Effect And Mechanism Of Insular Oxytocin On Social Behavior Of SCN1A Gene Knockout Heterozygote Mice

Objective To study the effect and the possible mechanism of exogenous oxytocin for social interaction behaviors in the SCN1 A gene knockout heterozygous mice.In order to provide new animal experimental evidence for the clinical diagnosis and treatment of autism.Methods 1.The F1 generation mice were genotyped by DNA gene identification.2.The social interaction disorder and anxiety-like behavior of SCN1 A KO heterozygous mice were verified by three-box interaction test and open field test.3.These mice were randomly assigned according to the different drug to be injected: Normal saline wild type group(WT),normal saline SCN1 A +/-KO group(KO),oxytocin SCN1 A +/-KO group(O),oxytocin + baclofen SCN1 A +/-KO group(O+B),oxytocin + CGP35348 SCN1 A +/-KO group(O+C),baclofen SCN1 A +/-KO group(B),and CGP35348 SCN1 A +/-KO group(C),with 10 rats in each group.Oxytocin: 10 mg/kg,Baclofen: 2mg/kg,CGP35348: 100 mg/kg.All these drugs that used in the experiment were diluted with saline.The total intraperitoneal dose of the diluted drug solution was 2 ml per kilogram body weight.All these NS groups,every mouse was only given 2 ml saline per kilogram of body weight by intraperitoneally injecting.Mice from PND22 to PND24 were given the drug intraperitoneally for three days.The behavioral experiment was performed 1 hour after the injection of the drug in each mouse.All the behavioral experiments took place between 8 p.m.and 12 p.m.4.The plasma BDNF concentrations of every mouse was measured by the mouse BDNF ELISA kit.5.Morphological number of insular neurons was observed by Nissl staining.6.IF and WB were used to measure the expressions of oxytocin receptor protein,GABAb receptor protein,CREB and p-CREB in insula and hippocampusResults 1.SCN1 A +/-KO mice and WT mice were obtained by DNA gene identification.2.In the three-box interaction experiment,SCN1 A +/-KO mice had no novelty preference,while their wild-type sibling mice had novelty preference.The total time of SCN1 A +/-KO mice in the two mice regions was reduced compared with that of the wild-type mice(253.1 ± 23.32 s VS 344.6 ± 19.52 s,p = 0.010).They show problems with social interaction.The total amount of self-grooming of these SCN1 A +/-KO mice was more than those WT mice(269.9 ± 24.84 VS 104.7 ± 9.86,p = 0.001),showing an increase in stereotyped movements.In open field experiments,SCN1 A +/-KO mice spent less time in the intermediate region than their wild-type counterparts(36.33 ± 8.47 s VS 104.8 ± 21.06 s,p = 0.010).SCN1 A +/-KO mice reared more times(60.8 ± 12.7 s VS 23.7 ± 8.4 s,p = 0.034),showing anxiety-like behavior.SCN1 A +/-KO mice had a lower travel distance than their wild-type counterparts(2294.0 ± 184.1 cm VS 4322.0 ± 797.2 cm,p = 0.042),indicating a decreased ability of motor exploration.3.After the drug intervention,SCN1 A +/-KO mice in the oxytocin group,SCN1 A +/-KO mice in the oxytocin + baclofen group,and SCN1 A +/-KO mice in the baclofen group spent more time with Stranger 2 in the three-box interaction experiment,showing a novelty preference.SCN1 A +/-KO mice in the CGP35348 group spent no more time in the stranger domain(Stranger 2)than they did in the relatively familiar domain(Stranger 1)SCN1A +/-KO mice in the oxytocin group,SCN1 A +/-KO mice in the oxytocin + baclofen group,SCN1 A +/-KO mice in the baclofen spent more time in the two mice area than normal saline group,showing that its social communication disorders improved.The time of SCN1 A +/-KO mice in the oxytocin + CGP35348 group and CGP35348 group showed no more than the NS group.The total number of self-grooming in SCN1 A +/-KO mice in the oxytocin group,SCN1 A +/-KO mice in the oxytocin + baclofen group and SCN1 A +/-KO mice in the baclofen group was less than that in the normal saline group,indicating a decrease in the stereotypical repetitive