Effect Of P53 Gene Knockout On Morphological And Electrophysiological Characteristics Of Layer V Pyramidal Neurons In The Barrel Cortex In Juvenile Mice

Objective:p53 is a tumor suppressor gene and its encoded protein is a transcription factor that regulates DNA damage and repair and maintains the stability of the genome.Therefore,it is known as the"genome guardian".In recent years,more and more studies have shown that p53 is closely related to neurological diseases.For example,in children with autism,p53 levels are increased in frontal and cerebellar cortical neurons.Moreover,p53 knockout(KO)mice exhibited autistic-like behavior.In in vitro studies about cultured neurons,p53 is demonstrated to be necessary for nerve cell growth and axon regeneration.However,it is unclear what is the role of p53 in neuron morphology and function in intact animal.Our previous studies showed that the whisker sensitivity was injured in p53KO mice,but the underlying mechanism is unknown.Since the central projection area of whisker is barrel cortex,and to observe the effect of p53 deletion on neuron morphology and function,we investigated the morphology and electrophysiological characteristics of layer V pyramidal neurons(L5PC)in barrel cortex of p53KO mice.The results may provide new direction and target for the treatment of p53 related neurological diseases.Methods:In this study,juvenile(20-22 days)male p53KO mice and(wild-type)WT mice(both C57BL/6)were recruited.Acute brain coronal slices(300μm)were prepared,and Neurobiotin(1%)was included in the intracellular electrode fluid.L5PCs,including thick tuft(L5TT)and slender tuft(L5ST)neuron,were record by whole cell patch clamp technique which was used to record the characteristics of active membrane(resting membrane potential,membrane capacitance,input resistance)and passive membrane(action potential threshold,membrane resistance amplitude,membrane resistance half-width,rheobase currents,I_h and sag,etc.)and compare the electrophysiological characteristics of the two genotypes of neurons.The whole cell recordings were continued for at least 25 min,which allowed neurobiotin to completely spread into the nerve cells.After the electrophysiological recording was completed,the slice was fixed with 4%PFA.In the next morning,the slice was incubated with the second antibody rhodamine and then was mounted in the evening.Images were taken by a confocal microscope to observe the neuronal morphological characteristics(That include these measures of apical dendrite height,apical tuft,apical tuft width,soma size and normalized cortical depth from pia)of the two genotypes of mice.Results:(1)Major morphological characteristics of L5TT and L5ST neurons in barrel cortex of p53KO mice,including apical dendrite height,dendritic tuft height,dendritic tuft width,soma size,normalized cortical depth from pia were not significantly different from those of the WT mice.(2)Dendritic spine density of L5ST neurons in p53KO mice was not significantly different from that of WT mice,while the proportion of dendritic spine maturity was lower than WT mice.(3)Dendritic spine density of L5TT neurons in p53KO mice was lower than WT mice,but no significant difference was observed in the proportion of dendritic spine.(4)Compared with WT mice,L5ST neuron in p53KO mice had increased rheobase currents,reduced action potential(AP)amplitude,and decreased I_h amplitude.(5)Compared with WT mice,the frequency and amplitude of spontaneous excitatory post-synaptic current(sEPSC)of L5TT neurons in p53KO mice were decreased,and the AP half-width decreased,without any significant difference in L5ST neurons.Conclusions:In summary,this study demonstrated that p53 deletion play an important role in the morphological and electrophysiological characteristics of L5PCs in mouse barrel cortex.These results may provide theoretical basis for the disease of p53 abnormality associated nervous system.