| Fragile X syndrome (FXS) is one of the most common forms of mental Retardation, Whose prevalence rate is only inferior to Down syndrome. It accounts for 2%-6% of non-special mental retardation and 40% of X-linked mental retardation. A trinucleotide repeat sequence expansion in the FMR1 gene leads to the decrease or lack of the encoded protein, called fragile X mental retardation protein (FMRP). The patients of FXS have the following abnormal symptom of different degrees: mental retardation, developmental delay, attention deficit and hyperactivity, epilepsy, loneliness, anxiety with mood lability, obsessive–compulsive and autistic behaviors, macroochidism, and special feature or worries. The diversity of the above-mentioned symptoms has connection with the following factors: 1. the production change of FMRP caused by the change of expression degree of FMR1; 2. direct or indirect influence to FMRP by other background genes; 3. the environment. So, in order to find out the complicated system of FXS, the patients'intelligence, behaviours and other related issues are now becoming the research focus.At present, the behaviour study of FXS mainly focuses on the followings: ability of study and remembrance, hyperactivity, social relationship, worriment, movement abilities, unusual reaction to feeling or stimulation, etc. In view of the patients of FXS prone to epilepsy, it is also currently focus that people carry on epilepsy research exploiting the mouse model with FXS. The first part we carry through a behaviour observation separately to the minor and adult knock-out mice. The second part we observed the change of glutamate decarboxylase (GAD) expression in brain of FMR1 knockout mice. Here we also explore internal relationship between abnormal behavior of FMR1 knockout mice and GAD, in order to find bases for the pathogenesis and treatment programs about FXS.Materials and MethodsAfter the genotype identification, FMR1 knock-out mice (KO mice) and wild type mice (WT mice) will be bred and reproduced by Experiment Animal Center of Guangzhou Medical College, this research the first part adopts experiments of audiogenic seizure, autonomic activities, step-through test, step-down test, and open field test to carry through the behaviour observation. The second part we adopt two methods about immunohistochemistry and Western blot to observe the numbers of GAD65/67-positive neurons and expression and variation of GAD protein in each region of the hippocampus and cortex of FMR1 knock-out mice. Results1.The results of audiogenic seizure experimentKO mice at different ages all have outbreak of AGS. The AGS susceptibility of three-week KO mice is the strongest, but there is a low-rated seizure severity score(SSS). However, WT mice have high AGS susceptibility and no status epilepticus. As age grows, the outbreak percentage of AGS and SSS of KO mice will decrease gradually, and WT mice have no outbreak of AGS.2. The results of Morris Water Labyrinth Experiment(1) Space navigation experimentFrom the first day to the third day, KO mice have no Obviously difference with the WT mice in the Latency and number of crossing platform (P>0.05), but on the fourth day, there is a statistical significance (P<0.05); the six-weeek mice have no difference with the four-week mice of the same type in the latency and number of crossing platform (P>0.05).(2) Space search experimentThe four-week and six-week WT mice will stay longer than the other mice at the target quadrants; the six-week KO mice stay at the fourth quadrant longer; and the four-week KO mice stay at the second quadrant longer.3. The results of the open field testKO mice and WT mice of same weeks have no different stay time at the first area (nearby area) (P>0.05), but there is difference at the fourth area (central area) (P<0.05); KO mice of same weeks have longer movement distance at the four areas than the WT mice (P<0.05);the times of KO mice crossing the central area are obviously more than WT mice(P<0.05); the activity and ambulation times of KO mice at the central area are obviously more than the WT mice(P<0.05), but they have no big difference at speed and standing still (P>0.05), and the same tpye of mice at two ages have no difference in various index(P>0.05).4. The results of autonomic activities, step-through test and step-down test(1) results of autonomic activitiesThe activity times of KO mice are obviously more than the WT mice of the same week (P<0.05), but the standing times of KO mice are obviously less than WT mice (P<0.05). The same type of mice of different weeks have no difference in activity and standing times (P>0.05).(2) results of step-through testKO mice obviously have the shorter Latency than the WT mice of the same week, but KO mice obviously have more number of errors than WT mice (P<0.05); On the first day, the Latency and number of errors of KO mice have no difference compared with the second day (P>0.05); On the first day, the Latency and number of errors of WT mice have distinct difference compared with the second day (P<0.05).(3) results of step-down testKO mice obviously have the shorter Latency than the WT mice of the same week, but KO mice obviously have more number of errors than WT mice (P<0.05); On the first day, the Latency and number of errors of KO mice have no difference compared with the second day (P>0.05); On the first day, the Latency and number of errors of WT mice have distinct difference compared with the second day (P<0.05).5. The results of Expression of GAD in brain of FMR1 knockout mice(1) results of GAD65/67 immunohistochemistry method KO mice obviously have the fewer number of GAD65/67-positive neurons (in region of the hippocampus and cortex) than the WT mice of the same week (P<0.05). The six-week KO mice and WT mice have the greater number of GAD65/GAD67-positive neurons (in region of the hippocampus and cortex) than the two-week counterpart (P<0.05).(2) results of GAD65/67 Western blot methodKO mice have the more expression of GAD65/67 protein (in region of the hippocampus and cortex) than the WT mice of the same week (P<0.05). Between the different ages, the six-week KO mice and WT mice have the more expression of GAD65/67 protein (in region of the hippocampus and cortex) than the one-week counterpart (P<0.05).Conclusions1. The KO mice have audiogenic seizure susceptibility, learning disorder, increase of athletic ability, increase of excitement, increase of exploring behaviour, increase of autonomic activities, and low ability of learning and memorizing. Many behaviours of FMR1 Knock-out mice are quite similar to that of the patients of Fragile X syndrome.The above-mentioned KO behaviours have connection with the FMRP expression.2. The changes of the numbers of GAD65/67-positive neurons and the GAD protein in KO mice may play an important role in theirs own epilepsy and abnormal behavior in the FXS... |
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