movements.SCN1 A +/-KO mice licked and groomed more in the baclofen group than in the oxytocin + baclofen group,but the difference was not statistically significant.4.In the open field experiment,SCN1 A +/-KO mice in the oxytocin group,SCN1 A +/-KO mice in the oxytocin + baclofen group,and SCN1 A +/-KO mice in the baclofen group,whom spent more time in central zone than those in the NS group,indicating an improvement in their anxiety-like behavior.Oxytocin + Baclofen group spent more time in central region than the oxytocin group(p = 0.041),showing the synergistic effect.SCN1 A +/-KO mice in the CGP35348 group did not spend more time in the central region than those in the NS group.The total moving distance of SCN1 A +/-KO mice in the oxytocin group,and CGP35348 group and the oxytocin + CGP35348 group was more than the NS group,and the moving distance of the oxytocin + CGP35348 group was more than the CGP35348 group.Both oxytocin and CGP35348 promoted the movement of the mice.SCN1 A +/-KO mice in the oxytocin + baclofen group and in the baclofen group did not increase the running distance compared with SCN1 A +/-KO mice in NS group.The total rearing number of SCN1 A +/-KO mice in the oxytocin group,SCN1 A +/-KO mice in the oxytocin + baclofen group and SCN1 A +/-KO mice in the baclofen group,which was less than the NS group.The rearing number in the oxytocin + baclofen group were not less than the oxytocin group.SCN1 A +/-KO mice in the oxytocin + CGP35348 group and SCN1 A +/-KO mice in the CGP35348 group did not stand up more than the NS group.5.The plasma concentrations of(BDNF)of the NS WT group,SCN1 A +/-KO mice in the oxytocin group,SCN1 A +/-KO mice in the oxytocin + baclofen group,and SCN1 A +/-KO mice in the baclofen group were less than the NS group.SCN1 A +/-KO mice in the oxytocin + baclofen group was lower than the oxytocin group(p = 0.014).Both oxytocin and baclofen can reduce plasma BDNF concentration in SCN1 A +/-KO mice,and the combination of the two indicated synergistic effect.The concentrations of oxytocin + CGP35348 group and CGP35348 group in SCN1 A +/-KO mice were more than the NS group in SCN1 A +/-KO mice.The results implied that CGP35348 increased plasma BDNF concentrations in SCN1 A +/-KO mice.6.The number of insular neurons in SCN1 A +/-KO mice were less than its litter wild-type mice.After treated with oxytocin and baclofen,the number of insular neurons in SCN1 A +/-KO mice increased.7.The relative amount of oxytocin receptor protein and GABAb receptor protein in insula and hippocampus of SCN1 A +/-KO mice compared with wild-type mice of their lair were decreased;The amount of receptor protein increased after the administration of receptor agonists and decreased after the administration of receptor inhibitors.Immunofluorescence localization showed that both oxytocin receptor and GABAB receptor were membrane receptors.8.The amount of CREB and p-CREB of SCN1 A +/-KO mice was more than the WT group in insula and hippocampus.Oxytocin,baclofen,and CGP35348 did not increased the amount of CREB and in the insula and hippocampus of SCN1 A +/-KO mice.But the amount of p-CREB and in the insula and hippocampus of SCN1 A +/-KO mice increased.The IF density also showed the same trend as that of WB.Conclusion 1.SCN1 A +/-KO mice showed social dysfunction and anxiety-like behavior.2.Oxytocin can improve the social interaction disorder of SCN1 A +/-KO mice,and the combination of oxytocin and baclofen has a synergistic effect,while CGP35348 has an antagonistic effect.Oxytocin can improve the anxiety-like behavior of SCN1 A +/-KO mice,and the combination of oxytocin and baclofen has a synergistic effect.The insular as well as the hippocampus may be involved in the pathophysiological process of autism.3.Oxytocin,Baclofen and CGP35348 may exert biological effects by regulating CREB and its phosphorylation through G-protein coupled receptors,but further research is needed.