CGMP Signalling In O₂ Sensory Neurons Promotes The Development Of Acute Functional Ethanol Tolerance In Caenorhabditis Elegans

Ethanol is a widely used beverage and abused drug.Alcoholism causes severe damage to human health and creates serious social problems.Understanding the mechanisms is important for the development of effective therapies.Alcohol has a wide spectrum of effects on physiological activities and behaviours from sensitization to sedation and even intoxication with increasing concentrations.Animals develop acute functional tolerance to ethanol,it means that blood or tissue concentrations of ethanol are kept constant,animals'behavior can be observed in the form of decreased intoxication in a short time.However,the underlying mechanisms are not well understood.Due to feasible genetic and neuronal manipulations,rich genetic tools and vertebrate neuronal protein homologues,the nematode C.elegans is a useful model animal to study the function of genes in nervous system and analyze neural circuit.Here,we used C.elegans as a model animal to study the molecular mechanism and neural circuit which involve in acute functional tolerance to ethanol.In Caenorhabditis elegans,NPR-1 negatively regulates the development of acute tolerance to ethanol.In N2,carrying the high-activity form of the NPR-1 receptor,the development of acute functional tolerance to ethanol was suppressed,but the C.elegans strains with a lower function or loss-of-function npr-1 alleles,such as CB4856 and npr-1?ad609?,recover more quickly from exposure to ethanol than N2 animals.NPR-1also negatively regulates oxygen sensation.We speculated that the different rates at which N2 and npr-1 animals recovered from ethanol exposure reflect differences in the functions of this O₂ sensing circuit.We performed experiments to test this possibility.We found that GCY-35/GCY-36-TAX-2/TAX-4 signalling pathway is involved in the rapid development of ethanol acute tolerance in npr-1 worms,by using behavioral experiments.Unexpected,the O₂ levels have no effect on acute functional tolerance of worms.By using in vivo Ca²⁺imaging,we showed that URXs respond to ethanol in a cell-autonomous manner,and GCY-35/GCY-36--TAX-2/TAX-4 signalling pathway mediates ethanol activating O₂ sensory neurons.We ectopically co-expressed gcy-35 and gcy-36 cDNA in the neurons ASI and ASG that express tax-2/tax-4 and no gcy-35/gcy-36.However,ectopic co-expression of gcy-35 and gcy-36 did not confer ethanol sensitivity on ASIs or ASGs.Whether this complex is an ethanol receptor still needs further study,it's also not certain that there are other alcohol receptors in the URX neurons.Finally,by using chemogeneticmanipulation,we demonstrated that activation of the O₂-sensory neurons URX,AQR and PQR and the downstream neural circuit is indispensable for acute functional ethanol tolerance in npr-1 worms.C.elegans lives in rotting fruit and vegetation.Due to metabolic pathways of bacteria,bacterial flourishing produces alcohol and reduces ambient oxygen levels in microenvironments.The nematodes develop sensations and behavioural responses to oxygen and ethanol.This endows worms with advantages for survival and population propagation in the wild.