well as total distance traveled (Figure S4),

indicating that neuron-specific Igfbp2 knockdown in the PFC did not affect anxiety-

like behavior or locomotor activity. In addition, body weight remained unaffected by

Igfbp2 deficiency in PFC (Figure S5).

Collectively, these findings demonstrate that Igfbp2 knockdown in PFC neurons

selectively impairs cognitive function without affecting mood-related behaviors,

locomotor activity, or body weight.

Neuron-specific Igfbp2 deficiency in the PFC leads to synaptic dysfunction

Synaptic dysfunction in the PFC is strongly implicated in cognitive decline,

14, 15

Previous studies have demonstrated that Igfbp2 can modulate synaptic function by

regulating synaptic connectivity and dendritic spine density.

16, 17

Therefore, we

investigated whether Igfbp2 deficiency in PFC neurons alters synaptic transmission

properties. Whole-cell patch-clamp recordings were performed on virus-infected PFC

neurons to assess synaptic function. We found that both the frequency and amplitude of

spontaneous excitatory postsynaptic currents (sEPSC) were significantly reduced in the

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AAV-shRNA group compared with the AAV-Scramble group (Figure 3A-C), indicating

that Igfbp2 deficiency in PFC neurons impairs excitatory synaptic transmission. In

contrast, no significant differences were detected in the frequency or amplitude of

spontaneous inhibitory postsynaptic currents (sIPSC) between the two groups (Figure

3D-F). Furthermore, PFC neurons in the AAV-shRNA group exhibited markedly

reduced intrinsic excitability compared with those in the AAV-Scramble group, as

evidenced by decreased action potential (AP) firing frequency

in response to

depolarizing current injections (Figure 3G-H). Importantly, the overall neuronal density

in the PFC remained unchanged following neuron-specific Igfbp2 knockdown (Figure

S6), ruling out neuronal loss as a confounding factor.

To further investigate the structural correlates of impaired synaptic transmission, we

assessed dendritic spine densities in the PFC using Golgi-Cox staining. The AAV-

shRNA group showed a significantly lower dendritic spine density compared with the

AAV-Scramble group (Figure 4A-B), suggesting that reduced spine density may

contribute to the observed alterations in synaptic transmission. Given that synapsin-1

(a presynaptic marker) and PSD-95 (a postsynaptic marker) are key regulators of

synaptic plasticity

through their respective roles in neurotransmitter release and

glutamate receptor trafficking,

18-20

we next examined their expression levels. Consistent

with the reduction in spine density, Western blot analysis revealed that the protein

expression levels of both synapsin-1 and PSD-95 were significantly decreased in the

AAV-shRNA group compared with the AAV-Scramble group (Figure 4C-D).

Taken together, these findings indicate that Igfbp2 knockdown in PFC neurons

contributes to synaptic dysfunction characterized by impairing excitatory synaptic

transmission, reducing neuronal excitability, and decreasing synaptic structural

integrity, which may underlie the cognitive deficits observed following Igfbp2

depletion.

Exogenous supplementation of Igfbp2 in the PFC rescues cognitive deficits

induced by neuron-specific Igfbp2 deficiency.

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Building upon these findings, we next investigated whether exogenous Igfbp2

supplementation in the PFC could ameliorate cognitive deficits induced by neuron-

specific Igfbp2 deficiency. Two weeks after viral injection, we stereotaxically

implanted bilateral guide cannulas into the PFC. One week later, recombinant Igfbp2

protein or vehicle (saline) was locally infused 24 hours prior to behavioral testing

(Figure 5A). Representative coronal brain sections confirmed successful viral

transduction and accurate cannula placement within the PFC (Figure 5B). Western blot

analysis further demonstrated that Igfbp2 infusion significantly increased Igfbp2

protein levels in the PFC compared with saline infusion (Figure 5C).

Behavioral analysis showed that the Igfbp2-treated group spent significantly more time

exploring and made more approaches to the novel object compared with the saline-

treated group in the novel object recognition test (Figure 5D-G). Similarly, in the Y-

maze test, Igfbp2-treated mice exhibited significantly increased time in and more

entries into the novel arm relative to the saline group (Figure 5H-K). By contrast, mice

infused with heat-denatured Igfbp2 did not show increases in exploration time or

approaches to the novel object in the novel object recognition test, nor increases in time

spent in or entries into the novel arm in the Y-maze test, compared with saline-treated

mice (Figure S7). These findings indicate that exogenous Igfbp2 supplementation in

the PFC effectively rescues cognitive dysfunction induced by neuron-specific Igfbp2

knockdown.

Exogenous supplementation of Igfbp2 in the PFC restores synaptic function

impaired by neuron-specific Igfbp2 deficiency.

Given that Igfbp2 deficiency impaired synaptic function, we next examined whether

exogenous Igfbp2 supplementation could reverse these deficits. Whole-cell patch-

clamp recordings revealed that the Igfbp2-treated group exhibited significantly

increased frequency and amplitude of sEPSC compared with the saline-treated group

(Figure 6A-C), indicating restoration of excitatory synaptic transmission. No

significant differences were observed in either the frequency or amplitude of sIPSC

between the two groups (Figure 6D-F). Furthermore, Igfbp2 supplementation markedly

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increased AP firing frequency in PFC neurons compared with saline treatment (Figure

6G-H), demonstrating recovery of neuronal excitability.

