Preliminary Investigation Of The TMC-7 Knockout Rat Phenotypes And TMC-7 Function

The TMC(transmembrane channel-like gene)family is a family of conserved genes in mammals.There functions have long been unknown.Until 2012 and 2013,studies reported that TMC1 and TMC2 are mechanosensitive ion channels expressed in the cochlear hair cells and participate in the development of auditory sensation.Our preliminary experiments found that TMC7 m RNA is highly expressed in the peripheral ganglion DRG(dorsal root ganglion).Therefore,we speculate that the TMC7 gene may be involved in sensory neuron perception of the external environmental stimuli.In order to further identify the function of TMC7 gene,we first started our study from doing behavioral phenotype screening using general TMC7 knockout rats.Three groups of nervous system relevant behavioral tests were performed,(1)tests for motor function: elevated plus maze test,open field test,and rotarod test;(2)testing for sensory function: Von Frey test and thermal Hargreaves test,auditory brainstem response(ABR),and distortion product to acoustic emission(DPOAE)experiments;(3)advanced neurobehavioral tests: forced swimming test,water maze test and Maximal electroshock seizure(MES)test.It was found that TMC7 knockout rats have certain abnormal phenotypes in sensory behavior including a significant lower threshold of hind paw for mechanical stimulation and an obvious hearing lost at auditory stimulation from medium to high frequency spectrum.Then,we validated the TMC7 m RNA expression in the phenotype-associated tissue by various methods such as Basescope and qPCR,and found that TMC7 m RNA is highly expressed in DRG and spiral neurons of the cochlea.Finally,we compared the excitability of spiral neurons in TMC7 knockout rats a wild type rats and got a preliminary conclusion that TMC7 has a certain function in regulating the membrane potential of spiral neurons from the BASE of cochlea.These results could be the foundation for the further study.Part one Screening of neurophenotypes of TMC7 Knockout Rats with multiple behavioral testsObjective: To investigate the neurophenotypes of TMC7 knockout rats by comparing the nervous related behaviors of TMC7 knockout rats and littermates,and to determine the direction for the further research.Methods: Elevated cross labyrinth test,open field test,rotarod test;Von Frey test and Hargreaves test,ABR and DPOAE test;forced swimming,water maze experiment,Maximal electroshock seizure(MES)test.Statistical method: t test,Mann-Whitney U test.Results:1.The rat's exploration ability,exercise ability,endurance and balance ability were evaluated through the elevated plus maze experiment,open field experiment and rotarod experiment.The results showed no statistical difference between TMC7 knockout mice and wild type mice.2.The basic thresholds of mechanical and thermal stimuli were measured with Von Frey test and Hargreaves test..The results showed that the mechanical threshold of TMC7 knockout rats was significantly reduced.3.ABR test were used to evaluate the hearing response to different frequencies spectrum of sound stimuli.It was found that the hearing of TMC7 knockout rats was significantly damaged in the middle and high frequency spectrum stimuli.However,no statistically significant differences were found in the DP experiments representing function of hair cells.It is suggested that the knockout of TMC7 gene may have a significant effect on the function of auditory primary sensory neurons(spiral neurons).4.The forced swimming test was used to detect the behavior of despair and depression in rats.There was no significant statistical difference between TMC7 knockout rats and wild type rats.By comparing the number of shuttling through platforms in water maze,it was found that the learning and memory abilities of TMC7 knockout rats were not significantly affected.5.Electroshock seizure test showed no significant difference in the epileptogenic latency and duration of epilepsy between TMC7 knockout rats and wild type rats.Part two Eexpression in spiral neurons and its effect on excitabilityObjective: To investigate the expression of TMC7 in spiral neurons by qPCR and Basescope methods,and to explore effect of TMC7 knockout on membrane potential and excitability of spiral neurons in rats by patch-clamp technique.Methods: qPCR technique,Basescope detection RNA technique,whole cell patch clamp technique.Results:1.The cochlear tissue of wild-type rats was taken to observe the expression of TMC7 m RNA through qPCR method.The results showed that TMC7 m RNA was highly expressed in the cochlea.Using Basescope technique,it was revealed that TMC7 m RNA was expressed in spiral neurons.2.The excitability of spiral neurons in TMC7 knockout and wild rats was recorded and compared by whole cell patch clamp technique.It was found that the membrane potential of spiral neurons located at the base of cochlea of TMC7 knockout rats was significantly hyperpolarized.Conclusion:1.There is a significant reduction in mechanical threshold in hind paw.2.There is an auditory ability damage of hearing in the mid-high frequency spectrum in TMC7 knockout rats.This dissertation will focus on the phenotype on hearing lost.3.TMC7 is highly expressed in spiral neurons of the he cochlea and may play an important role in regulating the membrane potential and excitability of spiral neurons,and thus affect auditory conduction.