Auditory Brainstem Response And Distribution Of GAD In Olivocochlear And Cochlear Nuclei In FMR-1 Knockout Mice

Introduction:Fragile X syndrome (FXS) is a common form of mental retardation, and it is caused by a trinucleotide expansion in the fragile X mental retardation 1 (fmr1) gene that prevents the expression of the encoded protein, called fragile X mental retardation protein (FMRP). In addition to the hallmark cognitive defect, other symptoms are also apparent including hyperactivity, seizures and sensory abnormalities including a characteristic increase in sensitivity to auditory, tactile, visual, and olfactory stimuli. Fragile X is a developmental disorder with the first symptoms apparent in the first year of life but little is known about the role of FMRP in developmental processes. The Fmr1 knockout (KO) mouse is characterized by an increased audiogenic seizure (AGS) susceptibility and is considered a good animal model for epilepsy and seizures in the human fragile-X (FRAX) syndrome. Recent evidence suggests that the lack of FMRP leads to aberrant synaptic plasticity which may be a seminal mechanism underlying mental retardation and other FXS phenotypes. Synaptic plasticity which either potentiates or depresses synaptic strength is essential to learning and memory and has been well characterized in the hippocampus. Depression of synaptic strength is mediated by at least two different pathways involving either N-methyl-Daspartate(NMDA) receptor or metabotropic glutamate receptor (mGluR) signaling. In addition, Fmr1 KO mice show an increased susceptibility to audiogenic seizures (AGS) at all ages. A recent study showed reduced GABAA receptor expression could result in increased GAD expression. This study was designed to identify Fmr1 knockout mice, AGS susceptibility, the ABR threshold and the distribution of GAD in auditory cortex, olivocochlear and cochlear nuclei of these mice was examined and compared to wild type at different ages.Materials & Methods:1. Experimented animalsFVB strain mice, Fmr-1 knockout (FMR-/-) and their wild type (FMR+/+) counterparts were used. They were genotyped by PCR to confirm the lack of the FMR-1 gene .Experimented animals were divided into 5 groups according to their genotype and age: KO group(3w,4w,6w,8w,10w) and WT group(3w,4w,6w,8w,10w), each group has 30 FVB mouse.2. Fmr1 knockout mice were identified using the PCR technical and audiogenic seizures were used in the study . The intensity of response was scored and the results were analyzed by means of one-way analysis of variance to evaluate the effects of age and genetic condition.3. ABR test and Immunohistochemistry: ABR was detected after anesthesia. The cryostat sections were utilized for Streptavidin-Peroxidase immunohistochemistry with antibodies.4. Image analysis and statisticsThe mean optical density of immunostaining signal of the expression of GAD protein in Olivocochlear and cochlear nuclei were determined by Image J system. All the data were calculated and analysis with statistic software SPSS 16.0. The accepted level of significance was 0.05. Results:1. The Fmr1 knockout mice exhibited increased excitability at different ages (P<0.05).2. ABR threshold of KO group(3w,4w) was increased remarkably compared with the control group(P<0.01). There were no differernce between KO group and WT group in 6w, 8w and 10w, respectively(P>0.05).3. The mean optical density values of immunostaining signal of GAD were obtained from immunohistochemistry in Olivocochlear and cochlear nuclei were significantly increased in KO group compared to WT group in all ages (P<0.01).Conclusion:1. Mice lacking the gene encoding fragile X mental retardation protein (FMR-1) are susceptible to audiogenic seizures. The Fmr1 knockout (KO) mouse is characterized by an increased audiogenic seizure (AGS) susceptibility.2. ABR threshold has significantly difference between immature KO mice and immature WT mice. ABR threshold has no significantly difference between mature KO mice and mature WT mice.3. The expression of GAD of olivocohlear and cochlear nuclei is increased in KO mice and this change may suggests that FMRP negatively regulates the expression of GAD.