Shank3 deficiency elicits autistic-like behaviors by activating p38α in hypothalamic AgRP neurons

Background SH3 and multiple ankyrin repeat domains protein 3 (SHANK3) monogenic mutations or deficiency leads to excessive stereotypic behavior and impaired sociability, which frequently occur in autism cases. To date, the underlying mechanisms by which Shank3 mutation or deletion causes autism and the part of the brain in which Shank3 mutation leads to the autistic phenotypes are understudied. The hypothalamus is associated with stereotypic behavior and sociability. p38α, a mediator of inflammatory responses in the brain, has been postulated as a potential gene for certain cases of autism occurrence. However, it is unclear whether hypothalamus and p38α are involved in the development of autism caused by Shank3 mutations or deficiency. Methods Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and immunoblotting were used to assess alternated signaling pathways in the hypothalamus of Shank3 knockout (Shank3−/−) mice. Home-Cage real-time monitoring test was performed to record stereotypic behavior and three-chamber test was used to monitor the sociability of mice. Adeno-associated viruses 9 (AAV9) were used to express p38α in the arcuate nucleus (ARC) or agouti-related peptide (AgRP) neurons. D176A and F327S mutations expressed constitutively active p38α. T180A and Y182F mutations expressed inactive p38α. Results We found that Shank3 controls stereotypic behavior and sociability by regulating p38α activity in AgRP neurons. Phosphorylated p38 level in hypothalamus is significantly enhanced in Shank3−/− mice. Consistently, overexpression of p38α in ARC or AgRP neurons elicits excessive stereotypic behavior and impairs sociability in wild-type (WT) mice. Notably, activated p38α in AgRP neurons increases stereotypic behavior and impairs sociability. Conversely, inactivated p38α in AgRP neurons significantly ameliorates autistic behaviors of Shank3−/− mice. In contrast, activated p38α in pro-opiomelanocortin (POMC) neurons does not affect stereotypic behavior and sociability in mice. Limitations We demonstrated that SHANK3 regulates the phosphorylated p38 level in the hypothalamus and inactivated p38α in AgRP neurons significantly ameliorates autistic behaviors of Shank3−/− mice. However, we did not clarify the biochemical mechanism of SHANK3 inhibiting p38α in AgRP neurons. Conclusions These results demonstrate that the Shank3 deficiency caused autistic-like behaviors by activating p38α signaling in AgRP neurons, suggesting that p38α signaling in AgRP neurons is a potential therapeutic target for Shank3 mutant-related autism. Supplementary Information The online version contains supplementary material available at 10.1186/s13229-024-00595-4.


Background
Autism spectrum disorders (ASD) are associated with severe stereotypical behavior and social impairments [1] and primarily caused by gene mutations.Shank3 mutation or deficiency frequently occurs in autism cases [2][3][4].Shank3 deficiency impairs the structure and function of synapses, which affects the neural networks that are vital for individuals, thereby leading to ASD [4][5][6].Indeed, restored SHANK3 expression in animal models reverses some of the deficits in synaptic function and autistic-like behaviors [3].Notably, Shank3 is a core excitatory postsynaptic protein that regulates synaptic function in multiple brain regions including the medial prefrontal cortex (mPFC), striatum, and hippocampus [7][8][9].Shank3altered mice and children with autism often have eating disorders [3,10,11], and the center of feeding regulation is hypothalamus [12].However, the role of the hypothalamus in Shank3 deletion or mutation-caused autism is still poorly understood.
AgRP neurons are a class of neurons located in the arcuate nucleus of the hypothalamus and primarily recognized for controlling feeding and energy metabolism [13].Substantial literatures have shown that AgRP neurons are implicated in regulating core symptoms of autism beyond feeding.For example, activation of AgRP neurons in the absence of food drives stereotypic behaviors [14].Consistently, the activation of melanocortin 4 receptor (MC4R), a downstream molecular of AgRP, induces excessive stereotypic behaviors [15].On the other hand, AgRP neurons also participate in regulating social behaviors due to their critical role in controlling the structure and function of the mPFC [16,17].Notably, AgRP neurons are 5-hydroxytryptamine (5-HT) receptor-positive neurons, which are strongly associated with autism [18][19][20].Thus, whether AgRP neurons are associated with the Shank3 deficiency-caused autism is still unknown.
Previous studies have shown that 5-HT transporter (SERT) mutations cause alterations in SHANK3 signaling pathway, and inhibition of p38α cures autistic-like behaviors in SERT mutant mice [19,21].p38α, a member of the mitogen-activated protein kinase (MAPK) family, is involved in various cellular processes, including inflammation, cell differentiation, and response to stress [22].In particular, p38α is a key mediator of inflammatory responses in the brain, which has been postulated as a potential risk factor for certain cases of autism occurrence [23][24][25].Indeed, alterations in the upstream or downstream signaling of p38α affect autistic symptoms [26][27][28].Of note, p38α in 5-HT neurons drives autisticlike phenotypes directly in the ASD model [18,19].However, it is still unknown whether and how p38α plays a crucial role in ASD development due to its wide expression in multiple tissues and differential functions [29][30][31].
Here, we demonstrate that hypothalamic p38 is activated in Shank3 deficiency mice.Overexpression of p38α in ARC or activating p38α in hypothalamic AgRP neurons elicits autistic behaviors.Conversely, inactivating p38α in AgRP neurons ameliorates autistic behaviors in Shank3 −/− mice.Our results demonstrate that p38α signaling in AgRP neurons is one of the SHANK3 downstream pathways in controlling autistic behaviors.

