Characterization of Dentate Mossy Cell-restricted Genetic Manipulation Mice

早些时候，我们建立了一个条件转基因小鼠系，其中白喉毒素受体在苔藓细胞中使用Cre/loxP系统选择性表达。白喉毒素注射后一周内，我们观察到80%的苔藓细胞在纵轴上消失。在活体局部场电位记录中，我们在海马区没有发现明显或持续的癫痫样放电。有趣的是，TIMM染色未检测到苔藓纤维发芽。这些结果表明，与以前的报道相反，整个肺门区的损伤会导致大量苔藓纤维发芽和癫痫，体内选择性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，在体外切片制备中，DT处理的突变体中的齿状颗粒细胞对传入刺激变得过度兴奋，并在这种过度兴奋状态下检测到上下文模式分离的缺陷。我们还评估了海人酸(KA)注射后即刻早期基因(IEG)的表达，假设与对照相比，兴奋性刺激会导致突变体中更多的颗粒细胞释放并激活IEG表达。KA注射诱导突变组Zif268在颗粒细胞中的表达高于对照组。我们还检测了KA诱导的癫痫发作强度。突变组在注射KA后1小时内的累积癫痫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的兴奋性增加。综上所述，我们得出结论，体内苔藓细胞的丢失使颗粒细胞过度兴奋。然而，与休眠篮子细胞假说中隐含的癫痫发生预测相反，它不足以触发苔藓纤维发芽和癫痫放电。也许，除了苔藓细胞的丧失外，其他边缘区域的神经退化，如内嗅觉皮质，也是诱发内侧颞叶癫痫所必需的。这些发现为研究边缘皮质癫痫的发生机制提供了新的见解。 今年，对于第二版和第三版的手稿，我们又进行了一些额外的实验。首先，我们通过尼氏染色、荧光Jade B染色和双重免疫染色来评估DT处理后苔藓细胞的变性程度。我们发现，许多苔藓样细胞在DT后7天已经退化；然而，这些细胞，甚至退化，似乎至少在DT后6周才出现结构上的存在。因此，我们决定用苔藓细胞变性来描述细胞死亡的过程，而不是苔藓细胞的删除。其次，针对文献中提出的变异性高的观点，我们在注射DT 5周(慢性期)后重新检测了红藻氨酸诱导的Zif268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，突变的动物数量为n=7。我们发现，与急性期数据相比，两个IEG IR阳性细胞的总体变异性并不那么高。基于IEG数据，我们的结论仍然有效，即苔藓细胞变性时齿状颗粒细胞短暂过度兴奋。这份手稿再次提交，正在接受审查。

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.