| Autism spectrum disorder(ASD)is a severe behavioral disorder characterized by pervasive impairments in social interactions,deficits in verbal and nonverbal communication,and stereotyped,repetitive patterns of behavior and interests(Diagnostic and Statistical Manual of Mental Disorders-?,DSM-?)(Makushkin,Makarov,& Pashkovskiy,2019).According to the World Health Organization(WHO),the number of autistic patients in China increasing rapidly and reached one million.the autistic patients lack the ability to take care of themselves,which has caused a great burden to the family and the society.The cause of autism is not clear,however,many reports claimed that autism is caused by genetic and environmental interaction.Studies from monozygotic twins and heterozygotic twins have found that the incidence of homozygous twins and heterozygotic twins is 60% and 5%,respectively.These studies show that genetic factors play an important role in the pathogenesis of autism(Masi,DeMayo,Glozier,& Guastella,2017).On the other hand,in a study of 192 pairs of twins,it was found that genetic factors account for 37% of the incidence of autism,while environmental factors account for 55%(Hallmayer et al.,2011),indicating that environmental factors may also play an important role in the pathogenesis of autism.In the aspect of environmental factors,some studies have found that many factors are related to the onset of autism,such as brain organic lesions(prefrontal lobe damage),neurobiochemical factors(environmental toxicants such as mercury,lead,arsenic),pregnancy and childbirth diseases(gestational diabetes),infection(gestational infection with valproic acid)and immunity(maternal immune response caused by pregnancy infection virus)(Sandin et al.,2014).Therefore,currently there is no effective specific drug treatment available for autism.At present,the intervention of autism is mainly through daily training and behavior intervention to improve social adaptability and certain self-care ability of patients.Therefore,it is urgent and important to find out the etiology and mechanism of autism for the prevention,diagnosis and treatment of autism.In 2013,DSM-V,the fifth edition of diagnostic and Statistical Manual of mental disorders,changed the three characteristics of autistic patients described by DSM-IV into two characteristics: social interaction disorder;limited interest and repetitive stereotyped behavior(DSM-?,2013).Normal social behavior plays an important role in individual's social and environmental adaptation,while autistic patients are difficult to integrate into the group due to their social barriers,so they can't adapt to the society.Although the researchers have done some behavioral descriptive research on the social deficit behaviors of autistic patients,they still know little about the mechanism behind the social deficit behaviors of autistic patients,and need further research.Repetitive stereotyped behavior includes "focusing on one or more stereotyped and limited interest patterns,which are abnormal in intensity and focus".This is usually manifested as "object solidification" behavior enhancement and "excessive attachment to favorite objects".This "object solidification" behavior may interfere with social,emotional and memory functions,complicating the relationship between autism symptoms.However,the pathological mechanism that leads to the enhancement of "object solidification" behavior of autism is not well understood,which needs to be further explored.Although it is the most direct method to study the patients with loneliness,there are technical limitations and moral and ethical problems in the direct study of human subjects.It shows some advantages to explore the pathological mechanism of autism by using animal models for preclinical research.It can be seen that it is an effective way to find out the prevention and treatment of autism disease to build an animal model that can well imitate autism and further explore the pathological mechanism of autism.Therefore,in the present study,we used non genetic method to construct the autism animal model using valproic acid exposure in pregnant mice.On the basis of verifying the validity of the two core symptoms of simulated autism: social defect and limited repetitive stereotyped behavior investigated.Further,the brain basis of the abnormal expression of 5-HT receptor in autistic rats was explored from the perspective of 5-hydroxytryptamine development,and the specific mechanism of 5-HT1 A receptor was studied.When pregnant rats are exposed to valproate acid(VPA),their offspring will show similar social defects to those of autistic patients(T.Schneider & R.Przew?ocki,2005).However,whether this method can simulate another core symptom of autism,repetitive stereotyped behavior,needs further studies.Research on human autism patients found that autism generally starts before the age of 3 and lasts for a lifetime,and with the advancement