Morphological and molecular analyses further supported these electrophysiological

findings. Golgi-Cox staining revealed that the Igfbp2-treated group showed a

significantly higher dendritic spine density compared with the saline group (Figure 7A-

B). Consistently, Western blot analysis showed that protein expression levels of both

synapsin-1 and PSD-95 were significantly increased in the Igfbp2 group relative to the

saline group (Figure 7C-D).

Collectively, these results demonstrate that exogenous Igfbp2 supplementation

effectively restores synaptic structure and function impaired by Igfbp2 knockdown in

PFC neurons, providing a mechanistic basis for the observed cognitive rescue.

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Discussion

Igfbp2 has been shown to play important roles during brain development, including the

regulation of neuronal growth and synaptic formation. However, its contribution to

cognitive function has remained incompletely understood. In this study, we identified a

previously unrecognized role for Igfbp2 in PFC neurons as a critical regulator of

cognition (Figure 8). Neuron-specific deficiency of Igfbp2 led to significant cognitive

deficits in male mice, accompanied by impaired excitatory synaptic transmission and

compromised synaptic structural integrity, which collectively disrupted synaptic

function. Importantly, local supplementation with recombinant Igfbp2 protein in the

PFC reversed both synaptic and cognitive deficits, establishing Igfbp2 as an essential

factor for sustaining PFC-dependent cognitive processes.

Our study demonstrates that Igfbp2 is predominantly expressed in neurons within the

PFC, with minimal expression in astrocytes or microglia. To determine whether

neuronal Igfbp2 in the PFC plays a critical role in cognitive function, we employed an

adeno-associated virus vector expressing Igfbp2 short hairpin RNAs under the neuron-

specific hSyn promoter to achieve targeted Igfbp2 knockdown, followed by

comprehensive behavioral characterization. We validated Igfbp2 knockdown efficiency

by collecting PFC tissue 24 h after the final behavioral assay, three weeks after AAV

injection, and confirmed a significant reduction in Igfbp2 expression by quantitative

RT-PCR, Western blot analysis, and immunofluorescence.

Although we did not assess

this longitudinally beyond our testing window, a prior intracranial rAAV study reported

that expression is detectable within approximately 1 week, becomes robust by

approximately 3 weeks post-injection, and remains relatively stable thereafter,

supporting stable knockdown throughout our behavioral testing period.

We then

assessed the behavioral consequences of Igfbp2 reduction in the PFC and found that

neuron-specific Igfbp2 knockdown selectively impaired cognitive performance, as

evidenced by deficits in both novel object recognition and Y-maze tests. Notably,

anxiety-like behavior, depression-like behavior, and locomotor activity remained

unaffected, indicating that the effects of Igfbp2 deficiency were specific to cognitive

domains.

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Although the PFC is implicated in both cognitive and affective regulation, Igfbp2

knockdown in our study resulted in selective cognitive deficit without significantly

altering anxiety- or depression-like behaviors.

Specifically, Igfbp2 deficiency reduced

dendritic spine density, decreased synaptic protein expression, and impaired excitatory

synaptic transmission and neuronal excitability in PFC neurons. These Igfbp2-

dependent synaptic and intrinsic changes are fundamental for PFC-dependent

information processing and are likely rate-limiting for working memory and

recognition performance. By contrast, affect-related behaviors often rely on broader,

distributed networks and are more context- or state-dependent. Thus, the extent of

Igfbp2 reduction achieved here may be sufficient to disrupt cognitive computations

under baseline conditions but insufficient to produce measurable changes in baseline

anxiety- or depression-like assays. Consistent with this interpretation, our findings align

with and extend previous observations. Earlier work demonstrated that Igfbp2 in

hippocampal neurons regulates early cognitive development through modulating

neuronal plasticity.

More recently, Igfbp2 in basolateral amygdala (BLA) astrocytes

was shown to be essential for fear memory expression without affecting locomotor

activity or anxiety-like behaviors, consistent with the behavioral specificity observed

in our findings.

Furthermore, another study demonstrated that astrocyte-derived

Igfbp2 in the amygdala forms a multiday molecular trace that stabilizes fear memory

and sustains long-term synaptic plasticity.

Our study builds upon this foundation by

providing direct evidence that Igfbp2 expression in PFC neurons is indispensable for

adult cognitive function, thereby extending the role of Igfbp2 to higher-order executive

processes beyond hippocampal- and amygdala-dependent learning and memory.

Synaptic dysfunction in the PFC is a cardinal feature of cognitive decline in

neurodegenerative disorders such as Alzheimer’s disease.

24-26

Previous studies have

demonstrated that Igfbp2 is critical for maintaining synaptic function

through its

contributions to synaptic structural organization and neurotransmission regulation.