Genotyping
Genotyping was conducted as previously report [33].In brief, 2 mm mouse tails were harvested at 3 weeks of age.Primers are as follow: For p38α flox/flox : Forward

Home-cage monitoring test
Mice were kept in individual cages for behavior monitoring, with a 12 h light/12 hours dark cycle and an ambient temperature of 24 ± 2 °C in a silent room.Mice were acclimated to the cages for 24 h before starting records, and their behaviors were recorded for 24 h from 7 postmeridiem to 7 postmeridiem the next day.Fluorescent lights simulate daytime and lights off simulate night, without disrupting the normal circadian rhythm of mice.Mice were monitored by an infrared camera (Shanghai Vanbi Intelligent Technology Co., Ltd.) mounted horizontally on the side of the cage for 24 h.Video data were analyzed by Tracking Master V3.1.56software (Shanghai Vanbi Intelligent Technology Co., Ltd.), and behavioral definitions were as described previously [34].Tracking Master V3.1.56software monitored parameters as follows: contour erosion (2 pixels), contour expansion (2 pixels), animal size (500-10000 pixels), motionless (0-4 cm/s), active ( > = 4 cm/s), slow active (4-10 cm/s), fast active ( > = 20 cm/s), move between two frames (1-10000 pixels) [35].

Three chamber tests
The test was performed as previously described [36].Before the three-chamber test, each mouse was acclimatized in three chambers for five minutes, and three hours later, the first and second phases of the experiment were conducted.In the first stage, an unfamiliar mouse (stranger 1) was placed in chamber (1) The mouse was placed in the middle chamber and allowed to travel freely in the three chambers.The trajectory of mice was recorded by an automatic video tracking system (Tracking Master V3.0, Shanghai Vanbi Intelligent Technology Co., Ltd.) for ten minutes and the time spent in each chamber was counted.In the second phase of the experiment, the mouse in chamber 1 from the first phase was maintained, while a new unfamiliar mouse (stranger 2) was placed in chamber (2) Again, the trajectories of mice were recorded for ten minutes, and the time spent in each chamber was counted.

Transcriptomic analysis
Transcriptomic data of primary neurons transduced with Shank3 short hairpin ribonucleic acid (shRNA, GSE47150) and hypothalamus in Shank3-overexpressing (Shank3 TG ) mice (GSE120609) were downloaded from https://www.ncbi.nlm.nih.gov.Data were imported into the R-package 'limma' for differential gene screening.Fold change was set at 1.2.Then, the KEGG pathways were enriched by 'enrichKEGG' in the R-package 'clus-terProfiler' .These pathways were sorted by count and selected the first 20 pathways for presentation.

