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

基本信息

批准号：
8745729
负责人：
Kazutoshi Nakazawa
金额：
$ 6万
依托单位：
NATIONAL INSTITUTE OF MENTAL HEALTH
依托单位国家：
美国
项目类别：
财政年份：
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/8745729
关键词：
Ablation Address Animals Area Back Behavior Behavioral Brain Cell physiology Cells DTR gene Diphtheria Toxin Electrodes Epilepsy Epileptogenesis Episodic memory Frequencies Gene Expression Genes Genetic Genotype 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 Neurons Pattern Perforant Pathway Play Preparation Property Publishing Pyramidal Cells Recurrence Reporting Research Role Seizures Slice Staining method Stains Stimulus System Techniques Temporal Lobe Epilepsy Transgenic Mice Transgenic Organisms dentate gyrus diphtheria toxin receptor entorhinal cortex frontier genetic manipulation granule cell in vivo insight interest mossy fiber mutant neural circuit neuron loss response selective expression tool

项目摘要

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).

早些时候，我们生成了一个条件转基因小鼠系，其中白喉毒素受体使用 Cre/loxP 系统在苔藓细胞中选择性表达。注射白喉毒素后一周内，我们观察到整个纵轴的苔藓细胞损失了 80%。通过体内局部场电位记录测量，我们发现海马体中没有明显或持续的癫痫样放电。有趣的是，蒂姆染色没有检测到苔藓纤维发芽。这些结果表明，与先前的报告显示整个肺门区域的病变诱发大量苔藓纤维出芽和癫痫相比，选择性体内消除苔藓细胞不会引发行为性癫痫或苔藓纤维出芽。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，并且在这种过度兴奋状态期间检测到上下文模式分离的缺陷。我们还评估了红藻氨酸 (KA) 注射后立即早期基因 (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放电并激活 IEG 表达。注射 KA 后，突变体中比对照组更多的颗粒细胞中产生了 Zif268 表达。我们还检查了 KA 诱导的癫痫发作强度。注射 KA 后一小时内突变体的累积癫痫评分显着高于对照组。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，体内苔藓细胞的损失导致颗粒细胞过度兴奋。然而，与休眠篮细胞假说中隐含的预测癫痫发生相反，它不足以触发苔藓纤维发芽和癫痫放电。也许，除了苔藓细胞的丧失之外，其他边缘区域（例如内嗅皮层）的神经变性也是诱发内侧颞叶癫痫所必需的。这些发现为边缘皮层癫痫发生机制提供了新的见解。今年，为了对手稿进行第四次修订，为了分析苔藓细胞丢失对齿状回体内大脑活动的影响，我们在齿状回放置电极来记录局部场电位（LFP）。与 DT 治疗前和 4 周后相同的动物/电极/行为状态相比，在 DT 暴露一周后对突变体进行探索期间，LFP 在 theta 频率 (712 Hz) 下的振荡功率增强。 DT 处理的 fDTR 对照中没有发生此类变化，这表明突变体中 θ 功率的短暂增加不是由于 DT 处理造成的。无论基因型如何，DT 注射在不动期间对 LFP 功率谱没有影响。由于输入到体内齿状回的 θ 输入是从内嗅皮层通过穿通路径传递到颗粒细胞的，因此 θ 振荡功率的瞬时升高可能反映了颗粒细胞兴奋性的瞬时增加。这一结果再次支持我们的结论，即齿状苔藓细胞在颗粒细胞活性中总体上发挥抑制作用。手稿终于发表了（Jinde S, Zsiros V, Jiang Z, Nakao K, Pickel J, Kohno K, Belforte JE, Nakazawa K. (2012) Hilar mossy cell degenesis Causes instant dentate 管理颗粒细胞过度兴奋和受损的模式分离。神经元76：1189-2000）。我们还发表了关于该主题的评论文章，题为“控制齿状颗粒细胞兴奋性的海拉尔苔藓细胞电路”（Jinde S、Zsiros V 和 Nakazawa K (2013) Frontier Neural Circuits 7:14）。