of time,the symptoms of autism patients will become more and more serious(Varghese et al.,2017).Whether,the animal model constructed by exposure to valproic acid during pregnancy become more and more serious with the increase of age? To address these two problems,we used Schneider's exposure to valproic acid on the 12.5th day of gestation to induce the autism like behavior in offspring.Specifically,the first study attempts to solve:(1)whether exposure to valproic acid during pregnancy is effective in simulating the two core symptoms of autism;social defects and "object solidification" behavior enhancement;(2)whether the symptoms of autism like rats produced by valproic acid change with age in the early adolescence and adolescence.As early as 1961,schain and Freedman found that the 5-hydroxy tryptamine(5-HT)system in autistic patients was abnormal(Schain & Freedman,1961).It is found that 5-hydroxytryptamine concentration in peripheral platelets is very high in about one third of autistic children(Nickl-Jockschat & Michel,2011).Therefore,the abnormality of serotonin system may be one of the causes of autism.5-HT is an important neurotransmitter,which can regulate many physiological processes of animals(such as reproduction,emotion,attack,pain,appetite vasoconstriction,hormone release,etc.).Through the regulation of these physiological functions,the behavior can be regulated.The serotonin system dominates almost all X areas of the brain.Tryptophan(TP)is the precursor of 5-HT biosynthesis in the brain.TP generates 5-hydroxy tryptophan(5-HTP)under the action of tryptophan hydroxylase(TPH),and 5-HT under the action of 5-thp decarboxylase(5-htpdc).The 5-HT synthesized in the cytoplasm was absorbed(bound to 5-HT binding protein)and stored in the vesicles of 5-HT nerve endings.5-HT released into the synaptic space binds to the receptor and plays a role.Central 5-HT neurons are widely distributed in the prefrontal cortex,including the prefrontal cortex.5-HT receptors are widely distributed in the cerebral cortex,limbic system,hippocampus and other regions,and participate in various cognitive functions of the brain.Many researchers believe that 5-HT receptor function may be related to the occurrence of autism.There are 14 subtypes of 5-HT receptor in the brain.Whether the expression of each subtype of 5-HT receptor in the brain of autistic patients is abnormal is still unclear.Among them,5-HT1 A receptor is widely distributed in the brain,mainly in the brain stem,frontal cortex,limbic system,which provides nutrition for the brain.5-HT2 A receptor is mainly distributed in many forebrain regions,especially in cortex,caudate nucleus,accumbent nucleus and hippocampus,which may play an important role in synaptic connection of brain(Duman,Heninger,& Nestler,1997).5-HT2 B receptors are mainly distributed in cerebellum,lateral septum,hypothalamus and amygdala,which may play a role in regulating 5-HT mitosis and anti anxiety in social behavior.5-HT2 C receptor is mainly distributed in choroid plexus,cortex(olfactory nucleus,pyriform nucleus,cingulate gyrus and spleen),limbic system(accumbent nucleus,hippocampus,amygdala)and basal ganglia(caudate nucleus,substantia nigra).Activation of the receptor can cause behavioral responses including lack of exercise,dysphagia,anxiety and hyperthermia(Koek,Jackson,& Colpaert,1992).The medial prefrontal lobe is a brain region related to social cognition,which is considered to be one of the main brain regions of autistic patients.It is mainly dominated by 5-HT axons and rich in various 5-HT receptor subtypes,especially 5-HT1 A receptor and 5-HT2 A receptor.This brain area controls the activity of neurons in many subcortical areas through the excitatory axons of the vertebral neurons,including the antinergic neurons in the midbrain(ventral tegmental area,raphe nucleus and locus coeruleus),which in turn project to the prefrontal cortex(Groenewegen & Uylings,2000).Amygdala is involved in the regulation,learning and memory of emotion and fear.It receives a large number of sensory inputs in a highly processed form and can respond to somatosensory,visual,auditory and all types of visceral inputs.Disruption of the amygdala's excitatory / inhibitory balance has recently been recognized as a cause of autism(Marín,2012).As hippocampus plays an important role in learning and memory,social behavior and repetitive stereotyped behavior also involve recognition and memory of objects and social companions.As it is not clear whether the expression of 5-HT receptor in the brain of the patients with loneliness is abnormal,whether the abnormal expression of 5-HT receptor is related to the symptoms of autism,and which brain regions have abnormal expression of 5-HT receptor,therefore,in Study 2,the relatively rich expression of 5-HT1 A,5-HT2 A,5-HT2 B,5-HT2 C in medial prefrontal lobe,hippocampus,amygdala were selected for exploration.Specifically,on the basis of the