The Role And Mechanism Of IGF2 In The Nucleus Accumbens In Anxiety-like Behavior Induced By Restraint Stress In Mice

Objective: Insulin-like growth factor-2(IGF2)is a member of the insulin-like growth factor family and is one of the most complex growth factors by far.Increasing evidence has demonstrated that nucleus accumbens(NAc)plays an important role in stress response.NAc integrates excitatory and regulatory inputs from multiple brain regions,and is mainly composed of two types of γ-aminobutyric acid(GABA)neurons D1-medium spiny neurons(D1-MSN)and D2-medium spiny neurons(D2-MSN),which play a different role in stress response.Previous results indicate that IGF2 involves in learning and memory processes and antidepressant treatment,but the role and mechanism of IGF2 in anxiety behaviors are rarely studied.In this study,we investigated the role and mechanism of IGF2 signal in the NAc in anxiety-like behavior induced by restraint stress(RS).Methods: Gene chip data were acquired from gene expression omnibus(GEO)to screen for repeated differentially expressed genes,and functional enrichment was performed through Gene Ontology and pathway analysis.The signal pathway and protein function interaction analysis on IGF2 were performed using Reactome and String database.Restraint stress model was constructed and anxiety-like behavior was evaluated by open field test(OFT),and the expression of IGF2 in the brain area of NAc was detected by quantitative real-time PCR(QPCR).IGF2 was administered exogenously through the NAc to mice,and its effect on mice behavior was observed.Q-PCR was used to detect the expression levels of D1-MSN marker genes tachykinin precursor 1(Tac1)and D2-MSN marker genes proenkephalin(Penk)after restraint stress in mice.The D1 or D2 cre mice were injected with AAV-DIO-sh RNA-IGF2 virus to specifically knock down IGF2 of the NAc to evaluate open field test.Open field test was detected after regulation of D1-MSN or D2-MSN activity by designer receptor exclusively activated by designer drugs(DREADD).Results:(1)The GSE53987,GSE54563 and GSE54564 gene chips were obtained from the brain regions of depression patients and healthy people through GEO,and a total of 3513 differentially expressed genes were obtained,of which 312 genes were expressed in two or more repeated differential genes,and 17 genes were expressed in three repeated genes.(2)Functional enrichment analysis of differentially expressed genes found the main related functions such as myelin formation and glial cell regeneration.The insulin-like growth factor binding protein 2(IGFBP2)was selected,and string was used to find the interaction between IGFBP2 and IGF2.(3)The results from reactome and string analysis showed that the pathways most relevant to IGF2 included insulin-like growth factor 2 m RNA binding protein binding RNA and IGF1 R signal cascade.(4)The obvious anxiety-like behavior was induced by 3 d restraint stress in male mice.Compared with the control group,the duration in central area(Control: 18.78 ± 1.90 sec,3 d: 11.25 ± 1.02 sec,P <0.01 vs Control),and the entry numbers into the central area were decreased significantly(Control: 21.13 ± 1.83,3 d: 13.83 ± 1.14,P <0.01 vs Control).However,the total distance was unchanged.The IGF2 in the NAc was increased significantly(Control: 1.01 ± 0.06,3 d: 1.48 ± 0.08,P <0.001 vs Control).The duration in central area,the entry numbers into enter the central area and the total distance of movement did not change significantly in female mice.(5)Microinjection of recombinant IGF2 into NAc significantly decreased total distance(PBS: 23.02 ± 3.30 m,2.5 ng: 9.92 ± 1.94 m,P <0.01 vs PBS),the entry numbers into the central area(PBS: 16 ± 3.15,2.5 ng: 5.33 ± 1.38,P <0.01 vs PBS),and the duration in central area(PBS: 15.69 ± 3.13 sec,2.5 ng 8.5 ± 2.79 sec).(6)Restraint stress significantly increased the level of D1-MSN marker gene Tac1(Control: 0.95 ± 0.02,RS: 1.11 ± 0.05,P <0.05 vs Control)and D2-MSN markers gene Penk(Control: 0.98 ± 0.08,RS: 1.41 ± 0.04,P <0.01 vs Control)in the NAc of mice.(7)Microinjection of AAV-DIO-sh RNA-IGF2 into NAc provided the cell-specific knockdown of IGF2 in D1-cre or D2-cre mice,and reduced the expression level of IGF2 in D1-MSN(GFP: 0.69 ± 0.06,sh RNA-IGF2: 0.49 ± 0.04,P <0.05 vs GFP).Meanwhile,the anxiety-like behavior of D1-cre mice was improved,including the increased entry numbers into the central area(GFP: 10.5 ± 1.36,sh RNA-IGF2: 17.41 ± 2.98).The expression level of IGF2 decreased in D2-MSN(GFP: 0.88 ± 0.10,sh RNA-IGF2: 0.65 ± 0.06,P = 0.06 vs GFP),but the entry numbers of D2-cre mice into the central area did not change.(8)Microinjection of AAV-DIO-h M3Dq-m Cherry into NAc actived D1-MSN activity.The expression level of c-fos increased(D1-m Cherry: 1.22 ± 0.10,D1-h M3Dq: 4.36 ± 1.27,P < 0.001 vs D1-m Cherry),and the expression level of IGF2 reduced in NAc(D1-m Cherry: 1.03 ± 0.06,D1-h M3Dq: 0.72 ± 0.05,P <0.01 vs D1-m Cherry),and the anxiety-like behavior of mice were improved,including increased the total distance of movement(D1-m Cherry: 27.83 ± 3.70 m,D1-h M3Dq: 41.32 ± 4.28 m,P <0.05 vs D1-m Cherry),the entry numbers into the central area(D1-m Cherry: 17.2 ± 3.06,D1-h M3Dq: 32.92 ± 5.34,P <0.01 vs D1-m Cherry)and the duration in central area(D1-m Cherry: 13.56 ± 2.07 sec,D1-h M3Dq: 28.98 ± 6.28 sec,P <0.05 vs D1-m Cherry).(9)Microinjection of AAV-DIOh M3Dq-m Cherry into NAc actived D2-MSN activity.The expression level of c-fos significantly increased(D2-m Cherry: 1.66 ± 0.34,D2-h M3Dq: 8.51 ± 0.56,P <0.0001 vs D2-m Cherry).Mice show anxiety-like behavior,and total distance of movement(D2-m Cherry: 20.59 ± 2.08 m,D2-h M3Dq: 4.98 ± 1.41 m,P <0.05 vs D2-m Cherry),entry numbers into the central area(D2-m Cherry: 19.8 ± 2.75,D2-h M3Dq: 2.6 ± 0.68,P <0.01 vs D2-m Cherry)and the duration in central area(D2-m Cherry: 18.05 ± 2.10 sec,D2-h M3Dq: 6.5 ± 1.69 sec,P <0.05 vs D2-m Cherry)were reduced,but the mice IGF2 expression level did not change.Conclusion: RS could induce anxiety-like behavior in mice,increase IGF2 expression in the NAc.Exogenous administration of IGF2 caused anxiety-like behavior in mice.Cell-specific reduction of IGF2 expression in D1-MSN produced anxiolytic effects.Activation of NAc D1-MSN by h M3 Dq could down-regulate the expression of IGF2 and produce anxiolytic effects,suggesting that IGF2 signaling in D1-MSN is a new target for the treatment of anxiety.