| Background Synaptic plasticity and changes in learning and cognitive function were found in both epileptic patients and animal models.Our previous studies have shown that the increased expression of Rac1 in the brain is associated with epilepsy.Whether Rac1 is associated with synaptic plasticity and changes in learning and cognitive function in animal models of epilepsy remains unclear.In this study,in vitro brain slices and animal models of epilepsy were used.In vitro brain slices were divided into control group,Rac1 inhibitor group and Rac1 agonist group.Postsynaptic potential(PSP)and HFS-induced LTP were recorded.Y-maze and Morris water maze were used in behavioral study.The experiment was divided into control group,epilepsy group,epilepsy group + Rac1 inhibitor,epilepsy group+ Rac1 agonist group.The effects of Rac1 inhibitors and agonists on synaptic plasticity in hippocampus of normal rats and learning and cognitive function in epileptic animal models were studied.The results show that Rac1 plays an important role in synaptic plasticity.Rac1 agonists enhance the learning and memory function of epileptic animals.Rac1 inhibitors impair the learning and memory function.Objective The effects of Rac1 agonists and inhibitors on synaptic plasticity of hippocampal slices in vitro and on learning and cognitive function in epileptic animal models were observed.Methods 1.Electrophysiology Postsynaptic potential(PSP)recording and LTP induction In SD rats of 4 weeks old,10% chloral hydrate was injected into abdominal cavity for anesthesia.The thoracic cavity was opened quickly with a scalpel and perfused with pre-frozen slice solution.After seeing limbs turn white,the brain tissue was cut off and placed in the slice solution immediately.The hippocampus was cut horizontally with a vibration slice machine and the slices containing hippocampus were quickly transferred tothe recording trough.The artificial cerebrospinal fluid was perfused continuously with mixed gases(95% O 2 + 5% CO 2)at a temperature of 30 ~32.Brain slices were moved to the recording trough.Stimulation electrodes were placed in Schaffer collateral of CA3 area and stimulated the pathway.Radiation layer(SR)of CA1 area of glass electrode was recorded and PS was recorded.After 15-30 minutes of stable recording,LTP was induced by high frequency stimulation(HFS)for 1 hour.In order to test the effects of specific Rac1 agonists or antagonists on LTP,brain slices were pretreated for 15-20 minutes before HFS.2.Ymaze Y maze is widely used in functional decline,directionality and spatial working memory.Y maze is mainly used to study the working memory of rodents in dynamic space.It explores new and different environments based entirely on the nature of experimental animals.Because every time an animal changes the direction of detection,it needs to remember the direction of the previous detection.Y-maze experiment can effectively determine the ability of the animal to work in space.The time and distance of the memory damage detector in the new arm will be shortened.On the contrary,the improvement of learning cognitive ability increases the time and distance of novice arm exploration.3.Morris water maze experiment The specific methods of water maze experiment are as follows: 1.Location navigation experiment: rats were put into water facing the wall of the pond at a fixed point of four quadrants and the platform was the end point.The time from the beginning to the end point of the swimming was recorded,that is,the escape latency.The maximum escape latency allowed in the experiment was 120 seconds.Each group of rats entered the water once from four quadrants,and the escape latency was recorded.Every day at a fixed time,a total of 5 days.And keep the environment temperature and other conditions.(2)Space exploration experiment: Remove the platform,put rats into the water in the relative quadrant of the platform,record the number of times crossing the platform area in 120 seconds and the swimming time in the platform quadrant.Data acquisition and processing are accomplished by water maze image automatic monitoring and processing system.The results of each subgroup were compared.The escape latency is an important experimental index.Its duration reflects the learning and memory ability of animals.The shorter the escape time,the better the learning and memory ability.Results Figure 1: Rac1 inhibitors affect late LTP in a concentration-dependent manner.Rac1 inhibitor NSC23766(60 umol/l)reduced LTP in rats.Especially in 55-60 minutes after HFS(from 1.463±0.117 to 0.99± 0.22 P < 0.002 * P < 0.05,** P < 0.01,and *** P < 0.001).After univariate analysis of variance,paired t test was performed.Normal control group: n = 5,30 micromol / L group: n = 6;60 micromol / L group: n = 6).Figure 2: Rac1 agonist significantly increased LTP.Rac1 agonist ML099 significantly increased LTP in rats.Especially in 55-60 minutes after HFS,60 nmol/l was compared with the control group(from 1.463±0.117 to2.078±0.254 P < 0.01),300 nmol/l was compared with the control group(from 1.463±to0.117 to 1.869±0.167 P < 0.001).After univariate analysis of variance,paired t test was performed.Normal control group: n = 5,60 nmol/L group: n = 5;300 nmol/L group: n = 7.Figure 3: The exploratory time of Y-labyrinth new heterodyne shows:(1)The learning and memory abilities of epilepsy group were impaired compared with the control group.Specifically,the exploration time of new heterodyne in epilepsy group was significantly shorter than that in the control group,which decreased from 156.0±14.85)to 107.02± 10.13 P < 0.05.