Preparation Of GABRG2 Conditional Knockout Mice In Hippocampus And Neocortex And Its Effect On Learning And Memory

Objective To establish and identify the neocortex and hippocampus specific GABA_A receptorγ2 subunit（GABRG2）knockout mice via Cre/Loxp conditional gene knockout technology,and provide the important animal model for further investigating the functional role of GABRG2 in hippocampus and neocortex in epileptogenesis.Evaluating the effects of different temperature on seizures,and also further explore the impact of the GABRG2 gene on learning,memory and cognitive function in mice.Methods 1.The constructed GABRG2^fl/wt mice and the mice that express Cre recombinase to approximately 88%of the neurons of the neocortex and hippocampus were bred and authenticated,respectively.Mating and identification of GABRG2^fl/fl mice with Cre mice were carried out,and the GABRG2^fl/wt Cre⁺mice were screened.The Real-Time qPCR and Western Blotting were used to detect the expression of GABRG2 mRNA and protein levels in mouse neocortex and hippocampus.2.The seizures model of heat induced by rectal temperature probe.We observed the seizures of the mice,recorded the seizure classification and the number of jumps during heating process,and also evaluated the effect of temperature rising on seizures,and the seizures was recorded by electroencephalogram.3.Smart 3.0 software system was used to record and analysis animal behaviors in light/dark box（LDB）,open field（LF）,new object recognition test（NORT）,new location test（OLT）,and three chamber test（TCT）of mice in the control group and GABRG2 knockout group.The mice behavioral changes were used to analyze the anxiety,learning,memory and cognitive functions of mice.Results1.The genotype of GABRG2^fl/wtCre⁺mice were identified by agarose gel electrophoresis.Real-Time qPCR and Western blot results showed that the mRNA and protein level of GABRG2were significantly lower in hippocampus and neocortex in GABRG2^fl/wtCre⁺mice than those of control group.2.During the heat induced seizures process,the spike wave of cortical EEG in the experimental group was significantly higher than that in the control group.GABRG2 KO mice were more sensitive to heat induced seizures compared with control normal mice（0.75±0.09 vs.0.50±0.06,P<0.001）.The seizures became more pronounced with increasing temperature,and temperature elevation produced generalized tonic clonic seizures（GTCSs）in KO mice.3.In LDB experiment,the distance travelled（5 min）of GABRG2 knockout mice in the dark area（1115.62±283.76 cm vs.1880.53±309.42 cm,P<0.001）and the number of transitions（9.38±2.77 vs.32.13±10.11,P<0.001）were significantly reduced compared with WT mice,while the percentage of time spent in the dark area was higher in KO group than the control group（69.82±7.16%vs.60.34±3.34%,P<0.01）after heating at 37℃for 30 min.After incubating at 39.5℃for 30 minutes,the distance of KO mice in the dark box（646.32±106.32cm vs.1835.27±278.14 cm,P<0.001）and the number of transitions（8.00±2.27 vs.29.13±11.26,P<0.001）were significantly decreased,and the percentage of time in the black（53.27±4.76%vs.65.34±6.34%,P<0.01）was higher than that of the control group.4.The OF test results suggested that the distance travelled（5 min）of GABRG2 knockout mice in marginal zone had significantly higher than that of wild mice（3791.80±630.67 cm vs.1552.63±581.86 cm,P<0.001）and（3043.45±451.22 cm vs.1368.41±412.79 cm,P<0.001）,however the distance in the center zone was lower than that of wild