Development And Application Of AAV In Neural Circuit Tracing

Neurons are the basic units of the human brain that connect to each other to form a complex,replicating circuit.Exploring these circuits has important implications for understanding how the brain works.Adeno-associated virus(AAV)is a common tool to mark the neural circuits and an important vector for gene therapy.There are few kinds of AAV tools with reverse tracer ability,and few neural circuits that can be labeled.The efficiency of marking neurons needs to be improved.These deficiencies limit the application of AAV vector in retrograde labeling of neural circuits.In this paper,the genomes of different AAV virus serotypes were modified mainly by directional mutation and random mutation to improve the reverse tracer efficiency of AAV vector in different circuits.The modification involves AAV1,AAV2 and AAVDJ virus variants.The AAVDJR vector was engineered by site-directed mutation to acquire a strong ability to reverse-trace the striatum to the cortex,thalamus and amygdala.The AAV2R-9M virus variant modified by directed mutation has a more efficient reverse tracer capability in the striatum pathway than the original rAAV2-retro.This paper also compares the labeling effect of AAV2R-9M virus variant injected in the substantia nigra and amygdala regions.In this paper,we also established the mutation library of the Cap protein gene of AAVDJR virus by random mutation,and screened out the candidate mutants that might have stronger reverse labeling effect in the striatum-nigra pathway by in vivo evolution.In this paper,the genomes of different AAV virus variants were modified,a variety of mutants were obtained and tested,and the virus mutants with higher reverse labeling efficiency were obtained,which provided more application options of AAV tools for neuroscience research.