Behavioural Phenotype And Analysis Of Mice Knocking Out The TMC7 Gene In The Central Associated Nucleus Cluster

The transmembrane channel-like gene family(TMC)encodes a group of transmembrane proteins with eight transmembrane regions(TM1-TM8),including the eight members TMC1-TMC8,which are evolutionarily conserved and are predicted to have important cellular functions.However,research on the TMC protein family is limited due to difficulties in exogenous expression,the lack of effective specific antibodies and unknown protein structures.The TMC protein family has been a bottleneck for research for several reasons.TMC channels form three subfamilies: TMC1-3(subfamily 1),TMC5 and 6(subfamily 2),and TMC4/7 and 8(subfamily 3).Among them,TMC1 and TMC2 are components of mechanotransduction channels in mammalian inner ear hair cells,suggesting that members of the transmembrane ion channel-like TMC family are likely to be ion channel proteins and play an important role in the animal’s perception of external stimuli,for example.According to the database and our previous studies,high levels of TMC7 m RNA were detected in the nervous system and peripheral primary sensory ganglia of DRG,suggesting that TMC7 may play an important role in the perception of stimuli from the external environment by sensory neurons.The2017 genome-wide association study of psychotic tendencies in the Finnish population pointed to an intron on the TMC7 gene that was significantly associated with mild mania.To further validate the function of the TMC7 gene,this thesis first started with local knockout of the TMC7 gene within the central system and observed phenotypic alterations in animals with local knockout of the TMC7 gene through basic behavioural experiments and related experimental model behaviouralexperiments.Behavioural experiments include the open field experiment,which measures the animals’ locomotor abilities,and the elevated cross experiment,the hanging tail experiment and the forced swimming experiment,which measure anxiety.Experimental models include 24 h short-term sleep deprivation,social isolation and continuous ketamine injection models.Objective: The effects of central local knockout of the TMC7 gene on mouse behaviour were determined by local knockout of the TMC7 gene in the ventral tegmental area(vta),prelimbic cortex(Pr L)and substantia nigra(SNc),and the direction of the next study was determined.Methods:The basic behavioural results on day 28 of intra-VTA virus injection showed that mice in the AAV5-CMV-scramble-GFP virus group and mice in the AAV5-CMV-TMC7 shRNA-GFP virus group showed no differences in locomotor performance or anxiety.Mice in the AAV5-CMV-TMC7shRNA-GFP virus group showed a significant increase in locomotion and anxiolytic mood after 28 days of 24 h short-term sleep deprivation following intra-VTA injection of virus,and mice in the AAV5-CMV-scramble-GFP virus group showed no significant changes.The results suggest that mice with intra-VTA injection of knockdown TMC7 gene are more prone to manic-like behaviour after 24 h short-term sleep deprivation.Mice in the AAV5-CMV-TMC7 shRNA-GFP virus group showed a significant increase in locomotion and no significant change in anxiety after28 days of intra VTA injection of virus in social isolation.mice in the AAV5-CMV-scramble-GFP virus group showed no significant change in locomotion and anxiety.The results suggest that mice injected with the TMC7 gene in the VTA are more likely to develop manic-like behaviour after social isolation.After 28 days of continuous intra-VTA virus injection with ketamine,mice in the AAV5-CMV-scramble-GFP virus group and the AAV5-CM V-TMC7 shRNA-GFP virus group showed no differences in locomotion,anxiety and stereotyped behaviour.Basic behavioural results on day 28 of intra-PrL virus injection sho wed that mice in the AAV5-CMV-scramble-GFP virus group and the AA V5-CMV-TMC7 shRNA-GFP virus group showed no differences in locom otor performance or anxiety.Mice in the AAV5-CMV-TMC7 shRNA-GFP virus group showed a significant increase in locomotor ability and anxiolytic mood after 28 day s of 24 h short-term sleep deprivation by intra-Pr L injection of virus,w hile mice in the AAV5-CMV-scramble-GFP virus group showed no signi ficant changes.The results suggest that mice with intra-Pr L injection of knock down TMC7 gene are more prone to manic-like behaviour after24 h short-term sleep deprivation.After 28 days of intra-PrL injection of virus in social isolation,mic-e in the AAV5-CMV-scramble-GFP virus group and the AAV5-CMV-T MC7 shRNA-GFP virus group showed no differences in locomotor perfor mance or anxiety.After 28 days of continuous ketamine injection within PrL,mice in the AAV5-CMV-scramble-GFP virus group and the AAV5-CMV-TMC7 s hRNA-GFP virus group showed no differences in locomotor performance,anxiety and stereotyped behaviour.After a 28-day 24 h short-term sleep deprivation experiment with intra-SNc injection of virus,mice in the AAV5-CMV-TMC7 shRNA-GFP virus group showed a significant increase in locomotor ability and no si gnificant change in anxiety,while mice in the AAV5-CMV-scramble-GFP virus group showed no significant change in locomotor ability and anxi ety.The results indicated that mice injected with the TMC7 gene wer-e more likely to develop manic-like behaviour after 24 h short-term sleep deprivation.Mice in the AAV5-CMV-TMC7 shRNA-GFP virus group showed a significant increase in locomotion and no significant change inanxiety after 28 days of social isolation following intra-SNc injection of virus.mice in the AAV5-CMV-scramble-GFP virus group showed no significant c-hange in locomotion or anxiety,and the results s-uggest that mice with intra-SNc injection of the knockdown TMC7 gene are social isolation w-ere more likely to develop manic-like behaviour.After 28 days of continuous ketamine injection of virus within SNc,the wood stiffness scores of AAV5-CMV-TMC7 shRNA-GFP mice in the ketamine group were significantly higher than those of AAV5-CMV-scra mble-GFP mice at days 5 and 7.The results suggestthat mice with int-r a SNc injection of knockdown TMC7 gene were more prone to mani-c li ke behavior after continuous ketamine injection.Conclusion:The TMC7 gene may play an important role in the centre,and del-etion of the TMC7 gene may be more likely to lead to the onset of mania.