(2)The learning and memory abilities of epilepsy + NSC23766 group were impaired compared with the control group,and the exploration time of new allograft in epilepsy + NSC23766 group was significantly shorter than that in the control group,from156.0 + 14.85 to 62.50±13.03,P < 0.05.(3)Compared with epilepsy group,learning,memory and cognitive function of epilepsy + ML099 group were significantly improved,and the exploration time of epilepsy+ ML099 group was significantly increased compared with epilepsy group.The exploration time of epilepsy + ML099 group was 147.8 ±20.12 higher than 107.02 ±10.13,P < 0.05.These results suggest that Rac1 agonist ML099 improves learning,memory and cognitive function in epileptic mice.(4)Compared with epilepsy + NSC23766 group,epilepsy + ML099 group significantly increased learning,memory and cognition,and epilepsy + ML099 group significantly increased the exploration time of new allobrachium compared with epilepsy +NSC23766 group,147.8±20.12 significantly increased compared with 62.50±13.03 P <0.05.Figure 4: Total number of Y-labyrinth arms(1)Compared with the control group,the learning and memory ability of epilepsy group was impaired.The total number of arm strikes in epilepsy group was significantly reduced from 24.25(+1.500)to 15.25(+2.630),P < 0.05.(2)The learning and memory ability of epilepsy + NSC23766 group was impaired compared with the control group,and the total number of arm strikes in epilepsy +NSC23766 group was significantly reduced compared with the control group.It decreased from 24.25±1.500 to 8.000±0.8165 P < 0.05.(3)The learning,memory and cognitive function of epilepsy + ML099 group were significantly improved compared with epilepsy group.The total number of arm strikes in epilepsy + ML099 group was significantly increased compared with epilepsy group,21.50±3.416 was significantly increased compared with 15.25±2.630,P < 0.05.These results suggest that Rac1 agonist ML099 improves learning,memory and cognitive function in epileptic mice.(4)Compared with epilepsy + NSC23766 group,epilepsy + ML099 group significantly increased learning,memory and cognition,and epilepsy + ML099 group significantly increased the total number of arm movements compared with epilepsy +NSC23766 group,21.50±3.416 significantly increased than 8.000±0.81653 group,P <0.05.Fig.5: Morris water maze escape latency:(1)Learning and memory impairment in epilepsy group compared with control groupThe escape latency time of epilepsy group was longer than that of control group on the 2nd,3rd,4th and 5th day,and increased from 52.97±13.94 on the 2nd day to 76.80±11.86 P < 0.01 on the 2nd day in epilepsy group,from 39.65±10.74 on the 3rd day in control group to 59.94±6.239 P < 0.05 on the 3rd day in epilepsy group,from 23.16 ±8.906)on the 4th day in control group to 42.21±6.273 P < 0.05 on the 4th day in epilepsy 7.63±1.360 increased to 28.97±3.932 P < 0.05 on the fifth day in epilepsy group.(2)Learning and memory impairment in epilepsy + NSC23766 group compared with control groupThe escape latency of epilepsy+NSC23766 group was longer than that of control group on the 2nd,4th and 5th day,and increased from 52.97±13.94 on the 2nd day to71.96±11.61 P<0.05 on the 2nd day of epilepsy+NSC23766;from 23.16±8.906 on the 4th day of control group to 42.37±6.208 P<0.05 on the 4th day of epilepsy+NSC23766 group;from 9.763±1.360 on the 5th day of control group to 29.80±+3.313 P<0.05 on the 5th day of epilepsy group.(3)The learning and memory abilities of epilepsy group+ML099 were similar to those of control group.The escape latency time of epilepsy group + ML099 and control group on the 1st,2nd,3rd,4th and 5th day was P > 0.05,which had no statistical significance.These results suggest that Rac1 agonist ML099 can improve the learning and memory ability of epileptic mice.Conclusions 1.Rac1 affects synaptic plasticity in rat hippocampus.The Rac1 inhibitor NSC23766 significantly reduced LTP in a dose-dependent manner.The Rac1 agonist ML099 significantly increased LTP.2.Rac1 agonists and inhibitors have an effect on learning and memory function.Compared with the control group,the epilepsy group and epilepsy+NSC23766 group had impaired learning and memory ability.The epilepsy group+ML099 was equivalent to the learning and memory of the control group.The epilepsy group+ML099 had significantly improved cognitive function compared with the epilepsy group. |
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