Objective: To investigate the role of glutamatergic neurons in the dorsomedial periaqueductal grey (dmPAG) in regulating excessive defensive behaviors in mice with post-traumatic stress disorder (PTSD).
Methods: Eight-week-old male C57BL/6 mice were subjected to stereotactic injections of different recombinant adeno- associated viral vectors (rAAV2/9-CaMKII-mCherry, rAAV2/9-CaMKII-hM3Dq-mCherry and rAAV2/9-CaMKII-hM4Di-mCherry) into the bilateral dmPAG for chemogenetic activation or inhibition of the glutamatergic neurons, followed 2 weeks later by PTSD modeling by single prolonged stress. The looming test, response to whisker stimulation test and contextual fear conditioning (CFC) test were used to observe changes in defensive behaviors of the PTSD mice. The activity of glutamatergic neurons in the dmPAG were observed using immunofluorescence staining.
Results: Compared with the control mice, the mouse models of PTSD showed a shortened latency of flights with increased time spent in the nest, response scores of defensive behaviors and freezing time (all P<0.01). Immunofluorescence staining revealed significantly increased c-fos-positive glutamatergic neurons in the dmPAG of PTSD mice with defensive behaviors. Activation of the glutamatergic neurons in the dmPAG (in PTSD hM3Dq group) did not cause significant changes in the latency of flights or time in nest but obviously increased response scores of defensive behaviors and freezing time of the mice, whereas inhibiting the glutamatergic neurons in the dmPAG (in PTSD hM4Di group) caused the reverse changes and obviously alleviated defensive behaviors in the PTSD mice (P<0.05 or 0.01).
Conclusion: Inhibiting the activity of glutamatergic neurons in the dmPAG can alleviate defensive behaviors in mice with PTSD.
目的: 探索背内侧中脑导水管周围灰质（dmPAG）谷氨酸能神经元对创伤后应激障碍（PTSD）小鼠过度防御行为的调控作用。
方法: 84只雄性C57BL/6J小鼠随机分为12组（7只/组）。研究PTSD后过度防御反应时，分为CON组和PTSD组；研究激活dmPAG区谷氨酸能神经元对PTSD后过度防御反应的影响，分为CON NS组，CON mCherry组，PTSD mCherry组和PTSD hM3Dq组；研究抑制dmPAG区谷氨酸能神经元对PTSD后过度防御反应的影响，分为CON NS组，CON mCherry组，PTSD mCherry组和PTSD hM4Di组。构建单效长时程应激模型（SPS）模拟PTSD状态，通过视觉本能恐惧实验、触须刺激实验、条件恐惧实验测试小鼠的防御行为，免疫荧光染色观察dmPAG区谷氨酸能神经元活性变化。
结果: 与CON组相比，PTSD组回巢潜伏期缩短（P<0.001），巢内停留时间百分比增加（P<0.01），防御评分增加（P<0.001），僵直时间百分比增加（P<0.01）。免疫荧光染色结果可见PTSD小鼠防御行为过程中，dmPAG区c-fos阳性的谷氨酸能神经元占比明显增加（P<0.001）。与PTSD mCherry组相比，激活dmPAG区谷氨酸能神经元后，PTSD hM3Dq组回巢潜伏期、巢内停留时间百分比无差异，防御评分升高（P<0.05），僵直时间百分数增加（P<0.01）。与PTSD mCherry组相比，抑制dmPAG区谷氨酸能神经元后，PTSD hM4Di组回巢潜伏期延长（P<0.05），巢内停留时间百分比降低（P<0.05），防御评分降低（P<0.01），僵直时间百分比降低（P<0.01）。
结论: 抑制dmPAG区谷氨酸能神经元活性可以明显减轻PTSD的过度防御行为。
Keywords: defensive behavior; dorsomedial periaqueductal grey; glutamatergic neurons; midbrain neural circuits; post-traumatic stress disorder.