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，在手稿第四版中，为了分析苔藓细胞丢失对齿状回活体脑活动的影响，我们在齿状回放置电极以记录局部场电位(LFP)。与DT处理前和处理后4周相同的动物/电极/行为状态相比，在DT暴露一周后，突变体在theta频率(712赫兹)的LFP振荡功率在探索过程中增强。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theta能力的瞬时增加不是由于DT处理。静止期注射DT对LFP功率谱无影响，与基因型无关。由于体内齿状回的theta输入是通过穿支通路从内嗅皮层传递到颗粒细胞，因此theta振荡功率的瞬时升高可能反映了颗粒细胞兴奋性的一过性增加。这一结果再次支持了我们的结论，即齿状苔藓细胞总体上对颗粒细胞的活动起到了抑制作用。手稿终于出版了(金德S，齐西罗斯五，蒋志，中尾K，Pickel J，Kohno K，Belforte Je，Nakazawa K(2012年))肺门苔藓细胞变性导致一过性齿状颗粒细胞过度兴奋和模式分离障碍。神经元76：1189-2000)。我们还发表了一篇关于这一主题的综述文章，题为《海门区苔藓细胞电路控制齿状颗粒细胞兴奋性》(金德·S，Zsiros V，和Nakazawa K(2013)前沿神经电路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).