Our findings revealed that Igfbp2 knockdown in PFC neurons impaired excitatory

synaptic transmission and reduced neuronal excitability. In addition, Igfbp2 depletion

disrupted synaptic structural integrity, as evidenced by decreased dendritic spine

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density and reduced expression of key synaptic markers (synapsin-1 and PSD-95) in

the PFC. Notably, Igfbp2 knockdown did not alter the overall neuronal density in the

PFC, ruling out neuronal loss as a contributing factor to the observed deficits. These

findings are consistent with recent studies demonstrating that Igfbp2 enhances neuronal

excitability and promotes dendritic spine maturation through autocrine or paracrine

signaling mechanisms.

16, 17, 27

Overall, these results strongly suggest that Igfbp2 in PFC

neurons is essential for cognitive function through its regulation of synaptic structure

and activity.

Recent evidence has shown that an Igfbp2-derived peptide promotes neuroplasticity

and rescues cognitive and behavioral impairments in a mouse model of Phelan-

McDermid syndrome.

Consistent with these findings, our results suggest that

exogenous Igfbp2 supplementation in the PFC reverses the deficits caused by Igfbp2

knockdown, as evidenced by restoration of excitatory synaptic transmission, neuronal

excitability, dendritic spine density, and synaptic protein expression. As a modulator of

insulin-like growth factor (IGF) signaling, Igfbp2 binds to IGFs to regulate their

bioavailability and localization.

Previous studies have shown that Igfbp2 can

potentiate IGF2 receptor signaling, leading to activation of downstream ERK1/2

pathways that promote dendritic spine maturation and upregulates synaptic protein

expression in forebrain neurons, including postsynaptic scaffold proteins such as PSD-

95 and other synaptic components.

These mechanisms collectively explain how

Igfbp2 may regulate these synaptic proteins in PFC neurons.

The restoration of synaptic integrity and excitatory activity by exogenous Igfbp2

suggests that this protein serves dual functions in the nervous. First, Igfbp2 acts as a

structural regulator of synaptic architecture by maintaining of dendritic spines and

stabilization of synaptic proteins. Second, it functions as a functional modulator of

neuronal excitability and synaptic transmission. These dual roles are consistent with

previous reports showing that Igfbp2 can interact with extracellular matrix components

to modulate growth factor bioavailability while simultaneously influencing intrinsic

neuronal membrane properties.

Notably, Igfbp2 deficiency in hippocampal pyramidal

neurons has been shown to cause cognitive impairments that can be ameliorated by

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recombinant Igfbp2 supplementation.

Our findings parallel these observations in the

hippocampus, demonstrating that Igfbp2 supplementation can similarly rescue

cognitive dysfunction resulting from Igfbp2 deficiency in PFC neurons. Collectively,

these findings underscore the importance of Igfbp2 across multiple brain regions for

maintaining synaptic function and cognitive integrity.

In summary, our study reveals that Igfbp2 in PFC neurons is critical for cognitive

function through its dual roles in maintaining synaptic structural integrity and

modulating excitatory neurotransmission. Igfbp2 deficiency disrupts these processes,

leading to cognitive impairment, whereas exogenous Igfbp2 supplementation

effectively restores synaptic function and rescues cognitive deficits. These findings

advance our understanding of Igfbp2 function in the brain and suggest that targeting

Igfbp2 may hold therapeutic potential for treating cognitive disorders associated with

synaptic dysfunction.

Limitations of the study

Despite these strengths, several limitations warrant consideration. First, our study

exclusively examined male mice, which may constrain the generalizability of our

findings. Therefore, future studies including both sexes will be necessary to determine

whether these effects extend to females and to directly compare males and females.

Second, while recombinant Igfbp2 protein effectively rescued behavioral and synaptic

deficits, the precise downstream molecular pathways mediating these effects remain

incompletely defined. Future studies employing cell-type-specific transcriptomic and

proteomic approaches will be necessary to elucidate the signaling cascades through

which Igfbp2 regulates synaptic function and cognition. Third, the long-term

consequences of Igfbp2 modulation on cognitive performance and synaptic plasticity

are unknown. Given the dynamic regulation of Igfbp2 expression by developmental

stage, neuronal activity, and environmental factors, it will be important to determine

whether transient Igfbp2 supplementation yields sustained cognitive benefits or

whether continuous treatment is required. Finally, although exogenous supplementation

of Igfbp2 significantly improved behavioral and synaptic deficits in the present study,

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RESOURCE AVAILABILITY

Lead contact

Further information and requests for resources and code should be directed to the lead

contact, Fuhai Ji (jifuhaisuda@163.com), and will be fulfilled by the lead contact.

Materials availability

This study did not generate new unique reagents.

Data and code availability

•All data reported in this paper will be shared by the lead contact upon request.

•This study does not report original code.

•Any additional information required to reanalyze the data reported in this paper is

available from the lead contact upon request.

ACKNOWLEDGMENTS

This work was supported by the National Natural Science Foundation of China (grant

number: 82471281, 82072130, 82001126 and 82302465), Key Medical Research

Projects in Jiangsu Province (grant number: ZD2022021), Key R&D Program Projects

in Jiangsu Province (grant number: BE2023709), Suzhou Key Laboratory of

Anesthesiology (SZS2023013), Suzhou Clinical Medical Center for Anesthesiology

(grant number: Szlcyxzxj202102), Health Talent Plan Project in Suzhou (grant number:

GSWS2022007) and Suzhou Basic Research Pilot Youth Project (grant number:

SSD2024065). The funders had no role in the study design, data collection and analysis,

decision to publish, or preparation of the manuscript.