Lipopolysaccharide (LPS) challenge assay
LPS was used to activate hypothalamic p38α [37,38], the mouse was intraperitoneally injected with 1 mg/kg LPS for 30 min, and then the hypothalamus was collected for immunofluorescence staining.

Hypothalamic p38 is activated in ASD mouse model
Shank3 may play a role in the hypothalamus due to the altered food intake in Shank3 deletion and overexpression mice [3,10].Notably, the hypothalamus regulates not only feeding behaviors but also stereotypic behavior and sociability [14,16,39], the latter being the typical symptoms of autism [1].Thus, it raises a possibility that the hypothalamus may be an important brain site in Shank3-related autism.To explore whether the hypothalamus is involved in the regulation of Shank3 deletion-caused autistic-like behaviors, we analyzed the alternations of signaling pathways profiles in Shank3 knockdown primary cortical neurons (GSE47150) [40] and hypothalamus of mice with overexpressing Shank3 (GSE120609), respectively (Fig. 1A, B) [41].Of note, the overlapping of KEGG pathways enrichment from the two datasets showed that the phosphoinositide 3-kinase (PI3K/Akt) signaling pathway and MAPK signaling pathway are significantly altered (Fig. 1A, B, C).Indeed, the PI3K/Akt and MAPK signaling pathways are strongly associated with neurodevelopment in children with autism [18,19,42,43].Although there is a strong correlation between MAPK and PI3K/Akt signaling pathways and autism [21,44], we are more interested in whether MAPKs pathway are involved in regulating Shank3-associated autism.
Next, we determined the phosphorylation levels of typical MAPKs [45]: p38, ERK1/2, and JNK in the hypothalamus by immunoblotting.Shank3 −/− mice displayed higher levels of p-p38, p-Erk1/2, and p-JNK compared to WT mice (Fig. 1C), indicating an inhibitory effect of SHANK3 on the MAPK pathway.Consistently, substantial literatures have shown that SHANK3 in synaptic components is regulated via signaling cascades p38α [19,46] and ERK2 [47].Notably, the level of hypothalamic p-p38α was also higher in BTBR mice (another ASD mouse model) than in WT mice (Fig. 1D).Furthermore, other labs and our previous studies found that the mutation of Shp2, an upstream molecule of p38α, leads to Noonan syndrome, accompanied by obesity and autism symptoms [26,28,48].Inhibition of MAP kinase interacting kinase (MNK1/2), a downstream effector of p38α, restores social behavior in ASD mouse model [27].Thus, these findings raise a possibility that hypothalamic p38α is a possible downstream molecule of SHANK3 to regulate stereotypic behavior and sociability.

p38α overexpression in ARC is sufficient to elicit excessive stereotypic behavior and impaired sociability in WT mice
Since ARC in the hypothalamus is a regulatory nucleus for behaviors [14,16,39].Next, we determined the expression of p38α by immunostaining and found that p38α highly expressed in ARC neurons (Fig. 2A).However, whether p38α in ARC neurons is sufficient to regulate stereotypic behavior and sociability is still unknown.To address this issue, we generated p38α ARC−OE mice, which allows for overexpressing p38α in ARC (Fig. 2B).As expected, p38α ARC−OE mice showed an increased trend in stereotypic behavior (Fig. 2C, D) and impaired sociability (Fig. 2E) compared to control mice, accompanied by an impaired trend of preference for social novelty (Fig. 2F).Total distance and activity time were unchanged (Fig. 2G, H).Thus, these results demonstrate that overexpressing p38α in ARC is sufficient to elicit excessive stereotypic behavior and impaired sociability.

