Abstract：
BACKGROUND: Repeated sevoflurane exposures in neonatal rats may lead to neuronal apoptosis affecting long-term cognitive function, the mechanism is unknown. Neuroligin1 (NL1) is essential for normal excitatory transmission and long-term synaptic plasticity in the hippocampus of intact animals. Herein, we explore the role of NL1 in hippocampal excitatory synapses on long-term cognitive impairments induced by repeated sevoflurane exposures in neonatal rats.
METHODS: From postnatal day six (P6) to P8, neonatal rats were exposed to 30% oxygen or 3% sevoflurane +30% oxygen for 2 h daily. Rats from each litter were randomly assigned to five groups: control group (Con), native control adeno-associated virus (NC-AAV) group (Con + NC-AAV), sevoflurane group (Sev), sevoflurane + recombinant RNAi adeno-associated virus targeting NL1 downregulation (NL1--AAV) group (Sev + NL1--AAV) and control + recombinant RNAi adeno-associated virus targeting NL1 upregulation (NL1+-AAV) group (Con + NL1+-AAV). Animals were injected with NC-AAV or NL1-AAV into the bilateral hippocampal CA1 area and caged on P21. From P35 to P40, behavioral tests including open field (OF), novel object recognition (NOR), and fear conditioning (FC) tests were performed to assess cognitive function in adolescent rats. In another experiment, rat brains were harvested for immunofluorescence staining, western blotting, co-immunoprecipitation, and real-time polymerase chain reaction (PCR).
RESULTS: We found that the mRNA and protein levels of NL1 were substantially higher in the Sev group than in the Con group. Immunofluorescence showed that NL1 and PSD95 were highly colocalized in hippocampal CA1 area and vesicular GABA transporter (vGAT) around neurons decreased after repeated sevoflurane exposures. Co-immunoprecipitation showed that the amount of PSD95 with NL1 antibody was significantly increased in the Sev group compared to the Con group. These rats had a poorer performance in the NOR and FC tests than control rats when they were adolescents. These results were reversed by NL1--AAV injection into the CA1 area. NL1+-AAV group was similar to the Sev group.
CONCLUSIONS: We have demonstrated that repeated neonatal sevoflurane exposures decreased inhibitory synaptic inputs (labelled by vGAT) around neurons, which may influence the upregulation of NL1 in hippocampal excitatory synapses and enhanced NL1/PSD95 interaction, ultimately leading to long-term cognitive impairments in adolescent rats. Injecting NL1--AAV reversed this damage. These results suggested that NL1 in excitatory synapses contributes to long-term cognitive impairments after repeated neonatal sevoflurane exposures.
METHODS: From postnatal day six (P6) to P8, neonatal rats were exposed to 30% oxygen or 3% sevoflurane +30% oxygen for 2 h daily. Rats from each litter were randomly assigned to five groups: control group (Con), native control adeno-associated virus (NC-AAV) group (Con + NC-AAV), sevoflurane group (Sev), sevoflurane + recombinant RNAi adeno-associated virus targeting NL1 downregulation (NL1--AAV) group (Sev + NL1--AAV) and control + recombinant RNAi adeno-associated virus targeting NL1 upregulation (NL1+-AAV) group (Con + NL1+-AAV). Animals were injected with NC-AAV or NL1-AAV into the bilateral hippocampal CA1 area and caged on P21. From P35 to P40, behavioral tests including open field (OF), novel object recognition (NOR), and fear conditioning (FC) tests were performed to assess cognitive function in adolescent rats. In another experiment, rat brains were harvested for immunofluorescence staining, western blotting, co-immunoprecipitation, and real-time polymerase chain reaction (PCR).
RESULTS: We found that the mRNA and protein levels of NL1 were substantially higher in the Sev group than in the Con group. Immunofluorescence showed that NL1 and PSD95 were highly colocalized in hippocampal CA1 area and vesicular GABA transporter (vGAT) around neurons decreased after repeated sevoflurane exposures. Co-immunoprecipitation showed that the amount of PSD95 with NL1 antibody was significantly increased in the Sev group compared to the Con group. These rats had a poorer performance in the NOR and FC tests than control rats when they were adolescents. These results were reversed by NL1--AAV injection into the CA1 area. NL1+-AAV group was similar to the Sev group.
CONCLUSIONS: We have demonstrated that repeated neonatal sevoflurane exposures decreased inhibitory synaptic inputs (labelled by vGAT) around neurons, which may influence the upregulation of NL1 in hippocampal excitatory synapses and enhanced NL1/PSD95 interaction, ultimately leading to long-term cognitive impairments in adolescent rats. Injecting NL1--AAV reversed this damage. These results suggested that NL1 in excitatory synapses contributes to long-term cognitive impairments after repeated neonatal sevoflurane exposures.