AUTHOR CONTRIBUTIONS

W-M. Z, Y-N. G and Y-C. W contributed equally to this work. F-H. J, H-Y. L and X-

W. M conception and design of research. W-M. Z, Y-N. G, Y-C. W, K. P, and S-Y. S

performed experiments. W-M. Z, B-J. Z, X-S. S, Y-F. Y and L. D analyzed data. W-M.

Z, K. P and S-Y. S prepared figures. W-M. Z, K. P and B-J. Z drafted the manuscript.

F-H. J, X-W. M, H-Y. L, and H. L edited and revised the manuscript. All the authors

have read and approved the paper.

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Figure Legends

Figure 1. Igfbp2 is predominantly expressed in the PFC neurons.

(A) Representative immunofluorescence image of Igfbp2 (red) and NeuN (green) in the

PFC.

(B)

Representative immunofluorescence image of Igfbp2 (red) and IBA1 (green) in the

PFC.

(D) Quantitative analysis of Igfbp2 colocalization with different cell types in the PFC.

Scale bar: 100 μm; n = 4 brain sections from 4 mice per group.

Figure 2. Neuron-specific Igfbp2 deficiency in the PFC induces cognitive

dysfunction.

(A) Schematic of the experimental design.

(B) Diagram of viral injection into the PFC (top) and representative image of viral

expression (bottom); scale bar: 500 μm.

in the PFC (bottom); n = 4 mice per group.

(D) Schematic of the novel object recognition test, depicting the training phase (top)

and the test phase (bottom).

(E) Representative trajectory plots from the novel object recognition test.

(F) Quantification of time spent investigating the novel object during the test phase; n

= 8 mice per group.

(G) Quantification of the number of investigations of the novel object during the test

phase; n = 8 mice per group.

(H) Schematic of the Y-maze test, depicting the training phase (top) and the test phase

(bottom).

(I) Representative trajectory plots from the Y-maze test.

(J) Quantification of time spent in the novel arm during the test phase; n = 8 mice per

group.

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(K) Quantification of the number of entries into the novel arm during the test phase; n

= 8 mice per group.

Analyzed by unpaired

test; **

< 0.01. Data are presented as means ± SEM.

Figure 3. Neuron-specific Igfbp2 deficiency in the PFC reduces excitatory synaptic

transmission and neuronal excitability.

(A) Representative traces of sEPSC recorded from PFC neurons

in AAV-Scramble (top)

and AAV-shRNA (bottom) groups.

(B) Cumulative probability plots (main panel) and quantification (insert panel) of

sEPSC frequency in AAV-Scramble and AAV-shRNA groups.

sEPSC amplitude in AAV-Scramble and AAV-shRNA groups.

(D) Representative traces of sIPSC recorded from PFC neurons in AAV-Scramble (top)

and AAV-shRNA (bottom) groups.

(E) Cumulative probability plots (main panel) and quantification (insert panel) of

sIPSC frequency in AAV-Scramble and AAV-shRNA groups

(F) Cumulative probability plots (main panel) and quantification (insert panel) of sIPSC

amplitude in AAV-Scramble and AAV-shRNA groups.

(G) Representative traces of AP elicited by 200 pA current injections in PFC neurons

from AAV-Scramble (left) and AAV-shRNA (right) groups.

(H) Quantification of AP firing frequency in response to a range of current injections in

PFC neurons

from AAV-Scramble and AAV-shRNA groups.

Analyzed by unpaired

test in B, C, E, and F, and two-way repeated-measure ANOVA

in H; n = 8 neurons from 4 mice per group; ns: not significance, **

< 0.01. Data are

presented as means ± SEM.

Figure 4. Neuron-specific Igfbp2 deficiency in the PFC disrupts synaptic integrity.

(A) Representative images of dendritic spines in the PFC from AAV-Scramble (left)

and AAV-shRNA (right) groups; scale bar: 10 μm.

(B) Quantification of dendritic spine density (number per 10 μm) in the PFC from AAV-

Scramble and AAV-shRNA groups; n = 9 brain sections from 3 mice per group.

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levels (bottom) in the PFC from AAV-scramble and AAV-shRNA groups; n = 4 mice

per group.

(D) Representative western blot bands (top) and quantification of PSD-95 protein levels

(bottom) in the PFC from AAV-Scramble and AAV-shRNA groups; n = 4 mice per group.

Analyzed by unpaired

test; *

< 0.05, ***

< 0.001. Data are presented as means ±

SEM.

Figure 5. Exogenous supplementation of Igfbp2 in the PFC rescues cognitive

deficits induced by neuron-specific Igfbp2 deficiency.

(A) Schematic of the experimental design.

(B) Diagram showing virus injection and cannula placement into the PFC (top), and a

representative image of cannula position and viral expression in the PFC (bottom); scale

bar: 500 μm.

in the PFC (bottom); n = 4 mice per group.