Overexpression of p38α in AgRP neurons elicits excessive stereotypic behavior and impaired sociability in WT mice
Previous studies unveiled that AgRP neurons in ARC regulate stereotypic behavior and sociability [14,16,39].Thus, these findings raise a possible regulatory effect of p38α in AgRP neurons on stereotypic behavior and sociability.For this reason, we first examined the expression of p38α in AgRP neurons and found that AgRP neurons highly express p38α (Fig. 3A).Next, we generated p38α AgRP−OE mice by expressing AAV9-p38α flox/flox in Agrp-cre mice, which allows for p38α specifically overexpressing in AgRP neurons (Fig. 3B).p38 AgRP − OE mice showed a significant increase in stereotypic behavior (Fig. 3C, D), sociability and preference for social novelty were dramatically impaired (Fig. 3E, F), accompanied by unaltered total distance and activity time (Fig. 3G, H).Consequently, these results demonstrate that p38α in AgRP neurons is sufficient to elicit excessive stereotypic behavior and impaired sociability.

Activated p38α in AgRP neurons elicits excessive stereotypic behavior and impaired sociability in WT mice
Since the levels of hypothalamic p-p38α were upregulated in Shank3 −/− mice (Fig. 1C), we next determined whether the activation of p38α is sufficient to regulate stereotypic behavior and sociability.For this, we generated a p38α AgRP−176/327 mouse line by p38α D176A−F327S flox knock-in mice (Fig. S3A) crossed with Agrp-Cre mice, in which D176A and F327S mutations lead to spontaneous and sustained activation of p38α in AgRP neurons [49] (Fig. 4A).Similar to p38α AgRP−OE mice, p38α AgRP−176/327 Groom time and bouts in 24 h of p38α ARC−con and p38α ARC−OE mice.E. The first phase of the three-chamber test, the time of p38α ARC−con and p38α ARC−OE mice in stranger1 chamber.F. The second phase of the three-chamber test, the time of p38α ARC−con p38α ARC−OE mice in stranger2 chamber.G.Total distance in 24 h of p38α ARC−con and p38α ARC−OE mice.H. Activity time in 24 h of p38α ARC−con and p38α ARC−OE mice.10-week-old WT male mice were injected with virus.The injection site is in the ARC area as an inclusion criterion.12 injected mice in the p38α ARC−con group, 3 mice were excluded and 9 mice were included in the experiment.11 injected mice in the p38α ARC−OE group, 3 mice were excluded and 8 mice were included in the experiment.Home-Cage monitoring test was performed 6 weeks after virus injection, three-chamber test was performed 8 weeks after virus injection.Statistical analysis: data were analyzed using unpaired two-tailed Student's t-test (Prism9, GraphPad Software Inc.).Data were represented as Mean ± SD.Significance levels are indicated with *p < 0.05.The first phase of the three-chamber test, the time of p38α AgRP−con and p38α AgRP−OE mice in stranger1 chamber.F. The second phase of the three-chamber test, the time of p38α AgRP−con and p38α ARC−OE mice in the stranger2 chamber.G.The total distance in 24 h of p38α