effective construction of the animal model of autism like rats in Study 1,study 2 step toward to solve whether the expression of 5-HT1 A,5-HT2 A,5-HT2 B and 5-HT2 C receptors in the medial prefrontal lobe,hippocampus and amygdala of autism like rats in early adolescence is abnormal.Finally,combined with the results of study 2,study 3 further explored the effects of activating or antagonizing 5-HT1 A receptor on the two core symptoms of autism like rats: social deficit and "object solidification" behavior from the perspective of manipulation and intervention.In Study 1(Experiment 1),This study first explored the effectiveness of valproic acid exposure during pregnancy in simulating two core symptoms of autism: social deficit and repetitive stereotyping.whether the rats were exposed to valproic acid as the independent variable and the time of exploring the companion in the two test stages of the classic social exploration test as the dependent variable,to explore whether the offspring rats exposed to valproic acid showed social defects;Taking the ratio of exploring objects in the object and peer competitive test as the dependent variable,we explored whether the offspring rats exposed to valproic acid showed the phenomenon of "object solidification".The results showed that after a single intraperitoneal injection of 600 mg / kg VPA on the 12 th.5th day of gestation,the offspring of SD pregnant rats had similar behavior patterns to those observed in autistic patients.Specifically,(1)compared with the control group,VPA treated rats significantly reduced the time of exploring peers in early adolescence and adolescence,they showed significant social defects,no matter in the social exploration test to detect social behavior alone,or in the object and peer competitive paradigm stimulated by objects;(2)In the object and peer competitive paradigm,compared with the control group,VPA treated rats significantly increased the time of exploring objects in early adolescence and adolescence,they showed obvious "object solidification" behavior.These results show that VPA is effective in modeling the two core symptoms of autistic rats(social deficit and repetitive stereotyped behavior).In Study 2(Experiment 2),after the behavioral test,the male rats in the autism like group and the control group were decapitated and the expression of 5-HT1 A,5-HT2 A,5-HT2 B and 5-HT2 C receptors in the medial prefrontal lobe,hippocampus and amygdala were quantify by Q-RT-PCR.The results showed that:(1)compared with the control group,the mRNA expression of 5-HT1 A and 5-HT2 A receptors in the medial prefrontal lobe of autistic rats in early adolescence was decreased,but the mRNA expression of 5-HT2 B and 5-HT2 C receptors was not affected;(2)The expression of 5-HT2 A,5-HT2 B and 5-HT2 C receptor mRNA in amygdaloid nucleus of autistic rats in early adolescence was decreased,but the expression of 5-HT1 A,5-HT2 B and 5-HT2 C receptor mRNA was not affected;(3)The expression of 5-HT1 A,5-HT2 A,5-HT2 B and 5-HT2 C receptor mRNA in the hippocampus of pre puberty autistic rats was not affected.These results suggest that exposure to valproic acid during pregnancy selectively disrupts the expression of 5-HT1 A and 5-HT2 A receptors in the medial prefrontal lobe and 5-HT2 A receptors in the amygdala of offspring rats,which may be one of the mechanisms leading to the occurrence of autism core symptoms.In Study 3,the specific mechanism of 5-HT1 A receptor on social deficit and XII "object solidification" behavior of autism was investigated by exposing 5-HT1 A receptor agonist 8-OH-DPAT(Experiment 3)and antagonist WAY-100635(Experiment 4).In Experiment 3,whether the rats were given 5-HT1 A receptor agonist 8-OH-DPAT or not was taken as the independent variable,and the time of exploring peers in the four test stages(sample stage: Test 1;object recognition stage: Test 2;position recognition stage: Test 3 and social recognition stage: Test 4)of object and peer competitive exploration paradigm was taken as the dependent variable,to investigate whether the 5-HT1 A receptor agonist could improve the social defects in the early adolescence of autistic rats.Taking whether the rats were given 5-HT1 A receptor agonist 8-OH-DPAT as the independent variable and the ratio of the rats to explore the objects in the object and the peer competitive exploration paradigm test 3 as the index of "object solidification"(the calculation method is: the sum of the time for the subject rats to explore two objects divided by the sum of the time for the subject rats to explore two objects and their companions),and taking this index as the dependent variable,the effect of 5-HT1 A receptor agonist on the "object solidification" behavior of pre puberty autistic rats was investigated.The results of Experiment 3 showed that:(1)5-HT1 A receptor agonist can improve the social deficit of autistic rats in early adolescence,but the effect of agonist