Characterization of Dentate Mossy Cell-restricted Genetic Manipulation Mice

齿状苔藓细胞限制性基因操作小鼠的表征

• 批准号: 8556960
• 负责人: Kazutoshi Nakazawa
• 金额: $ 6.9万
• 依托单位: NATIONAL INSTITUTE OF MENTAL HEALTH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8556960
• 关键词: Ablation; Acute; Address; Animals; Area; Back; Behavior; Behavioral; Cell Count; Cell Death Process; Cell physiology; Cells; Chronic Phase; DTR gene; Data; Diphtheria Toxin; Epilepsy; Epileptogenesis; Episodic memory; FOS gene; Gene Expression; Genes; Genetic; Goals; Hilar; Hippocampus (Brain); Hour; Human; Immediate-Early Genes; In Vitro; Injection of therapeutic agent; Interneurons; Kainic Acid; Knockout Mice; Lesion; Manuscripts; Measures; Medial; Mediating; Memory; Molecular; Mus; Myoepithelial cell; N-Methyl-D-Aspartate Receptors; Nerve Degeneration; Pattern; Phase; Preparation; Property; Pyramidal Cells; Recurrence; Reporting; Research; Role; Seizures; Slice; Staining method; Stains; Stimulus; System; Techniques; Temporal Lobe Epilepsy; Transgenic Mice; Transgenic Organisms; base; dentate gyrus; diphtheria toxin receptor; entorhinal cortex; fluoro jade; genetic manipulation; granule cell; in vivo; insight; interest; mossy fiber; mutant; neural circuit; neuron loss; research study; response; selective expression; tool

Earlier, we generated a conditional transgenic mouse line in which diphtheria toxin receptor was selectively expressed in mossy cells using the Cre/loxP system. Within one week after diphtheria toxin injection, we observed 80% loss of mossy cells throughout the longitudinal axis. We found no obvious or sustained epilepsy-like discharges in the hippocampus as measured by in vivo local field potential recordings. Interestingly, no mossy fiber sprouting was detected by Timm staining. These results suggested that, in contrast to previous reports showing that lesions of the entire hilar region induce massive mossy fiber sprouting and epilepsy, selective in vivo elimination of mossy cells does not trigger behavioral epilepsy or mossy fiber sprouting. This year, we found that dentate granule cells in the DT-treated mutants became hyperexcitable to afferent stimulation in in vitro slice preparation, and during this hyperexcitable state deficits in contextual pattern separation were detected. We also evaluated the immediate-early gene (IEG) expression in response to kainic acid (KA) injection under the assumption that an excitatory stimulus would cause more granule cells to discharge and activate IEG expression in mutants compared to controls. KA injection evoked Zif268 expression in more granule cells in mutants than in controls. We also examined the KA-induced seizure intensity. The cumulative seizure score of mutants for the hour following KA injection was significantly higher than controls. Together, these results all suggested an increase in granule cell excitability following mossy cell ablation. In summary, we concluded that mossy cell loss in vivo renders the granule cells hyperexcitable. Contrary to the predicted epileptogenesis implicit in the dormant basket cell hypothesis, however, it was insufficient to trigger the mossy fiber sprouting and epileptic discharges. Perhaps, in addition to the loss of mossy cells, neurodegeneration of other limbic areas, such as entorhinal cortex, is necessary to induce medial temporal lobe epilepsy. These findings provide new insights into the mechanisms of epileptogenesis in the limbic cortex. This year, for the manuscript second and third revisions, we conducted some more additional experiments. First, we assessed the degree of mossy cell degeneration after DT treatment by Nissl staining, by Fluoro-Jade B staining, and by double immunostaining. We found that many mossy cell-like cells are already degenerated by post-DT 7 days; however, these cells, even degenerated, appear to be structurally present at least by 6 weeks after DT. Therefore, we decided to use the term mossy cell degeneration, instead of mossy cell deletion when we describe the process of cell death. Second, in response to the reviewers claim that the variability is high, we re-examined the cell counting of kainic acid-induced Zif268 and c-Fos positive cells 5 weeks after DT injection (chronic phase). Now the number of animals are n=8 for controls and n=7 for mutants. We found that the overall variability for both IEG IR-positive cells was not so high compared to the acute phase data. And our conclusion that dentate granule cells are transiently over-excited upon mossy cell degeneration is still valid, based on the IEG data. The manuscript is submitted again and under review.

早些时候，我们产生了一个条件性的转基因小鼠系，其中白喉毒素受体选择性地表达在苔藓细胞中使用的Cre/loxP系统。 在白喉毒素注射后一周内，我们观察到80%的苔藓细胞损失整个纵轴。 我们没有发现明显的或持续的癫痫样放电在海马体中测量在体内局部场电位记录。有趣的是，通过Timm染色没有检测到苔藓纤维发芽。这些结果表明，与以前的报告显示，整个肺门区的病变诱导大量苔藓纤维发芽和癫痫相反，选择性地在体内消除苔藓细胞不会引发行为性癫痫或苔藓纤维发芽。今年，我们发现，DT处理的突变体中的齿状颗粒细胞在体外切片制备中对传入刺激变得过度兴奋，并且在此过度兴奋状态中检测到上下文模式分离的缺陷。 我们还评估了立即早期基因（IEG）的表达，在响应红藻氨酸（KA）注射的假设下，兴奋性刺激会导致更多的颗粒细胞放电和激活IEG表达突变体相比，控制。 KA注射诱发Zif 268在突变体中的颗粒细胞中的表达比在对照中的多。 我们还检查了KA诱导的癫痫发作强度。KA注射后1小时突变体的累积癫痫发作评分显著高于对照组。 总之，这些结果都表明苔藓细胞消融后颗粒细胞兴奋性增加。 总之，我们得出结论，苔藓细胞在体内的损失，使颗粒细胞过度兴奋。 然而，与休眠篮细胞假说中隐含的癫痫发生相反，它不足以触发苔藓纤维发芽和癫痫放电。 也许，除了苔藓细胞的损失，其他边缘系统区域的神经变性，如内嗅皮层，是必要的，以诱发内侧颞叶癫痫。这些发现提供了新的见解癫痫发生的机制在边缘皮层。 今年，在手稿的第二次和第三次修订中，我们进行了更多的实验。首先，我们通过Nissl染色、Fluoro-Jade B染色和双重免疫染色评估DT处理后苔藓细胞变性的程度。 我们发现，许多苔藓细胞样细胞已经退化后DT 7天，然而，这些细胞，即使退化，似乎是结构上存在的DT后至少6周。 因此，当我们描述细胞死亡的过程时，我们决定使用术语苔藓细胞变性，而不是苔藓细胞缺失。 其次，为了回应评论者声称变异性高的说法，我们在DT注射后5周（慢性期）重新检查了红藻氨酸诱导的Zif 268和c-Fos阳性细胞的细胞计数。现在，对照组的动物数量为n=8，突变体为n=7。 我们发现，与急性期数据相比，两种IEG IR阳性细胞的总体变异性并不高。基于IEG数据，我们关于苔藓细胞变性时齿状颗粒细胞短暂过度兴奋的结论仍然有效。 手稿再次提交并正在审查中。