AgRP−con and p38α AgRP−OE mice.H.The activity time in 24 h of p38α AgRP−con and p38α AgRP−OE mice.10-week-old control and AgRP-Cre male mice were injected with AAV9-p38α flox/flox .The injection site is in the ARC area as an inclusion criterion.12 injected mice in the p38α AgRP−con group, 4 mice were excluded and 8 mice were included in the experiment.12 injected mice in the p38α AgRP−OE group, 3 mice were excluded and 9 mice were included in the experiment.Home-Cage monitoring test was performed 4-6 weeks after AAV injection, three-chamber test was performed 8-10 weeks after AAV injection.Statistical analysis: data were analyzed using unpaired two-tailed Student's t-test (Prism9, GraphPad Software Inc.).Data were represented as Mean ± SD.Significance levels are indicated with *p < 0.05.mice displayed a significant increase in stereotypic behavior (Fig. 4B, C), slightly elevate total distance, increased activity time (Fig. 4D, E), and impaired sociability (Fig. 4F) accompanied by an impaired trend of preference for social novelty (Fig. 4G).Together, activation of p38α in AgRP neurons causes excessive stereotypic behavior and impaired sociability.
To investigate if the p38α activity in AgRP neurons is necessary for regulating stereotypic behavior and sociability, we generated a p38α AgRP−180/182 mouse line by p38α T180A−Y182F flox knock-in mice (Fig. S3B) crossed with Agrp-Cre mice, in which we specifically inactivated p38α in AgRP neurons by mutating residues both T180A and Y182F in p38α [50] (Fig. S4A, B).Consistent with the results of p38α deletion in AgRP neurons, p38α AgRP−180/182 mice displayed an unchanged stereotypic behavior (Fig. S4C, D), similar total distance (Fig. S4E), unaltered activity time (Fig. S4F), and unchanged sociability (Fig. S4G, H) compared to p38α AgRP−con mice.These results demonstrate that activation of p38α in AgRP neurons is not necessary for regulating stereotypic behavior and sociability in WT mice.mice and immunofluorescence staining of p-p38α (green, 488 nm, FITC).B and C, the groom time and bouts in 24 h of p38α AgRP-con and p38α AgRP-176/327 mice.D. the total distance in 24 h of p38α AgRP-con and p38α AgRP-176/327 mice.E. the activity time in 24 h of p38α AgRP-con and p38α AgRP-176/327 mice.F. the first phase of the three-chamber test, the time of p38α AgRP-con and p38α AgRP-176/327 mice in stranger1 chamber.G. the second phase of the three-chamber test, the time of p38α AgRP-con and p38α AgRP-176/327 mice in stranger2 chamber.p38α AgRP-con and p38α AgRP-176/327 male mice were used for the experiment, 8 mice in p38α AgRP-con and 9 mice in p38α AgRP-176/327 group.Mice were subjected to Home-Cage monitoring test at the age of 8-9 weeks, and subjected to threechamber test at the age of 9-10 weeks.Statistical analysis: data were analyzed using unpaired two-tailed Student's t-test (Prism9, GraphPad Software Inc.).Data were represented as Mean ± SD.Significance levels are indicated with *p < 0.05.

