Engineer The Rabies Virus To Explore Neural Circuits Of Mice Feeding Behavior Related Hypothalamic Nucleus

Exploring the feeding behavior of animal,has significant research value whether it is to enhance the efficiency of animal husbandry production,or research on human metabolic disease.Previous studies have shown that the central nervous system plays an important role in the process of animal feeding behavior,and the hypothalamus is a key site of the central nervous system given its role in regulating the animal metabolism.The aim of our project was to track the neural circuits of feeding behavior related hypothalamic nucleus by engineering the rabies virus.This experiment is divided into three parts: First,make use of the vaccine strain rabies virus knocked out the G protein and labeled with fluorescent protein(SAD?G-YFP)to trace the neurons,throughout the whole brain,which send axon to the five feeding-related nucleus located in the hypothalamus—hypothalamic paraventricular nucleus,dorsomedial hypothalamic nucleus,arcuate nucleus,ventromedial hypothalamic nucleus,the hypothalamic lateral area.Our results show that the main nucleus sending axons to the five nucleis of hypothalamus including amygdala,accumbens nucleus,bed nucleus of the stria terminalis,locus coeruleus,lateral septal nucleus and so on;second,construct Env A / TVA virus tracer system to help rabies virus infect neurons in "specific site,crossing synapses can be controlled" way.Finally,the tracer system is constructed successfully and verified on the mouse brain;third,in order to build B7B-VSV SAD G cell lines containing vesicular stomatitis virus and rabies virus chimeric G protein and help rabies virus infect neurons more effectively and more concentrated in the injected area.We use CRISPR / Cas9 technique to modify B7 GG cells' genome-transform the G protein of the cell line genome into chimeric G protein,and replace the blue fluorescent protein with green fluorescent protein,and finally use hygromycin pressure culturing and flow cytometry successively to purify the new cell line,and the cell line will be established by further expansion.Exploring the functional neural circuits' structure by using flexible and efficient neurotropic virus has been a hot topic in the field of neuroscience,and it is also one of the effective ways to solve the abnormal diseases related to the body metabolism.Our work has uncovered many neural sites,which has a projection relationship with the hypothalamus in our experiment,which is expected to play a role in the study of the feeding neural circuit.At the same time,the application of the rabies virus tracer in our study maybe can lay a certain experiment basis for exploration and the development of new tracer tools.