Developing A Novel FISH Technique To Explore The Response Mapping Of Key Brain Region Under Systemic Inflammation

Systemic inflammation,also known as systemic inflammation,is often caused by a large-scale infection of pathogenic micro-organisms.The systemic inflammatory process is often accompanied by a series of disease behaviors such as fever,decreased activity,loss of appetite,sleep disturbance,and cognitive impairment,which are important indicators in daily health monitoring.When large-scale systemic inflammatory infection occurs in animal husbandry,it will reduce the feed-to-weight ratio.And in severe cases,it will cause the death of sick animals,which will bring huge economic losses to farmers.Neuroimmune regulation is an important immune regulation method of the body.As the high-level regulatory center of the body,the brain can regulate the immune response through the coordination of various nuclei as well as the tissues and organs that dominate the body,and finally maintain the body’s homeostasis.The dorsal raphe nucleus(DR)is a nucleus with concentrated distribution of 5-HT neurons in the brain,which can widely regulate the behaviors of the body such as emotion,memory,cognition,learning,pain and food intake.Other studies have shown that DR plays an important role in immunity and thermoregulation,but the molecular mechanism and neural basis of DR in regulating disease behavior and immunity remain to be resolved.To explore the spatial transcriptional profile changes of DR nuclei under the systemic inflammation model,a new medium-throughput RCA-FISH technique was first developed,and based on this method,the fine spatial map of DR neurons was analyzed;Further,via indirect immunofluorescence,single-cell RNA sequencing,chemical genetics,and fluorescence in situ hybridization,we explored the transcriptome changes at the single-cell level in mouse DR under lipopolysaccharide(LPS)-induced systemic inflammation and discovered the potential molecules involved in inflammatory responses in DR;We initially explored the neurons involved in thermoregulation in DR,providing an important theoretical reference for the molecular mechanism of DR in regulating body temperature.The specific results are as follows:(1)In this study,we developed a medium-throughput FISH technology based on rolling circle amplification(RCA),namely medium-throughput RCA-FISH,which can interpret expression of 28 genes through two rounds of hybridization and imaging.Using the newly developed medium-throughput RCA-FISH technology,the spatial distribution map of DR neuron subpopulations and specific receptors was analyzed,which laid the foundation for the subsequent exploration of the changes in the spatial transcriptional profile of DR under systemic inflammation.(2)Immunofluorescent staining of c-Fos and i Ba1 showed that microglial cells in the DR nucleus were significantly activated and some neurons were activated under LPSinduced systemic inflammation,but the activated neurons were mainly non-5-HT neurons;Through single-cell RNA sequencing and analysis of transcriptome changes of DR under systemic inflammation,various types of cells in DR responded extensively,and vascular endothelial cells responded most strongly;The pathways of differential gene enrichment closely related to disease behavior and Gram-negative bacteria;Further analysis found that the expression of Tph2,a gene related to 5-HT synthesis,was down-regulated in 5-HT neurons,suggesting that 5-HT levels in 5-HT neurons were down-regulated.(3)Clustering of DR neuron subgroups revealed that Fev is not a specific marker gene for 5-HT neurons,and it is expressed not only in 5-HT neurons,but also in Vglut3 neurons.Subsequent transgenic mice and fluorescence in situ hybridization results confirmed the conclusion that Slc6a4 and Tph2 were strictly expressed in 5-HT neurons in DR and could serve as marker genes for 5-HT neurons,while Fev was expressed in both 5-HT neurons and Vglut3 neurons.(4)Further,we initially explored the role of 5-HT neurons in thermoregulation via chemical genetic methods.The results showed that specific activation of Fev-positive neurons(Vglut3 neurons and 5-HT neurons)in DR could alleviate the hypothermia under systemic inflammation,but specific inhibition of Fev-positive neurons in DR could also slow down the hypothermia under systemic inflammation to some extent.After further specific activation of Slc6a4-positive neurons(5-HT neurons)in DR,body temperature continued to decrease under systemic inflammation.The above results suggest that both 5-HT neurons and Vglut3 neurons in the DR nuclei may be involved in thermoregulation under systemic inflammation.In summary,we found that the transcriptome of DR was significantly changed during the systemic inflammation caused by LPS,and the differential genes were highly correlated with disease behavior.The 5-HT neuron marker gene was further verified,and the 5-HT neuron molecule was improved for research on markers.We developed medium-throughput fluorescence in situ hybridization technology to analyze DR spatial maps,and explored the role of specific neurons in thermoregulation,providing a certain basis for the prevention and treatment of animal infection and systemic inflammation.