Effects And Mechanism Of Conditional Knockout Hippocampal CA1 FGF13 In Murine Behaviours

It is well known that the integrality of brain function plays a crucial role in the development,and brain function and plasticity depend on the complex architecture of neural circuits,which are established during development.Dysplasia of nervous system can not only cause a variety of neurological diseases,such as autism,epilepsy,Huntington's chorea,spinocerebellar ataxia,mental development defects,and neuralgia,but cause other systems dysplasia and disorders.,such as neuroendocrine dysfunction.Recent studies show that X-linked mental retardation?XLMR?is an inherited intellectual disability,which is associated with disordered neural development.Interestingly,researchers find fibroblast growth factor 13?FGF13?plays a crucial role in XLMR.Therefore,the role of FGF13 in the X-linked mental ratardation is widely concerned.Fibroblast growth factor homologous factors?FHFs?belong to the fibroblast growth factors,including FGF11-FGF14.They form a distinct family and show 30%–50%amino acid sequence identity with other FGFs.Due to the lack of N-terminal signal peptide for secretion and function via their receptors,FHFs might play different roles in cells compared with other FGFs.Members of the FHF subfamily are expressed in the cerebral cortical neurons in the developing brain.The role of FHFs in the nervous system has been widely concerned.FGF11-and FGF14-deficient mice suffer from severe spincerebellar ataxia and other neurological disorders through regulating the voltage-gated sodium channels?VGSCs?and voltage-gated calcium channels?VGSCs?.FGF12 is necessary and sufficient for inducing the quiescent and differentiated phenotypes of VSMCs and that it acts as the key regulator of transdifferentiation into SMC-like cells.FGF13 plays a essential role in XLMR via regulating hippocampal neurons.The loss of FGF13 impairs neuronal polarization and increases the branching of axons and leading processes.The FGF13 knockout in mice results in neuronal migration defects in both the neocortex and the hippocampus.As we all known,glutamate receptors are highly expressed in hippocampal neurons,and their functions are closely related to learning and memory.The glutamate receptors-dependent LTP is considered to be the mechanism of learning and memory.However,the electrophysiology mechanism and the interaction between FGF13 and glutamate receptors are remains unclear.Therefore,we intend to complete the construction and behavioral evaluation of hippocampal CA1-specific knockout mice,and try to further explore the electrophysiological mechanism and the interaction between FGF13 and glumate receptors of FGF13 in XLMR.Part 1 Effects of conditional knockout FGF13 in hippocampal CA1neurons in behaviours.Objective:Using Cre-loxP technology to knock out FGF13 in hippocampal CA1 neurons to study the effects of FGF13 in learning and memory.Methods:?1?Mating CamK?-Cre mice[mice expressing Cre recombinase under regulatory element of mouse CamK?gene]with FGF13-loxP mice.Efficiency of FGF13 knockout was validated by genotyping,Real-time PCR.?2?Using Morris water maze,open-field test and elevated plus maze to evaluate the ability of learning and memory,anxiety and depression-like behaviour.?3?Statistic methods:t-test and one-way ANOVA analysis were used.Results:?1?Generation and genotyping of hippocampal CA1-specific knockout FGF13 mice:CamK?-Cre,FGF13^fl/+heterozygus mice were generated by crossing Camk?-cre mice with FGF13^fl/fll/fl mice.Then the CamK?-Cre,FGF13^fl/+heterozygus were crossed with FGF13^fl/+to get CamK?-Cre,FGF13^fl/fll/fl mice,which was the hippocampal CA1-specific knockout FGF13mice?CKO?.About ten days after birth,all the mice were marked by toe cutting,and genomic DNA was extracted from cutted tail of mice.CKO mice identification was done using PCR technique followed by gel electrophoresis.The target band of Cre was about 100 bp,while the WT band of FGF13 was about 183bp and FGF13^fl/fll/fl band of FGF13?loxP sites inserted?was about 241bp.?2?Efficiency of FGF13 conditional knock out in hippocampus:the mice hippocampus was isolated 15 days after birth.Then,using Real-time PCR technique to evaluate the expression of FGF13.The results indicates the FGF13 mRNA expression level are significantly reduced in CamK?-FGF13^fl/fl/f mice?P<0.05?.?3?Results of behavioral tests:Compared with WT mice,the loss of FGF13 impaired the ability of learning and memory but not anxiety and depression-like behaviour.The results indicate that the escape lantency time was significantly increased during the training process and a significantly reduced time in the target quadrant and a significantly reduced number of target crossing times?P<0.05?.Conclusions:These results suggested that FGF13 played an crucial role in spatial learning and memory.Conditional knockout hippocampal CA1neurons FGF13 significantly impaired the ability of spatial learning and memory but not anxiety and depression-like behaviour.Part 2 Effect of FGF13 in regulating hippocampal CA1 neuronsexcitabilityObjective:Using brain slice patch-clamp technology to identify the effects of FGF13 in regulating the hippocampal CA1 neurons excitability;trying to explore the elctrophysiology mechanism.Methods:?1?Using brain slice patch-clamp to identify the regulation of FGF13 in hippocampal CA1 neurons,such as action potential.?2?To identify the mRNA expression level of glutamate receptors via qPCR.?3?Statistic methods:two-sample t test,and one-way ANOVA analysis were used.Results:?1?The excitability analysis of WT and KO mice:compared with WT mice,there exists a both significantly reduced frequency of action potential in FGF13 decificent mice via two different protocol to evoke action potential?P<0.05?.The injected currents were significantly incressed,which the first action potential needed.But rest membrane potential do not change.?2?The mRNA expression level of glutamate receptors:compared with WT mice,the essential subunit of N-methyl-D-aspartate Receptor?NMDAR?GluN1 is significantly reduced?P<0.05?,not GluN2A and GluN2B.But the subunit of?-amino-3-hydroxy-5-methy receptor?AMPAR?auxiliary subunit GluA1 and GluA3 is significantly increased.Conclusions:The results show that FGF13-deficient in hippocampal CA1 neurons significantly impaired the excitability by reducing the frequency of action potential.While,GluN1 mRNA expression level was reduced in FGF13-deficient mice but GluA1 and GluA3 mRNA expression level were increased.