(D) Schematic of the novel object recognition test, depicting the training phase (top)

and the test phase (bottom).

(E) Representative trajectory plots from the novel object recognition test.

(F) Quantification of time spent investigating the novel object during the test phase; n

= 8 mice per group.

(G) Quantification of the number of investigations of the novel object during the test

phase; n = 8 mice per group.

(H) Schematic of the Y-maze test, depicting the training phase (top) and the test phase

(bottom).

(I) Representative trajectory plots from the Y-maze test.

(J) Quantification of time spent in the novel arm during the test phase; n = 8 mice per

group.

(K) Quantification of the number of entries into the novel arm during the test phase; n

= 8 mice per group.

Analyzed by unpaired

test; **

< 0.01. Data are presented as means ± SEM.

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Figure 6. Exogenous supplementation of Igfbp2 in the PFC restores excitatory

synaptic transmission and neuronal excitability impaired by neuron-specific

Igfbp2 deficiency.

(A) Representative traces of sEPSC recorded from PFC neurons in Saline (top) and

Igfbp2 (bottom) groups.

(B) Cumulative probability plots (main panel) and quantification (insert panel) of

sEPSC frequency in Saline (top) and Igfbp2 (bottom) groups.

sEPSC amplitude in Saline and Igfbp2 groups.

(D) Representative traces of sIPSC recorded from PFC neurons in Saline (top) and

Igfbp2 (bottom) groups.

(E) Cumulative probability plots (main panel) and quantification (insert panel) of sIPSC

frequency in Saline and Igfbp2 groups.

(F) Cumulative probability plots (main panel) and quantification (insert panel) of sIPSC

amplitude in Saline and Igfbp2 groups.

(G) Representative traces of AP elicited by 200 pA current injections in PFC neurons

from Saline (left) and Igfbp2 (right) groups.

(H) Quantification of AP firing frequency in response to a range of current injections in

PFC neurons

from Saline (left) and Igfbp2 (right) groups.

Analyzed by unpaired

test in B, C, E, and F, and two-way repeated-measure ANOVA

in H; n = 8 neurons from 4 mice per group; ns: not significance, **

< 0.01. Data are

presented as means ± SEM.

Figure 7. Exogenous supplementation of Igfbp2 in the PFC restores synaptic

integrity disrupted by neuron-specific Igfbp2 deficiency.

(A) Representative images of dendritic spines in the PFC from Saline (left) and Igfbp2

(right) groups; scale bar: 10 μm.

(B) Quantification of dendritic spine density (number per 10 μm) in the PFC from

Saline and Igfbp2 groups; n = 9 brain sections from 3 mice per group.

levels (bottom) in the PFC from AAV-Scramble and AAV-shRNA groups; n = 4 mice

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per group.

(D) Representative western blot bands (top) and quantification of PSD-95 protein levels

(bottom) in the PFC from AAV-Scramble and AAV-shRNA groups; n = 4 mice per group.

Analyzed by unpaired

test; *

< 0.05, ***

< 0.001. Data are presented as means ±

SEM.

Figure 8. Schematic summary of this study.

Neuron-specific Igfbp2 deficiency in the PFC impairs cognitive function by disrupting

synaptic integrity, excitatory transmission, and neuronal activity, whereas exogenous

Igfbp2 supplementation mitigates cognitive deficits by restoring synaptic integrity,

excitatory transmission, and neuronal activity. PFC prefrontal cortex, sEPSC

spontaneous excitatory postsynaptic currents, Igfbp2 Insulin-like growth factor-binding

protein 2.

Supplementary information

Document S1. Figures S1-S7.

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STAR

★

METHODS

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Animals

All experiments were performed in adult male mice to reduce potential sex-related

variability. C57BL/6J male mice (8 weeks old, 20-25 g) were purchased from Slaccas

Laboratory (Shanghai, China). Animals were housed in specific pathogen-free facilities

under standard laboratory conditions (23-25 °C, 40–60% relative humidity, 12-h light

/12-h dark cycle) with ad libitum access to food and water. Mice were randomly

assigned to experimental groups, and sample sizes were determined based on previous

behavioral, molecular, and electrophysiological studies conducted in our laboratory.

Investigators were blinded to

experimental conditions during both data collection and

analysis. All experimental procedures were approved by the Ethics Committee of

Soochow University (Suzhou, Jiangsu, China; approval No.: 202408A0217) and

conducted in strict accordance with the ARRIVE guidelines.

METHOD DETAILS

Behavioral tests

Mice were acclimated to the testing room for 1 h prior to each behavioral assay. All

behavioral tests were performed and analyzed in a blinded manner, with experimenters

unaware of group assignments throughout testing and data analysis.

Novel Object Recognition Test

The novel object recognition test was performed following procedures described in

previous study.

An open-topped arena (40 × 40 × 30 cm) was placed in a dimly lit

room. During the training phase, mice were placed in the arena and allowed to freely

explore two identical objects for 10 min.

