A Novel 4D Imaging System For Freely Behaving C.elegans And The Application To Dissect The Mechanisms Of Neural Circuits

In 2013,U.S.government and NIH launched the "BAM"(“Brain Project”),the first step to achieve the BAM's goal is to study the molecular mechanisms of neural circuits function and complete the model organism C.elegans.As the classic model organism C.elegans has only 302 neurons in the nervous system but at the same simple control the behavior of a relatively complex and diverse phenotypic characteristics,as well as its operation with the convenience of genetics and body imaging transparent and easy,making the study of neural circuits become nematodes excellent model cellular mechanisms and molecular mechanisms.This study includes two aspects,first established for monitoring the activity of the nervous system associated with nematodes and 4D imaging system analysis and behavioral phenotypes,making the recording neural activity and the state of freedom of movement under the nematodes associated with behavioral output as possible,With this novel 4D imaging system,we found a new intermediate inhibition of neuronal circuits in the regulation of feeding behavior of nematodes.4D imaging system established in this paper,4D specific meaning referring to x \ y \ z \ t 4D neuronal calcium imaging system combines 2D behavioral analysis,which is divided into three subsystems: a nematode behavioral phenotypes automatic tracking and analysis system;Second,nematodes and more neurons calcium imaging systems;Third,light-activated neurons with light nematode suppression system.Nematode behavior phenotype through automatic tracking and analysis system for machine vision technology for digital modeling after nematode,C.elegans can automatically analyze the behavior of the phenotypic behavior of more than 200 kinds of quantitative parameters,including the rate of movement,crawling track,forward and backward movement a variety of characteristic behavior frequency and speed,steering angle and eating rate and so on.C.elegans neurons and more calcium imaging system through the target tracking algorithm,can lock any single worm crawling freedom or more neuronal calcium signaling,tracking system allows C.elegans neurons signal with a microscope lens is relatively static,real-time monitoring and analysis of nematodes calcium activity in the nervous system.C.elegans neurons activated by light and light suppression system is to achieve a light stimulus signal and the aforementioned behavior will be regulated calcium imaging analysis system and associated systems,making light of genetics researchers can operate to achieve more complex neural circuits mechanism.This 4D imaging system established in this paper,the integrated use of optical,mechanical design,machine vision,automatic control and other technology,making function of the nervous system of nematodes become simple and convenient,more importantly,can not be achieved many previously experiments,the successful implementation of synchronous detection and analysis of MC,M3,ASH,AIB,RIM,NSM,RIA,and many other nematode pharyngeal muscle and neuronal signals associated with the behavior of calcium and muscle phenotype.Neurons in the environment signal processing mechanism,it has been an important problem in the field of neurobiology.In the worm's simple “brain”,how to calculate and process the external signal and generate the behavior output is poorly understood.By means of the 4D imaging system developed in this thesis,we found that the neurons in the middle of neurotransmitter glutamate on AIB have different affinity and GLR-1 GLR-5 of two kinds of glutamate receptor mediated the AIB neurons release different neurotransmitters,weak in the outside world under the action of stimulating signals and the strong stimulation,respectively,through the calculation of AIB neurons,RIM has realized the inhibition and activation of downstream neurons respectively with different signal intensity to make different behavioral responses.The molecular mechanism of the found that the calculation mechanism of neuron gives a new explanation,also demonstrated the little nematodes has a huge "brain".Feeding behavior is one of the most important basic biological behavior is the foundation of the organism to survive.Organisms in the environment when dealing with complex signals and stimulating the nervous system is how to feel signal processing and output feedback behavior is not clear.With 4D imaging system established in this paper,we study the regulation of eating behavior elegans neural circuits,and new findings by the middle of neurons AIB and RIM consisting of a neural feedback loop,played an important role in suppressing nematode regulation of eating behavior.The feedback loop is achieved through the release of neurotransmitter secretion RIM Tyramine Tyramine AIB feedback on the experience TYRA-3 receptors.AIB and the composition of this feedback loop RIM played a filter for the nematode stimulatory signals from the environment in the role.This paper also found that the function of this signal is filtered by the AIB metabotropic tyramine receptor TYRA-3 ionic glutamate receptor inhibition achieved.