摘要：
背景：新生大鼠反复暴露七氟醚可能导致神经元凋亡，影响长期认知功能，机制未知。Neuroligin1（NL1）对于完整动物海马的正常兴奋性传递和长期突触可塑性至关重要。在这里，我们探讨NL1在新生大鼠海马兴奋性突触中对七氟烷反复暴露引起的长期认知障碍的作用。
方法：从出生后第6天（P6）到P8天，新生大鼠每天暴露于30％氧气或3％七氟醚30％氧气2小时。每窝大鼠随机分为五组：对照组（Con），天然对照腺相关病毒（NC-AAV）组（Con+NC-AAV），七氟醚组（Sev），七氟醚+靶向NL1下调的重组RNAi腺相关病毒(NL1--AAV)组(Sev+NL1--AAV)和对照+靶向NL1上调的重组RNAi腺相关病毒(NL1+-AAV)组(Con+NL1+-AAV)。将NC-AAV或NL1-AAV注射到双侧海马CA1区并将其关在P21上。从P35到P40，包括开放场（OF）的行为测试，新颖的对象识别(NOR)，进行了恐惧条件（FC）测试以评估青春期大鼠的认知功能。在另一个实验中,收集大鼠大脑进行免疫荧光染色，西方印迹,免疫共沉淀,和实时聚合酶链反应（PCR）。
结果：我们发现Sev组NL1的mRNA和蛋白水平明显高于Con组。免疫荧光显示，反复七氟醚暴露后，NL1和PSD95在海马CA1区高度共定位，神经元周围的囊泡GABA转运蛋白（vGAT）减少。免疫共沉淀显示,与Con组相比,Sev组的PSD95与NL1抗体的量显著增加。这些大鼠在青少年时在NOR和FC测试中的表现比对照大鼠差。这些结果通过将NL1-AAV注射到CA1区域而被逆转。NL1+-AAV组与Sev组相似。
结论：我们已经证明，新生儿七氟烷反复暴露会降低神经元周围的抑制性突触输入（用vGAT标记），这可能会影响海马兴奋性突触中NL1的上调，并增强NL1/PSD95的相互作用，最终导致青春期大鼠的长期认知障碍。注入NL1--AAV逆转了该损伤。这些结果表明，反复暴露于新生儿七氟醚后，兴奋性突触中的NL1有助于长期认知障碍。
方法：从出生后第6天（P6）到P8天，新生大鼠每天暴露于30％氧气或3％七氟醚30％氧气2小时。每窝大鼠随机分为五组：对照组（Con），天然对照腺相关病毒（NC-AAV）组（Con+NC-AAV），七氟醚组（Sev），七氟醚+靶向NL1下调的重组RNAi腺相关病毒(NL1--AAV)组(Sev+NL1--AAV)和对照+靶向NL1上调的重组RNAi腺相关病毒(NL1+-AAV)组(Con+NL1+-AAV)。将NC-AAV或NL1-AAV注射到双侧海马CA1区并将其关在P21上。从P35到P40，包括开放场（OF）的行为测试，新颖的对象识别(NOR)，进行了恐惧条件（FC）测试以评估青春期大鼠的认知功能。在另一个实验中,收集大鼠大脑进行免疫荧光染色，西方印迹,免疫共沉淀,和实时聚合酶链反应（PCR）。
结果：我们发现Sev组NL1的mRNA和蛋白水平明显高于Con组。免疫荧光显示，反复七氟醚暴露后，NL1和PSD95在海马CA1区高度共定位，神经元周围的囊泡GABA转运蛋白（vGAT）减少。免疫共沉淀显示,与Con组相比,Sev组的PSD95与NL1抗体的量显著增加。这些大鼠在青少年时在NOR和FC测试中的表现比对照大鼠差。这些结果通过将NL1-AAV注射到CA1区域而被逆转。NL1+-AAV组与Sev组相似。
结论：我们已经证明，新生儿七氟烷反复暴露会降低神经元周围的抑制性突触输入（用vGAT标记），这可能会影响海马兴奋性突触中NL1的上调，并增强NL1/PSD95的相互作用，最终导致青春期大鼠的长期认知障碍。注入NL1--AAV逆转了该损伤。这些结果表明，反复暴露于新生儿七氟醚后，兴奋性突触中的NL1有助于长期认知障碍。
