Characterization of Dentate Mossy Cell-restricted Genetic Manipulation Mice

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，为了分析苔藓细胞丢失对齿状回体内脑活动的影响，我们在齿状回放置电极以记录局部场电位（LFP）。与相同的动物/电极/行为状态DT治疗前和4周后相比，LFP振荡功率在θ频率（712 Hz）在DT暴露一周的突变体探索过程中增强。在DT处理的fDTR对照中没有发生这样的变化，这表明突变体中θ功率的瞬时增加不是由于DT处理。DT注射不影响LFP功率谱在不动期，无论基因型。由于θ输入到齿状回在体内是从内嗅皮层的穿通路径颗粒细胞，瞬时海拔θ振荡功率可能反映了颗粒细胞兴奋性的瞬时增加。 这一结果再次支持了我们的结论，即齿状苔藓细胞在颗粒细胞活动中起着全面的抑制作用。 手稿终于出版了（Jinde S，Zsiros V，Jiang Z，Nakao K，Pickel J，Kohno K，Belforte JE，Nakazawa K.（2012）门苔藓细胞变性导致短暂的齿状颗粒细胞过度兴奋和受损的模式分离。Neuron 76：1189-2000）。我们还发表了一篇关于这个主题的综述文章，题为“Hilar mossy cell circuitry controlling dentate granule cell excitability”（Jinde S，Zsiros V，and Nakazawa K（2013）Frontier Neural Circuits 7：14）。

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 forth revisions, To analyze the impact of mossy cell loss on in vivo brain activity in the dentate gyrus, we placed electrodes in the dentate gyrus to record local field potentials (LFPs). In comparison with the same animals/electrodes/behavioral state before and 4 weeks after DT treatment, LFP oscillatory powers at theta frequency (712 Hz) were enhanced during exploration in mutants one week from DT exposure. That no such changes occurred in DT-treated fDTR controls suggests that the transient increase in theta power in mutants is not due to DT treatment. DT injection shows no effect on LFP power spectra during immobility periods regardless of genotype. Since the theta input to the dentate gyrus in vivo is conveyed from entorhinal cortex by the perforant path to granule cells, transient elevation of theta oscillatory power may reflect a transient increase in granule cell excitability. This result, again, support our conclusion that dentate mossy cells play overall an inhibitory role in granule cell activity. The manuscript has finally been published (Jinde S, Zsiros V, Jiang Z, Nakao K, Pickel J, Kohno K, Belforte JE, Nakazawa K. (2012) Hilar mossy cell degeneration causes transient dentate granule cell hyperexcitability and impaired pattern separation. Neuron 76:1189-2000). We also published a review article on this subject, entitled "Hilar mossy cell circuitry controlling dentate granule cell excitability (Jinde S, Zsiros V, and Nakazawa K (2013) Frontier Neural Circuits 7:14).