The Molecular And Circuit Mechanisms For Avoidance Of Rapid Cooling Stimuli In Caenorhabditis Elegans

The correct response to ambient temperature signal is the basis of organism’s survival.Previous studies have shown that the animal nervous system use different molecular and circuit mechanisms to respond to temperature changes in the environment.Caenorhabditis elegans,with its excellent neural circuit,high temperature resolution and chemotaxis to culture temperature,has become an ideal model organism for studying temperature stimulus input and behavior output.Because of the accurate control of the range and rate of thermal stimulation,the molecular and circuit mechanism of animal response to thermal stimulation have been analyzed,these results shows that C.elegans response to warming stimuli is depend on the temperature threshold.However,there are relatively few studies on the cold perception of C.elegans,and the mechanism of organisms responding to cold stimulation are still in infant.This is mainly due to the limitations of the cold stimulus temperature control method in the experiment.In the past,the cooling rate was usually controlled by liquid perfusion,which could not control the cooling rate and cooling time accurately.At the same time,this cooling method will introduce other stimuli in the process of stimulation.In view of the advance scientific problems and technical difficulties,our laboratory developed a cooling device based on semiconductor,which realized linear cooling at different rates through programmable feedback control circuit,use this device we investigated the molecular and circuit mechanisms of C.elegans in response to cooling stimuli.This study also combines engineering,calcium imaging,quantitative behavior,chemogenetics,and molecular biology techniques for genetically encoded calcium indicators.Results:1.The response of C.elegans to cooling stimulus is depend on cooling rate.That is,the faster the cooling rate,the more severe the avoidance behavior of worms.2.Using chemogenetic methods(blocking neurotransmitter release by expressing tetanus toxin TeTx,inhibiting neuronal activity by expressing histamine receptor His Cl1,and neuronal ablation by expressing Mini SOG)to control sensory neuron ASH neural signal transduction,the worm exhibited a significant decrease in reversal during rapid cooling,combined with in vivo calcium imaging,ASH showed robust transient calcium response,which indicated that sensory neuron ASH mediated avoidance response induced by rapid cooling.3.After screening the downstream first-layer interneurons,we found the avoidance of behavior in worms induced by rapid cooling decreased significantly after blocking AIZ neural signal transduction,and AIZ showed robust calcium activity.This result suggests that AIZ is involved in rapid cooling stimulus.Further experiments were performed by detecting calcium signaling in electrical synaptic mutants showed that ASH to AIZ signals pathway through gap junction.4.The above neuronal manipulation methods were also used to screen secondary-layer interneurons that were downstream of AIZ.The results showed that after blocking RIA neural signal transduction,the avoidance of behavior in worms decreased significantly during rapid cooling,and RIA neurons showed a significant calcium response detected by calcium imaging.Calcium activity was significantly decreased after AIZ inhibited.These results suggest that RIA act as downstream neurons of AIZ in response to rapid cooling stimuli.Further screening and genetic rescue of glutamate receptor molecules revealed that GLR-3 and GLR-6 on RIA neurons were involved in the regulation of AIZ-RIA signaling pathway.Altogether,ASH responds to worms rapid cooling response by activating first-layer interneuron AIZ through electrical synapses,and excited AIZ to activate RIA by releasing glutamate on GLR-3 and GLR-6 receptors,thus promoting worms’ avoidance of rapid cooling stimulus.The results of this study provide a novel neural circuit mechanism,elucidate a new model of C.elegans response to temperature stimulation,and provide a basis for understanding the rapid cooling response of higher animals.