Long-term Effects Of γ-aminobutyric Acid A Receptor α1and γ2Subunit Modulators On Immature Rats With Recurrent Seizures

Objective: To investigate the effects on the gamma-aminobutyric acid A (GABA(A))receptor subunit alpha1and gamma2and the impact of long-term neuroethology afterrecurrent seizures of immature rats used agonist clonazepam and antagonist flumazenil, inorder to understand the intervention mechanism of GABA(A) receptor modulators in theseizure-induced brain injury.Methods:160of postnatal5-day-old (P5) Waster rats were divided randomly intofive groups: normal control group (CONT group, n=32), the seizure group (RS group,n=32); normal saline intervention group after seizures (NS group, n=32); clonazepamintervention group after seizures (CZP group, n=32); flumazenil intervention group afterseizures (FMZ group, n=32). All rats except CONT group daily inhaled flurothyl inducedseizures five times, each time interval2h,5consecutive days; rats in CONT group, giventhe same intervention but not inhaled flurothyl. CZP group rats and FMZ group rats weregiven clonazepam6.0mg/(kg·d)、flumazenil1.0mg/(kg·d) intervention after the end ofseizures1d(P11), sharing two times intragastric administration,continuous5d. NS groupwere given equal weight of normal saline intervention after the end of seizures1d(P11),sharing two times intragastric administration,continuous5d. Then to examine neural reflex(Plane righting test, forelimb suspension test, cliff avoidance test, negative geotaxis test),open field test, balance beam experiments, Morris water maze test to eval1uate theneurobehavioral. Western blot and fluorescence quantitative RT-PCR was employed todetermine GABA(A) receptor subunits α1and γ2expression at different times points(0d,20d,55d) after the last intervention.Result:1. Behavior analysis:(1) Cliff avoidance: The time of cliff avoidance in RSgroup and NS group were both longer than CONT group (P<0.05); the time of cliffavoidance in CZP group was longer than NS group (P<0.05); the time of cliff avoidance inFMZ group was not different from the NS group (P>0.05).(2) Negative geotaxis: the RS group and NS group inverted180°required time were both significantly longer than theCONT group (P<0.05); the CZP group inverted180°required time was significantlylonger than the NS group (P<0.05); the FMZ group inverted180°required time was notdifferent from the NS group (P>0.05).（3）Open-field test: The central grid residence timeof CZP group was longer than the NS group(P<0.05); the across grid of the CZP groupcompared with the NS group was significantly reduced (P<0.01).（4）Balance beam test:The average score of the RS group was significantly lower than the CONT group (P<0.05);the average score of the CZP group was significantly lower than the NS group(P<0.05).The average score of the FMZ group was not different from the NS group(P>0.05).2. Learning and memory: Morris water maze test:①The average latencies of all ratswere reduced by degree from D1to D5(P61-P65). The Stage incubation period in RSgroup was longer than the CONT group at D3and D4(P<0.05); the Stage incubationperiod in the CZP group was longer than the NS group at D3and D4(P<0.05); the Stageincubation period in the FMZ group was not different from the NS group at D3and D4(P>0.05).②Search strategy: The CONT group mainly used straight lines and tendsstrategy, while the other four group used more of an edge and random type.③Thefrequency of through the platform: The frequency of through the platform in RS group wassignificantly reduced compared to the CONT group (P<0.05); the frequency of through theplatform in CZP group was significantly reduced compared to the NS group (P<0.05).3. Western blot anal1ysis: With the growth of day-old, the protein expression levelsof GABA(A) receptor alpha1and gamma2subunits gradually increased in all rats. Afterthe last day (P15) intervention, the protein expression of GABA(A) receptor alpha1subunit in CZP group was not significant difference compared to the NS group,the proteinexpression of GABA(A) receptor gamma2subunit in CZP group was significantly lowerthan the NS group(P<0.01). The protein expression of GABA(A) receptor alpha1andgamma2subunits in the FMZ group was not different from the NS group (P>0.05). Afterthe last intervention20d and55d (P35&P70), the protein expression of GABA(A) receptoralpha1and gamma2subunit in the CZP group was lower than the NS group (P<0.05), atthe same time the protein expression of GABA(A) receptor alpha1and gamma2subunitsin the FMZ group was not different from the NS group (P>0.05). 4. RT-PCR analysis:With the growth of day-old, the GABA(A) receptor alpha1andgamma2subunits mRNA gradually increased in all rats. After the last day (P15)intervention, the GABA(A) receptor alpha1subunit mRNA expression levels in the CZPgroup was significantly lower than the NS group (P<0.01), while gamma2subunit mRNAexpression levels in the CZP group was not different from the NS group (P>0.05). Afterthe last intervention20d and55d (P35&P70), the GABA(A) receptor alpha1and gamma2subunits mRNA expression levels in the CZP group were significantly lower than the NSgroup (P<0.05), at the same time the GABA(A) receptor alpha1and gamma2subunitsmRNA expression levels in the FMZ group was not different from the NS group (P>0.05).Conclusions:(1) Flurothyl can induced Waster rats recurrent seizures, the immature rats were givenGABA(A) receptor agonist CZP after recurrent seizures will become some damage in brain,resulting in the rat growth retardation, reflex delay, and generates adulthood decreasedability to adapt new environment, as well as long-term learning and memory abilitydamage.(2) In the hippocampal of immature rats given GABA(A) receptor agonist CZP afterrecurrent seizures, the GABA(A) receptor alpha1and gamma2subunits related genes andprotein expression were affected in short term, and the effect exist for a long time, whichmay be long-term impact on the brain development of neuronal function.(3) The immature rats given GABA(A) receptor agonist CZP after recurrent seizurescause long-term neurobehavioral changes and cognitive impairment, also changedadulthood GABA(A) receptor subunit alpha1and gamma2subunit expression; thelong-term change of subunit expression participate in learning and memory impairment,and produce long-term effects on brain function.(4) The immature rats given GABA(A) receptor antagonist FMZ after recurrentseizures compared to the normal saline intervention rats after seizures, the GABA(A)receptor alpha1and gamma2subunits related genes and protein expression were slight increased, but the difference was not significant, presumably the FMZ antagonismoccurred mainly in the case of the benzodiazepine class of drugs has been used.