After a 90-min inter-trial interval, one the

familiar object was replaced with a novel object during the test phase. The time spent

exploring the novel object and the number of novel object investigations were recorded

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over a 10-min period using the ANY-maze behavioral tracking system (Stoelting Co.,

USA).

Y-maze test

The Y-maze test was conducted as previously described.

The apparatus consisted of

three arms arranged at 120° angles (each arm: 30 × 4.5 cm, wall height: 15 cm). Distinct

visual cues were affixed to the walls at the distal end of each arm. The three arms were

designated as the start arm, familiar arm, and novel arm. During the training phase, the

novel arm was blocked, and mice freely explored the start and familiar arms for 5 min.

Following a 90-min inter-trial interval, the novel arm was opened during the test phase,,

allowing access to all three arms. The number of entries into and time spent in the novel

arm were recorded using the ANY-maze system.

Open field test

The open field test was performed as previously described.

Mice were gently placed

in a corner of an open-field chamber (40 × 40 × 30 cm), and locomotor activity was

recorded for 10 min using an overhead camera coupled with the ANY-maze tracking

system. Total distance traveled and time spent in the central zone (20 × 20 cm) were

quantified.

Elevated plus maze test

The elevated plus maze test was conducted as previously described.

The apparatus

consisted of two open arms (30 × 6) and two closed arms (30 × 6 cm, wall height: 15

cm), elevated 60 cm above the floor. Each mouse was placed at the center of the maze,

facing an open arm, and allowed to explore freely for 5 min. Time spent in the open

arms was recorded and analyzed using the ANY-maze system.

Forced swim test

The forced swim test was performed as previously described.

Mice were individually

placed in a transparent glass cylinder (height: 70 cm; diameter: 30 cm) filled with water

to a depth of 30 cm, maintained at 23 ± 1°C. Sessions were videotaped for 6 min, and

immobility time was quantified during the last 5 min.

Tail suspension test

The tail suspension test was conducted as previously described.

Each mouse was

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individually suspended by the tail using adhesive tape

positioned 1 cm from the tail tip,

at a height of 30 cm above the floor. The test duration was 6 min, and immobility time

during the final 5 min was quantified. Behavior was video-recorded throughout the

entire session.

Stereotaxic surgery

Stereotaxic procedures were performed as previously described.

Mice were

anesthetized with 1% sodium pentobarbital (100 mg/kg, intraperitoneally) and placed

in a stereotaxic frame (RWD Life Science Inc., Shenzhen, China). rAAV-hSyn-EGFP-

5′miR-30a-shRNA (Igfbp2)-3′miR-30a-WPREs (AAV2/9, 5.00E+13 vg mL

−1

, 150 nL;

BrainVTA Co. Ltd., Wuhan, China) or

rAAV-hSyn-EGFP-5′miR-30a-shRNA

(Scramble)-3′miR-30a-WPREs (AAV2/9, 5.00E+13 vg mL

−1

, 150 nL) were bilaterally

microinjected into the PFC (coordinates relative to bregma: anterior-posterior, + 2.20

mm; medial-lateral, ± 0.20 mm; dorsal-ventral, + 2.35 mm) at a flow rate of 20 nL/min

using a microinjection pump. Following injection, needles remained in place for 10 min

before being slowly withdrawn to minimize viral reflux. For cannula implantation

experiments, guide cannulas (internal diameter: 0.25 mm; RWD) were bilaterally

implanted into the PFC at identical stereotaxic coordinates immediately after viral

injection. Dummy cannulas (RWD), secured with dust caps were inserted into the guide

cannulas to maintain patency during the recovery period.

Drug administration

Recombinant Igfbp2 protein (MCE; Cat# HY-P74846) was dissolved in sterile saline at

10 μg/μL. Igfbp2 was bilaterally infused into the PFC (0.5 μL per side; 5.0 μg, 152

pmol per side) 24 h prior to behavioral testing in mice with neuron-specific Igfbp2

deficiency.

An equal volume of sterile saline was bilaterally infused into the PFC as a

vehicle control. Heat-denatured Igfbp2 was infused using the same volume and infusion

parameters as an additional protein control. Microinjectors extending 0.5 mm beyond

the guide cannula tips were inserted and left in place for 1 min to stabilize. Either sterile

saline, Igfbp2 solution, or heat-denatured Igfbp2 was then infused over 1 min.

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Microinjectors were kept in place for an additional 1 min to allow diffusion before being

replaced with obturators.

Western blotting

Western blot analysis was performed according to established protocols.

PFC tissues

were rapidly dissected and homogenized in ice-cold Radio-Immunoprecipitation Assay

(RIPA) lysis buffer supplemented with protease inhibitor cocktail. Protein

concentrations were quantified using the bicinchoninic acid (BCA) assay. Equal

amounts of protein were separated on 10-15% SDS-PAGE gels and electrotransferred

onto polyvinylidene fluoride (PVDF) membranes. Membranes were blocked with 5%

non-fat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 2 h at room

temperature, then incubated with primary antibodies overnight at 4 °C. Following three

15-min washes with TBST, membranes were incubated with horseradish peroxidase

(HRP)-conjugated secondary antibodies for 2 h at room temperature. Immunoreactive

bands were visualized using enhanced chemiluminescence (ECL) reagent (Thermo

Fisher Scientific) and quantified by densitometry using ImageJ software (NIH).

