The Role Of Gaba Neurons In The Pathogenesis Of Epileptic Fmr1 Ko Mice

BackgroundFragile X syndrome (FXS) is one of the most common forms of inherited mental retardation. The main symptoms of FXS include facial abnormality, macro-orchidism, mental retardation and high susceptibility to epilepsy. It is reported that epilepsy occurs in 20 to 25% of individuals with FXS and paroxysmal EEG abnormality presents in about 50% of prepubescent boys with FXS. Fragile X syndrome (FXS) results from a trinucleotide repeat (CGG) expansion in the 5-untranslated region of the gene FMR1 and subsequent hypermethylation which lead to transcriptional silencing of FMR1 and loss of its encoded protein, the fragile X mental retardation protein (FMRP). It has been found that FMRP is a brain specific mRNA binding protein that suppresses translation of target mRNAs. However, the exact mechanism remains elusive how the reduced FMRP causes the clinical manifestations.FMR1 knockout mouse has been served as a valuable tool in elucidating the mechanisms of FXS. The model mouse has shown the multifaceted phenotypes consistent with that in human, especially hyperactivity and susceptibility to auditory seizure. It was reported that limbic epileptogenesis markedly increased of mossy fibre sprouting in FMR1 KO mice. Our previous study also showed that FMR1 KO mice were more susceptible and more serious than wild type control mice during the patterns of hyperthermal-indused convulsion. FMR1 KO mouse has been generally accepted as an Serviceable epilepsy or seizures model. FMR1 knockout mouse, a single gene disease model, also provides a unique opportunity to study the molecular mechanism of epilepsy in hereditary mental retardation diseases.γ-aminobutyri acid(GABA), a major inhibitory neurotransmitter in the central nervous system, plays important role in the regulation of the excitability of neurons. Glutamic acid decarboxylase (GAD) is the rate-limiting enzyme for the coversion of glutamic acid intoγ-aminobutyri acid. Recent year's researches have confirmed that GAD65, GAD67 is closely related to epilepsy. It has been found that mutation of GABAA receptor gene may be predisposing factors of childhood absence epilepsy (CAE) and febrile seizures. Some study has shown that the GABA-immunoreactivity neurons and the expression of GABAARα5 subunit decreased after post-epilepticus.Voltage-gated sodium channels also play important role in the initiation and propagation of action potentials and are crucial regulators of neuronal excitability. However, it was been found that a loss-of-function mutation in human SCN1A gene causes severe infant myoclonic epilepsy. Further, the reduced sodium current was found in GABAergic inhibitory interneurons in SCN1A–heterozygotes mice. Immunohisto- chemistry reveals that Na (v)1.1 in the developing neocortex is predominantly clustered at the axon initial segments of parvalbumin-positive (PV) interneurons, suggesting that NaV1.1 plays critical role in the spike output from interneurons. It is speculated that abnormal inhibitory circuits caused by reduced function of NaV1.1 may contribute to epileptic seizures.The studies indicate that the amount of PV positive neurons and the GABAA receptors are significantly decreased in FMR1 KO mice when compared to the WT mice. In hippocampal slices from FMR1 KO mice, the prolonged epileptiform discharges was recorded by blocking GABAergic transmission with the GABAA receptor antagonist bicuculline. Therefore, we speculate that the decreased number and function of interneurons might lead to the increase of seizures susceptibility in FMR1 KO mice; and the sodium channel might be one of important roles in abnormal excitability of interneurons.Purpose By comparing with the changes of GAD, GABAARα5 expression between FMR1 KO mice and WT mice hippocampus, to investigat whether the changes numer and structure of GABA neurons in the FMR1 KO mice are susceptible to epilepsy. By comparing with co-expression NaV1.1 and GAD in FMR1 KO mice and WT mice and by detecting the electrophysiological changes of hippocampal GAD neurons and pyramidal neurons in FMR1 KO and WT mice , to find whether changes of neuronal excitability in the FMR1 KO mice plays a critical role in epilepsy.Methods1. Experimental animalsMale FMR1 KO mice of the FVB strain (The DutchBelgian Fragile X Consortium) and their WT littermates were used. All animals were maintained on a 12:12 h light/dark cycle with a constant room temperature, and with adlib food and water. The genotype of mice was identified before the experiment.2. Quantitative real-time-PCR for target genesThe bilateral hippocampus tissues were collected and homogenized for total RNA with Trizol.First strand cDNA was synthesized and real-time PCR cycles were carried out for amplification of GAD67, GABAARα5 and Nav1.1 respectively.The house-keeping gene was GAPDH.Real-Time PCR results were analyzed with ABI Prism SDS 7000 software.3. Western blottingTotal proteins were isolated from hippocampus with Trizol from each group, and the protein concentrations were determined. The proteins were separated by 7.5% SDS-PAGE and transferred to nitrocellulose membranes with a transblot SD apparatus, and incubated with primary antibody anti-NaV 1.1 , anti-GAD, Anti-GABARAα5 and anti-β-Actin respectively. Immunoblots were developed with enhanced chemiluminescence. Quantitative analysis was performed using Image Quant software. 