p38α in POMC neurons does not regulate stereotypic behavior and sociability
Since the POMC neuron is another primary neuron in ARC [13], we next explored whether POMC neurons are involved in regulating stereotypic behavior and sociability.For this reason, we generated a p38α POMC-176/327  mouse line (Fig. 6A) by p38α D176A-F327S flox knock-in mice crossed with POMC-Cre mice, in which p38α is spontaneously constitutively activated in POMC neurons due to mutating in p38α residues both D176A and F327S [49].Activated p38α in POMC neurons did not cause significant changes in stereotypic behavior (Fig. 6B, C) and social behavior (Fig. 6D, E), accompanied by unchanged total distance and activity time (Fig. 6F, G).Together, p38α in POMC and AgRP neurons play distinct roles in regulating autism-like behaviors (Fig. 6H).

Discussion
Shank3 is a critical gene for autism development [2].Understanding the neuronal and molecular mechanisms of Shank3-related autism could help in the treatment of some subset of autism.Here, we demonstrate that deletion of Shank3 promotes p38α signaling to elicit autistic-like behaviors.Furthermore, activated p38α in AgRP neurons elicits excessive stereotypic behavior and impaired sociability in WT mice, and inactivated p38α improves autistic-like behaviors in Shank3 -/-mice.Our results suggest that the internal SHANK3/p38α signaling pathway in AgRP neurons is an important signaling transduction cascade for autistic behaviors, as elucidated in Fig. 6H.
In this work, we unveiled an unexpected role for AgRP neurons in controlling stereotypic behavior and sociability behaviors via p38α signaling.AgRP neurons control non-feeding behaviors by affecting the medial prefrontal cortex and regulate feeding and satiety via expressing serotonin1B receptors (5-HT 1B R) [20].Notably, p38α in 5-HT neurons drives autistic-like phenotypes in ASD model [18,19] and 5-HT receptors are the therapeutic target of multiple antipsychotic medications [51], suggesting that 5-HT receptors are the downstream effector of SHANK3/p38α pathway.Consistently, inhibition of MNK1/2, a downstream of p38α, restores social behavior in ASD mouse model [27].Thus, the p38α signaling is a Fig. 6 Activated p38α in POMC neurons does not affect stereotypic behavior and sociability.A. schematic of generating p38α POMC-176/327 mice.B and C, the groom time and bouts in 24 h of p38α POMC-con and p38α POMC-176/327 mice.D. the first phase of the three-chamber test, the time of p38α POMC-con and p38α POMC-176/327 mice in stranger1 chamber.E. the second phase of the three-chamber test, the time of p38α POMC-con and p38α POMC-176/327 mice in stranger2 chamber.F. the total distance in 24 h of p38α POMC-con and p38α POMC-176/327 mice.G. the activity time in 24 h of p38α POMC-con and p38α POMC-176/327 mice.H. Model of p38α activity associated with stereotypic behavior and sociability.p38α POMC-con and p38α POMC-176/327 male mice were used for experiment, 9 mice in p38α POMC-con and 12 mice in p38α POMC-176/327 group.Mice were subjected to Home-Cage monitoring test at the age of 8-9 weeks, and subjected to three-chamber test at the age of 9-10 weeks.Statistical analysis: data were analyzed using unpaired two-tailed Student's t-test (Prism9, GraphPad Software Inc.).Data were represented as Mean ± SD.Significance levels are indicated with *p < 0.05.potential target for developing drugs for treating autism, especially for the Shank3-related subset population.
Inactivation of p38α in AgRP neurons did not significantly promote the groom bouts of Shank3 -/-mice (Fig. 5C), indicating that other signaling pathways are associated with the regulation of stereotypic behaviors.Shank3 deficiency leads to abnormalities in brain structure and molecular signaling pathways, suggesting that the effects of Shank3 deficiency are widespread, multifarious, and profound [52].Thus, modulation of a specific molecular pathway in AgRP neurons is impossible to rescue the complete effects of Shank3 deficiency.
Stereotypic behavior and sociability in WT mice are unchanged by the deficiency or inactivation of p38α in AgRP neurons (Figs.S1, S2, S4).There are several possible reasons for these results.First, WT mice hardly exhibit the improved effect on stereotypic behavior and sociability, as evidenced by the inactivation of p38α in AgRP neurons improves the autistic-like behaviors in Shank3 -/-mice (Fig. 5).The second possible reason is the presence of functionally redundant p38 isoforms or intracellular compensatory MAPK subfamilies similar to a previous report [9].Consistently, our immunoblotting data showed that Erk1/2 and JNK phosphorylation levels were also upregulated in the hypothalamus of Shank3 -/- mice (Fig. 1C).Thus, we cannot rule out the possibility that Erk1/2 and JNK act as a regulator for stereotypic behavior and sociability.Indeed, Erk signaling is essential for neural development in the brain [53] and ERK2 regulates SHANK3 stability in vivo [47].Furthermore, the JNK pathway appears to be associated with the autistic population with intellectual disability [54].The third possibility is that multiple types of neurons are orchestrated to regulate stereotypic behavior and sociability.Indeed, ARC contains multiple types of neurons and glial cells [13,55].Finally, it is also possible that all or some of these reasons are orchestrated to regulate autistic-like behaviors.

Limitations
This paper demonstrated that SHANK3 regulates the phosphorylated p38 level in the hypothalamus and inactivated p38α in AgRP neurons significantly ameliorates autistic behaviors of Shank3 −/− mice.However, we did not clarify the relationship between SHANK3 and p38α in AgRP neurons.Although the level of p38α phosphorylation was also elevated in the hypothalamus of BTBR mice, we did not determine whether controlling the activity of p38α in AgRP improved autistic-like behaviors in BTBR mice.Therefore, further experiments are required to identify whether the activity of p38α in AgRP neurons modulates all types of autism.

Conclusions
Our results unveil that p38α signaling in AgRP neurons plays a critical role in the development of autism, particularly in the SHANK3 pathway mutant-related population.These findings will provide unique insights into the molecular mechanism for autistic behaviors and better therapeutic targets for autism.