Primary antibodies included rabbit anti-Igfbp2 (1:500, ab188200, Abcam), rabbit anti-

PSD95 (1:500, ab238135, Abcam), rabbit anti-Synapsin-1 (1:500, ab64581, Abcam),

and rabbit anti-GAPDH (1:1000, AB-P-R001, GoodHere). The secondary antibody was

goat anti-rabbit IgG (1:50,000, 111-035-003, Jackson ImmunoResearch). Protein

expression levels were normalized to GAPDH as an internal loading control.

Whole-cell electrophysiological recording

Electrophysiological recordings were performed as previously described.

Mice were

deeply anesthetized with 1% sodium pentobarbital (100 mg/kg, intraperitoneally) and

transcardially perfused with 20 mL of ice-cold, oxygenated (95% O₂ and 5% CO₂) slice-

cutting solution containing (in mM): 93 N-methyl-D-glucamine, 93 HCl, 2.5 KCl, 1.25

NaH₂PO₄, 10 MgSO₄, 30 NaHCO₃, 25 glucose, 20 HEPES, 5 sodium ascorbate, 3

sodium pyruvate, and 2 thiourea (pH 7.3-7.4, 300-310 mOsm). Following decapitation,

brains were rapidly extracted and 300-μm

-thick coronal sections containing the PFC

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were prepared using a vibrating microtome (VT1200S; Leica, Germany). Brain slices

were initially incubated in oxygenated artificial cerebrospinal fluid (aCSF) at 32 °C for

at least 30 min, then gradually equilibrated to room temperature (24-26 °C) before

recording. The aCSF contained (in mM): 126 NaCl, 2.5 KCl, 1.25 NaH₂PO₄, 2 MgSO₄,

10 glucose, 26 NaHCO₃, and 2 CaCl₂ (pH 7.3-7.4, 300-310 mOsm).

For recordings, individual slices were transferred to a submerged recording chamber

continuously perfused with oxygenated aCSF (1-2 mL/min) at room temperature.

Neurons in the PFC were visualized using infrared differential interference contrast (IR-

DIC) video microscopy with a 40× water-immersion objective (BX51WI, Olympus,

Japan). Patch pipettes with resistances of 3-5 MΩ were fabricated from borosilicate

glass capillaries using a horizontal pipette puller (P-1000, Sutter Instrument, USA). The

internal pipette solution contained (in mM): 133 potassium gluconate, 18 NaCl, 0.6

EGTA, 10 HEPES, 2 Mg-ATP, and 0.3 Na₃-GTP (pH 7.2 adjusted with KOH, 280-300

mOsm). Whole-cell recordings were acquired using a MultiClamp 700B amplifier

(Molecular Devices, USA) controlled by Clampex 10.6 software. Signals were low-

pass filtered at 2 kHz and digitized at 10 kHz using a Digidata 1440A analog-to-digital

converter (Axon Instruments, USA). Recordings were excluded if series resistance

exceeded 20 MΩ or changed by more than 20% during recording.

For current-clamp recordings, action potentials (APs) were evoked by injecting

depolarizing current pulses of increasing amplitude (500 ms duration, 40-320 pA, in

40-pA increments). For voltage-clamp recordings, neurons were held at −70 mV, and

spontaneous excitatory and inhibitory postsynaptic currents (sEPSC and sIPSC) were

continuously recorded for 4 min.

Immunofluorescence staining

Mice were transcardially perfused with ice-cold 0.9% saline followed by 4%

paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Brain sections were post-fixed

overnight in 4% paraformaldehyde at 4°C, cryoprotected in 30% sucrose solution, and

sectioned coronally at 30 μm thickness using a cryostat microtome. Free-floating

sections were washed three times in phosphate-buffered saline (PBS; 10 min each),

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blocked in PBS containing 5% normal donkey serum and 0.3% Triton X-100 for 1 h at

room temperature, then incubated with primary antibodies diluted in blocking solution

overnight at 4 °C. Following three PBS washes (10 min each), sections were incubated

with fluorophore-conjugated secondary antibodies diluted in PBS for 1 h at room

temperature in the dark. After three additional PBS washes (10 min each), sections were

counterstained with 4',6-diamidino-2-phenylindole (DAPI; 1:5000) for nuclear

visualization, mounted on glass slides, and coverslipped with anti-fade mounting

medium.

Primary antibodies included mouse anti-Igfbp2 (1:200, sc-51534, Santa Cruz

Biotechnology), anti-Igfbp2 (1:500, MAB797, R&D System), rabbit anti-Sox9 (1:500,

AB5535, Sigma), rabbit anti-IBA1 (1:200, ab178847, Abcam), and rabbit anti-NeuN

(1:200, 24307, Cell Signaling Technology). Secondary antibodies were donkey anti-

rabbit Alexa Fluor 488 (1:500, Invitrogen) and donkey anti-mouse Alexa Fluor 555

(1:500, Invitrogen). Fluorescence images were acquired using a confocal laser scanning

microscope (Zeiss, LSM900) with appropriate excitation and emission filters.