4. ImmunohistochemistryMice were randomly obtained for immunofluorescence histochemistry to detect colocalization of GAD. sections were blocked and incubated with primary antibody anti-NaV 1.1, anti-GAD overnight at 4oC and with the secondary antibodies at room temperature for lh. Fluorescence signals were detected with a microscope at excitation/emission wavelengths of 495/519 nm (FITC,green) and 550/570 nm (Cy3,red).5. Patch ClampAcute isolation of neurons and sodium channel electrophysiological properties, including current density, activation, inactivation and recovery from inactivation were studied by the traditional whole cell recording6. Statistical AnalysisThe data were analyzed with SPSS 16.0 (SPSS Inc,Chicago, IL,USA). Results were compared by t-test analysis. The level of significance was set at P< 0.05.Results1. Increased expression of GAD and decreased number of GABAergic inhibitory interneurons in hippocampus in FMR-1KO miceNumber of GABAergic inhibitory interneurons in hippocampus : FMR1 KO mice 80±20.134 , WT mice95.11±14.186(P=0.007 ) ; CA1: FMR1 KO mice 32.24±7.710, WT mice 39.59±6.738 ( P=0.002 ) ; CA3: FMR1 KO mice 29.94±10.028,WT mice 35.07±6.324(P=0.043); DG: FMR1 KO mice 17.82±5.703, WT mice 20.44±3.545 (P=0.066); The expressions of GAD65 and GAD67 were significantly increased in the hippocampus tissues in FMR-1KO when compaired to that in WT mice (P=0.000). The level of GAD67-mRNA in FMR-1KO mice was also significantly increased when compaired to that in WT mice(P=0.006).2. Down- expression of GABAARα5 in FMR-1KO miceThe expressions of GABAARα5 were significantly decreased in the hippocampus tissues in FMR-1KO when compaired to that in WT mice (P=0.000). The level of GABAARα5-mRNA in FMR-1KO mice was also significantly decreased when compaired to that in WT mice(P=0.031).3. Increasing expression of NaV1.1 in FMR-1KO miceThe expressions of NaV1.1 were significantly increased in the hippocampus tissues in FMR-1KO when compaired to that in WT mice (P=0.000). The level of NaV1.1-mRNA in FMR-1KO mice was also significantly increased when compaired to that in WT mice(P=0.000).4. Co-localization of GAD and NaV1.1 in neuronsNav1.1 channel protein is localization of the two main types of projection neurons in the hippocampus: pyramidal neurons of pyramidal layer and granule cells of dentate gyrus granule cell body layer (GLU neurons) is weak expression, NaV1.1 protein in interneurons between the pyramidal layer and granular layer is a strong positive expression.5. Sodium channel properties in FMR-1KO mice5.1 FMR-1KO mice and WT mice pyramids neurons Voltage dependence of activation: V0.5 and k(slope factor) were: FMR-1KO mice(n=4)-34.56±2.23(mV),4.43±0.35 ; WT mice ( n=4 ) : -37.87±2.18(mV), 4.69±0.41 , (P > 0.05 ) ; FMR-1KOmice and WT mice interneurons Voltage dependence of activation: V0.5 and k(slope factor) were: FMR-1KO mice(n=4): -32.07±2.06(mV), 4.38±1.11; WT mice(n=4): -32.56±1.68(mV),5.02±0.4,(P>0.05).5.2 FMR-1KOmice and WT mice pyramids neurons Steady-stage inactivation : V0.5 and k(slope factor):FMR-1KO mice(n=4): -53.14±2.62(mV), 5.46±1.14; WT mice(n=4): -55.86±2.12 (mV), 5.68±0.91, (P>0.05);FMR-1KO mice and WT mice inter- neurons V0.5 and k(slope factor) : FMR-1KO mice ( n=4 ) : -55.15±2.99(mV),8.02±1.15;WT mice(n=4): -54.47±2.44(mV),6.42±1.85, (P>0.05).5.3 FMR-1KO mice and WT mice pyramids neurons recovery from inactivation: the time constant was : FMR-1KO mice ( n=4 ) : 6.25±1.66(ms) , WT mice(n=4):6.85±2.09(ms),(P>0.05);FMR-1KO mice and WT mice interneurons recovery from inactivation: FMR-1KO mice(n=4):6.79±1.55(ms),WT mice(n=4): 6.54±2.62(ms), (P>0.05) 5.4 FMR-1KO mice and WT mice pyramids neurons current density: KO(n=4):0.2932±0.106(nA/pF); WT mice (n=4): 0.1260±0.054 (nA/pF),P=0.037; FMR-1KO mice and WT mice interneurons current density: KO mice (n=4): 0.2668±0.047 (nA/pF); WT mice (n=4): 0.5163±0.058(nA/pF), P=0.044.Conclusion1. 1. The number of GABAergic inhibitory interneurons in hippocampus is reduced in the FMR-1KO mice, which might contribute to the weaken inhibitory action in interneurons.2. The expression level of GABAARα5 in hippocampus is down-regulation in the FMR-1KO mice, which might contribute to the weaken inhibitory action in interneurons.3. The expression level ofαsubunit of Voltage-gated sodium channel is increased in the FMR-1KO mice. The excitability of sodium channels is enhanced in the pyramidal neurons and it is reduced in the interneurons, which supports the interneurons might play an important role in the pathogenesis of epilepsy in FXS.4.The expressions of GAD and NaV1.1 in FMR-1KO mice are increased, which provides more evidence for that FMRP suppresses translation of target mRNAs.