has a certain timeliness.The effect of agonist is the best when the agonist is exposed around 50-60 minutes,and then the effect of agonist weakens;(2)5-HT1 A receptor agonist has no effect on the "object solidification" behavior of autistic rats in early adolescence.These results indicate that the mechanism of social defect and "object solidification" behavior may be different.Activation of 5-HT1 A receptor can improve the social defect of autism,but it has no effect on "object solidification" behavior.In Experiment 4,whether the rats were given the 5-HT1 A receptor antagonist 8-OH-DPAT or not was taken as the independent variable,and the time of exploring peers in the four test stages(sample stage: Test 1;object recognition stage: Test 2;position recognition stage: Test 3 and social recognition stage: Test 4)of object and peer competitive exploration paradigm was taken as the dependent variable to investigate whether the 5-HT1 A receptor antagonist had further aggravating effect on the social defects in the pre puberty stage of autistic rats.Taking whether the rats were given 5-HT1 A receptor agonist WAY-100635 as the independent variable and the ratio of the rats to explore the objects in the object and peer competitive exploration paradigm test 3 as the index of "object solidification"(the calculation method is: the sum of the time for the subject rats to explore two objects divided by the sum of the time for the subject rats to explore two objects and their companions),and taking this index as the dependent variable,the effect of 5-HT1 A receptor agonist on the "object solidification" behavior of autistic rats in early spring was investigated.The results of Experiment 4 showed that:(1)5-HT1 A receptor antagonist alone could aggravate the social disorder of autistic rats,and the effect of the antagonist was not weakened at the end of the test(1.5 hours of the antagonist exposure);(2)5-HT1 A receptor antagonists had no significant effect on the "object solidification" behavior of autistic rats in early adolescence.These results further indicate that the mechanism of social defect and "object solidification" behavior may be different.Antagonizing 5-HT1 A receptor further aggravates the social defect of autism,but it has no significant effect on "object solidification" behavior.In general:(1)a single intraperitoneal injection of 600 mg / kg VPA to SD pregnant rats on the 12.5th day of pregnancy can effectively simulate the two core symptoms of autism: social defects and stereotyped behavior("object solidification" behavior enhancement);VPA rats show social defects and "object solidification" enhancement phenomenon in both pre puberty and adolescence,and are more stable in the pre puberty period.(2)Exposure to valproic acid during pregnancy resulted in abnormal expression of 5-HT1 A and 5-HT2 A receptors in the medial prefrontal lobe of offspring rats and abnormal expression of 5-HT2 A receptors in the amygdala;(3)5-HT1 A receptor agonist can improve the social defects of autistic rats in early adolescence,but the effect of agonist has a certain timeliness.The effect of agonist is the best when the agonist is exposed around for about 50-60 minutes,and then the effect of agonist weakens;5-HT1 A receptor antagonist alone can aggravate the social disorder of autistic rats,and the effect of the antagonist is longer;5-HT1 A receptor agonists and antagonists had no significant effect on the "object solidification" behavior in early adolescence of autistic rats.These results suggest that the pathological mechanism of social deficit and "object solidification" behavior may be different.The results of this study not only provide support for the effective construction of autism animal model by exposing valproic acid during pregnancy,but also provide a new perspective and possible way to treat social deficit symptoms of autism.Secondly,study 2 proved that the expression of different receptors of 5-HT in different brain regions of autistic rats was abnormal,and the abnormal expression of different receptors of 5-HT in different brain regions was inconsistent,which also indicated the necessity of future research on the expression of each receptor of 5-HT in certain brain regions with key functions.Finally,the third study showed for the first time that the effects of 5-HT1 A receptor on the two core symptoms of autism like rats-social deficit and repetitive stereotype behavior are inconsistent.Activation of 5-HT1 A receptor can improve the social deficit behavior of autism like rats,and inhibit the experience to further aggravate the deficit,while activation or inhibition of 5-HT1 A receptor has no significant effect on repetitive stereotype behavior.Concisely,current findings indicates that 5-HT1 A receptor may play an important role in the pathogenesis of autism,and could be a potential candidate for the development of drugs for the treatment of autism. |
PDF Full Text Request