mice（505.29±187.01 cm vs.1038.26±173.75 cm,P<0.01）and（381.27±139.72 cm vs.1156.47±342.17cm,P<0.001）after heating at 37℃and 39.5℃for 30 min,respectively.After respectively incubation at 37℃and 39.5℃for 30 min,the time spent in center of KO mice was significantly lower than that of WT mice（13.41±8.16 s vs.53.29±7.31 s,P<0.001）and（10.18±5.27 s vs.55.73±6.61 s,P<0.001）,while the total rearing times of KO group were significantly higher than that in the WT group（163.47±51.28 times vs.22.78±5.72 s）,P<0.0001)and（131.37±22.79 s vs.26.37±7.13 s,P<0.0001）.5.The results of the NORT showed that the KO mice and wild mice had no significant difference in the exploration time to the same left and right objects after incubation at 37℃and 39.5℃for30 min,respectively（WT:59.27±11.41 s vs.61.39±8.74 s,P>0.05;WT±39.5℃:57.16±7.79 s vs.63.25±9.17 s,P>0.05;GABRG2 KO:63.30±6.19 s vs.60.31±7.33 s,P>0.05;GABRG2 KO+39.5℃:59.72±6.93 s vs.62.33±7.83 s,P>0.05）;for the novel object,the wild mice showed more obvious interest,and the exploration time was significantly longer than familiar objects（WT:27.54±9.37 s vs.64.62±7.40 s,P<0.01;WT+39.5℃:21.39±6.62 s vs.62.57±8.68 s,P<0.01）,while there is no significant difference between the novel objects and familiar objects in KO mice（GABRG2 KO:49.61±8.73 s vs.52.37±5.73 s,P>0.05;GABRG2 KO+39.5℃:51.32±5.92 s vs.54.21±5.33 s,P>0.05）.6.The OLT results indicated that the knockout mice and wild mice had no significant difference in the exploration time to the same left and right objects at the same horizontal position after incubation at 37℃and 39.5℃for 30 min,respectively（WT:53.47±5.27 s vs.55.14±5.73 s,P>0.05;WT+39.5℃:58.19±6.16 s vs.55.14±7.20 s,P>0.05;GABRG2 KO:57.21±5.36s vs.54.29±5.71 s,P>0.05;GABRG2 KO+39.5℃:49.97±6.72 s vs.52.31±7.37 s,P>0.05）;when one of the objects was placed in the new location,it was observed that the same object in the new location was visited longer in the wild group（WT:21.73±5.29 s vs.71.62±8.31 s,P<0.001;WT+39.5℃:20.77±8.24 s vs.66.48±6.73 s,P<0.001）,while there is no significant difference in GABRG2 KO group（GABRG2 KO:48.33±10.73 s vs.53.37±9.21s,P>0.05;GABRG2 KO+39.5℃:49.92±7.87 s vs.53.21±6.33 s,P>0.05）.7.In TCT,these results suggested that the wild type mice showed more significant social interaction,and the interaction time with stranger is longer than that with empty in the first trail familiarity phase（WT:87.60±11.18 s vs.23.46±8.57 s,P<0.001;WT+39.5℃:90.33±6.39 s vs.19.14±8.77 s,P<0.001;GABRG2 KO:29.24±13.26 s vs.33.10±7.71 s,P>0.05;GABRG2 KO+39.5℃:27.38±7.23 s vs.29.46±9.04 s,P>0.05）.A new mouse of different nests was placed in the empty cage as novelty object,the knockout mice and the wild group performed significant differences in the social novelty of the second trail,the wild group mice showed more obvious social interaction,and the interaction time with the novel mice was longer than that of familiar mice（WT:88.13±12.37 s vs.25.10±6.44 s,P<0.001;WT+39.5℃:90.71±9.22 s vs.21.37±9.77 s,P<0.001;GABRG2 KO:33.71±9.83 s vs.30.27±8.53 s,P>0.05;GABRG2 KO+39.5℃:29.71±8.14 s vs.26.31±7.57 s,P>0.05）.Conclusion Based on the Cre/loxp conditional gene knockout technology,we succeed in building a group of neocortex and hippocampus specific GABRG2 gene knockout mice,and it is more sensitive to heat induced seizures.GABRG2 gene knockout in hippocampal and neocortical in mice can decrease the cognitive function,which may be closed to the impairment of hippocampal and neocortical functions,and seizures in mice.