Golgi staining

Golgi staining was performed using the FD Rapid Golgi Staining Kit (PK401, FD

NeuroTechnologies, USA) according to the manufacturer’s instructions. Briefly, freshly

dissected brain samples were immersed in a 1:1 mixture of solutions A and B and

incubated in the dark at room temperature for 14 days with solution changes every 3-4

days. Samples were subsequently transferred to solution C and incubated in the dark at

room temperature for an additional 5 days. Brain tissues were then embedded in optimal

cutting temperature (OCT) compound and rapidly frozen at −80 °C for 24 h. Coronal

sections (100 μm) were cut using a cryostat microtome (Leica, Wetzlar, Germany), and

mounted on gelatin-coated slides, and processed according to the kit protocol. Stained

sections were dehydrated through graded ethanol series, cleared in xylene, and

coverslipped with Permount mounting medium.

Dendritic spine analysis was performed on layer II/III pyramidal neurons in the PFC.

For each neuron, three secondary or tertiary basal dendrite segments (minimum 30 μm

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in length, located >50 μm from the soma) were randomly selected for analysis. High-

magnification images were acquired using a confocal microscope equipped with a 63×

oil-immersion objective lens (Zeiss, LSM900). Dendritic spine density (number of

spines per 10 μm dendritic length) was quantified using ImageJ software (NIH) by an

experimenter blinded to experimental conditions.

QUANTIFICATION AND STATISTICAL ANALYSIS

Animals with misplaced viral injections or cannula implantations, as verified by post-

hoc histological analysis, were excluded from statistical analysis. All data are presented

as mean ± standard error of the mean (SEM). Normality of data distribution was

assessed using the Shapiro-Wilk test, and homogeneity of variance was evaluated using

Bartlett’s test. For comparisons between two groups, unpaired two-tailed Student’s t-

test were performed. For comparisons involving multiple groups or repeated measures,

two-way repeated-measures analysis of variance (ANOVA) followed by Sidak’s post-

hoc multiple comparisons test was used. All statistical analyses were conducted using

GraphPad Prism 10.0 (GraphPad Software). A probability value of

< 0.05 was

considered statistically significant.

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KEY RESOURCES TABLE

REAGENT or RESOURCE

SOURCE

IDENTIFIER

Antibodies

Rabbit anti-Igfbp2

Abcam

Cat# ab188200; RRID:
AB_2938998

Rabbit anti-PSD95

Abcam

Cat# ab238135; RRID:
AB_2895158

Rabbit anti- Synapsin-1

Abcam

Cat# ab64581; RRID:
AB_1281135

Rabbit anti-GAPDH

GoodHere

Cat# AB-P-R001; RRID:
AB_3096355

Goat anti-rabbit IgG

Jackson
ImmunoResearch

Cat#

111-035-003;

RRID: AB_2313567

Mouse anti-Igfbp2

Santa Cruz

Cat# sc-515134; RRID:
AB_3675206

Mouse anti-Igfbp2

R&D Systems

Cat# MAB797; RRID:
AB_2264599

Rabbit anti-Sox9

Sigma

Cat# AB5535; RRID:
AB_2239761

Rabbit anti-IBA1

Abcam

Cat# ab178847; RRID:
AB_2832244

Rabbit anti-NeuN

CST

Cat#

24307;

RRID:

AB_2651140

Alexa Fluor 488-anti-Rabbit
secondary antibody

Invitrogen

Cat# A21206; RRID:
AB_2535792

Alexa Fluor 555-anti-Mouse
secondary antibody

Invitrogen

Cat# A31570; RRID:
AB_2536180

Bacterial and virus strains

rAAV-hSyn-EGFP-5′miR-30a-
shRNA

(Igfbp2)-3′miR-30a-

WPREs

BrainVTA

N/A

rAAV-hSyn-EGFP-5′miR-30a-
shRNA (Scramble)-3′miR-30a-
WPREs

BrainVTA

N/A

AAV2/2-Retro-Vglut2-Cre

BrainVTA

Cat# PT-1011

Chemicals,

peptides,

and

recombinant proteins

Recombinant Igfbp2 protein

MCE

Cat# HY-P74846

Software and algorithms

ANY-maze

Stoelting

https://www.any-
maze.com

Journal Pre-proof

MATLAB 2017b

MathWorks

https://ww2.mathworks.
cn/en/products.html?s_ti
d=gn_ps

GraphPad Prism 10.0

GraphPad Software

https://www.graphpad.co
m/scientific-software/

ZEN

Zeiss

https://www.zeiss.com/
microscopy/us/products/
microscope-
software/zen.html

ImageJ

National Institutes of
Health

https://imagej.nih.gov/ij/
index.html

Clampfit

Molecular Devices

https://www.molecularde
vices.com/products/axon
patch-
clamp-
system/acquisition-and-
analysissoftware/
pclamp-software-suite

Continued

REAGENT or RESOURCE

SOURCE

IDENTIFIER

MiniAnalysis

Synaptosoft

http://www.synaptosoft.c
om/MiniAnalysis/

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