| Locomotion is the instinctive behavior of all animals.The exchange of information between cells is required for locomotion generation,which needs to be completed by ion channels on the cell membrane.Bioelectrical phenomena and cell excitability are also realized on the basis of ion channels and ions.Neural signal transmission refers to the process in which neurons transmit electrochemical signals through synaptic information to downstream neurons or neuromuscular cells.Patch-clamp electrophysiology is a common technique used to study the electrical signals of ion channels and electrical signals transmitted by synapses to study neuronal function and the molecular mechanism of neural circuits.As an ideal model for genetic screening,Caenorhabditis elegans(C.elegans)is also increasingly important in the study of neural function mechanisms.This topic will apply the electrophysiological technique of body patch clamp to the nematode motor neural circuit,and combine with the latest optogenetic technology to develop an effective scheme to study the functional mechanism of neurons and neural circuits.With the help of this joint technology,a feasible method to study the functional properties of ion channels in nematode neurons was discovered.First,using optogenetic technology,the photosensitizing protein Chrimson was specifically expressed on the motor neuron cells of C.elegans,and the motor neurons were activated with 540 nm wavelength light to confirm that the nerve plays an inhibitory role in the movement of C.elegans.Secondly,using the in vivo patch clamp technique,obvious transient post-synaptic electrical signals m PSCs under physiological conditions were successfully recorded in C.elegans;combined with the chemical blocking agent d-TBC to block the signal transmission of neuronal acetylcholine,GABA-mediated Inhibitory post-synaptic current was recorded;combined with optogenetic technology,the light-sensitive channel protein ChR2 is expressed through a neuron-specific promoter,specifically excitatory motor neurons are activated,and larger post-synaptic current EPSCs are recorded;further combined with tool proteins mini SOG,which expresses the blue-sensitive singlet oxygen-producing protein mini SOG through a neuron-specific promoter,which mediates specific motor neuron-specific damage.It is found that the recorded post-synaptic current is significantly reduced.Therefore,in the patch clamp experiment of C.elegans,we can use chemical reagents to block local excitatoryelectrical signals of neurons,and recorded the inhibitory neuronal signals of neuronal signals;we can use the optogenetic technology to specifically activate neurons,and recorded specific activation of neuronal signals;we can use mini SOG-mediated specific neuronal damage,and recorded neuromuscular electrical signals after specific removal of neuronal signals.these methods provide great help for understanding the function of motor neurons and their role in neural pathways.Finally,through molecular biology genetic expression technology,the XXX(protein to be published)potassium channel of Caenorhabditis elegans was heterologously expressed in HEK293 T cells,and the fluorescent protein of expressing potassium channel was detected.Combined in vivo patch clamp electrophysiology,it also recorded the outward potassium current generated by the XXX potassium channel and the more negative resting membrane potential.This shows that ion channel protein of the Caenorhabditis elegans can not only be heterologously expressed in HEK293 T cells,but also it can perform normal functions.Taken together,this project has developed a combined in vivo electrophysiology with optogenetics,and studied the mechanisms of motor circuit and rhythmic locomotion in C.elegans. |
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