Fig. 1 Fig. 2
Fig. 1 Phosphorylated p38 level in the hypothalamus is significantly enhanced in ASD mouse model.A. KEGG enrichment analysis of the transcriptome from Shank3 knockdown primary neurons (GSE47150).B. KEGG enrichment analysis for the hypothalamus of Shank3TG mice (GSE120609).C. Overlapping of signaling pathways of two datasets, the number represents the well-defined pathways, and non-defined pathways were excluded.D. Immunoblotting and intensity of phosphorylation of p38, ERK1/2, and JNK in hypothalamus from WT and Shank3 −/− mice (n = 3 per group).E. Immunoblotting and intensity of phosphorylated p38, ERK1/2, and JNK in hypothalamus from WT, BTBR mice (n = 3 per group).Statistical analysis: data were analyzed using unpaired two-tailed Student's t-test (Prism9, GraphPad Software Inc.).Data were represented as Mean ± SD.Significance levels are indicated with *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 3
Fig.3Overexpression of p38α in AgRP neurons elicits excessive stereotypic behavior and impaired sociability in WT mice. A. Immunofluorescence staining of DAPI (blue, 405 nm) and p38α (green, 488 nm, FITC) in ARC, AgRP-tomato mice were generated by mating Agrp-Cre mouse with Tomato-reporter mouse (red, 561 nm).B. Schematic and immunofluorescence image of the AAV9-p38α flox/flox stereotaxic injection to ARC in Agrp-Cre mice, GFP represents virus expression.C and D, Groom time and bouts in 24 h of p38α AgRP−con and p38α AgRP−OE mice.E. The first phase of the three-chamber test, the time of p38α AgRP−con and p38α AgRP−OE mice in stranger1 chamber.F. The second phase of the three-chamber test, the time of p38α AgRP−con and p38α ARC−OE mice in the stranger2 chamber.G.The total distance in 24 h of p38α AgRP−con and p38α AgRP−OE mice.H.The activity time in 24 h of p38α AgRP−con and p38α AgRP−OE mice.10-week-old control and AgRP-Cre male mice were injected with AAV9-p38α flox/flox .The injection site is in the ARC area as an inclusion criterion.12 injected mice in the p38α AgRP−con group, 4 mice were excluded and 8 mice were included in the experiment.12 injected mice in the p38α AgRP−OE group, 3 mice were excluded and 9 mice were included in the experiment.Home-Cage monitoring test was performed 4-6 weeks after AAV injection, three-chamber test was performed 8-10 weeks after AAV injection.Statistical analysis: data were analyzed using unpaired two-tailed Student's t-test (Prism9, GraphPad Software Inc.).Data were represented as Mean ± SD.Significance levels are indicated with *p < 0.05.

Fig. 4
Fig.4 Activated p38α in AgRP neurons elicits excessive stereotypic behavior and impaired sociability in WT mice. A. schematic of generating p38αAgRP-176/327  mice and immunofluorescence staining of p-p38α (green, 488 nm, FITC).B and C, the groom time and bouts in 24 h of p38α AgRP-con and p38α AgRP-176/327 mice.D. the total distance in 24 h of p38α AgRP-con and p38αAgRP-176/327 mice.E. the activity time in 24 h of p38α AgRP-con and p38α AgRP-176/327 mice.F. the first phase of the three-chamber test, the time of p38α AgRP-con and p38αAgRP-176/327 mice in stranger1 chamber.G. the second phase of the three-chamber test, the time of p38α AgRP-con and p38α AgRP-176/327 mice in stranger2 chamber.p38α AgRP-con and p38α AgRP-176/327 male mice were used for the experiment, 8 mice in p38α AgRP-con and 9 mice in p38α AgRP-176/327 group.Mice were subjected to Home-Cage monitoring test at the age of 8-9 weeks, and subjected to threechamber test at the age of 9-10 weeks.Statistical analysis: data were analyzed using unpaired two-tailed Student's t-test (Prism9, GraphPad Software Inc.).Data were represented as Mean ± SD.Significance levels are indicated with *p < 0.05.
22 ± 1 °C) and humidity (~ 40%) in a 12 h light/12 hours dark cycle, with free access to food and water.Mice were used for experiments at 10-12 weeks of age.All animal experiments were permitted by the Institutional Animal Care and Use Committee at Shandong Provincial Hospital and complied with the China National Regulations on the Administration of Experimental or Laboratory Animals (No.2, 20,170,301, SSTC, China), and the ARRIVE guidelines or the U.K.