Characterization of Dentate Mossy Cell-restricted Geneti

众所周知，海马在情景记忆的获得和提取中起着至关重要的作用，而齿状回是海马中感觉输入的中央信息处理器。齿状门的海马能苔藓细胞沿着海马的纵轴提供了与齿状颗粒细胞和门GABA能中间神经元的重要的反复兴奋性连接。虽然颗粒细胞和门部中间神经元的轴突投射被限制在一个板层平面内，苔藓细胞介导前馈抑制和反馈兴奋的远端颗粒细胞沿着纵轴，并可能发挥重要作用，在确定整体兴奋的齿状回。有人认为，虽然苔藓细胞驱动的translamellar抑制颗粒细胞是占主导地位的在正常条件下，从而保持稀疏编码的颗粒细胞，translamellar去抑制发生后的损失脆弱的苔藓细胞，这可能是损伤后海马功能障碍。事实上，门苔藓细胞是已知的最容易损伤的海马神经元。因此，可以推测，在损伤性刺激（如癫痫发作）期间，苔藓细胞丢失后会发生跨板层去抑制。然而，苔藓细胞在记忆形成或暴露于强烈刺激过程中对齿状回兴奋性的体内贡献尚未得到解决。 为了了解这种苔藓细胞介导的齿状回网络在体内的作用，我们首先通过使用携带5？小鼠降钙素受体样受体（Crlr）基因和Cre cDNA的转录调控区。其中一个品系Cre#4688在1-2月龄时仅在齿状门细胞中显示限制性Cre免疫反应性（IR），其不与GAD 65/67-IR共定位，而是与calretininin-IR共定位，表明Cre阳性细胞是苔藓样细胞。与Rosa 26 LacZ报告小鼠杂交的Cre系的X-Gal染色揭示了在齿状苔藓细胞和CA 3c细胞中的功能性Cre重组，在小脑核和视前核中具有稀疏的染色，这表明Cre蛋白在青春期之前在CA 3c细胞和其他区域中瞬时表达。我们建立了海马苔藓细胞限制性NMDA受体（NR）基因敲除小鼠的基础上的关键作用，神经元受体对海马依赖性记忆。在与一个封闭的NMDA受体R1（NR 1）菌株杂交后，在12周龄的突变体的齿状门中发现NR 1 mRNA和NR 1-IR的显着减少，使用原位杂交和免疫组织化学。令人惊讶的是，NR 1 mRNA的减少是不明显的CA 3区，小脑核，视前核，最有可能是由于相对无效的铬介导的重组的floxed NR 1位点。因此，我们的海马苔藓细胞限制性NR基因敲除细胞系可以为苔藓细胞功能的研究提供一个理想的动物模型。使用这些小鼠，Seiichiro Jinde等人正在开发行为研究策略，以阐明苔藓细胞在关联记忆中获得和检索上下文特异性的体内NR功能，例如区分上下文中两个相似输入模式的能力（模式分离）。 我们还产生了另一个突变株系，其在alphaCaMKII启动子的控制下具有Cre重组酶介导的诱导型白喉毒素受体（DTR）的转基因表达，该启动子限制了前脑神经元的表达。在与表达Cre的小鼠品系Cre #4688杂交后，所得的双转基因突变体将仅在前脑中表达Cre的细胞的表面中表达DTR。由于DTR的表达允许DT的B亚基的结合和随后的受体介导的内吞作用，白喉毒素（DT）的应用诱导Cre表达细胞的选择性消融。我们计划使用这些诱导苔藓细胞消融小鼠来评估苔藓细胞在齿状兴奋性和癫痫发生中的作用。

It is widely known that the hippocampus is critically involved in acquisition and retrieval of episodic memory and the dentate gyrus is the central information processor for sensory inputs in the hippocampus. The glutamatergic mossy cells of the dentate hilus provide an important recurrent excitatory connection to dentate granule cells and hilar GABAergic interneurons along the longitudinal axis of the hippocampus. While the axonal projections of granule cells and hilar interneurons are restricted within a lamellar plane, mossy cells mediate feedforward inhibition and feedback excitation of distal granule cells along the longitudinal axis and may play a significant role in determining the overall excitability of the dentate. It has been suggested that while mossy cell-driven translamellar inhibition of granule cells is dominant in the normal condition, thereby maintaining sparse coding of granule cells, translamellar disinhibition occurs following the loss of vulnerable mossy cells, which may underlie postinjury hippocampal dysfunction. In fact, hilar mossy cells are known to be the most injury-prone hippocampal neurons. Accordingly, it is presumed that translamellar disinhibition would occur following the loss of mossy cells during injurious stimuli, such as epileptic seizure. However, in vivo contribution of mossy cells to dentate gyrus excitability during either memory formation or exposure to intense stimuli has not been addressed. To understand the role of this mossy cell-mediated dentate gyrus network in vivo, we first generated Cre recombinase transgenic mice by using a BAC DNA fragment carrying 5?-transcriptional regulatory region of murine calcitonin receptor-like receptor (Crlr) gene and Cre cDNA. One of the lines, Cre#4688, showed restricted Cre immunoreactivity (IR) only in the dentate hilar cells at 1-2 months of age, which was co-localized not with GAD65/67-IR but with calretinin-IR, suggesting that the Cre positive cells are mossy cells. X-Gal staining of Cre lines crossed with Rosa26 LacZ reporter mice revealed a functional Cre recombination in dentate mossy cells and CA3c cells, with sparse staining in cerebellar nuclei and preoptic nuclei, suggesting that Cre protein is transiently expressed in CA3c cells and other regions before adolescence. We created hippocampal mossy cell-restricted NMDA receptor (NR) knockout mice based on the critical role of NRs on hippocampus-dependent memory. After crossing with a floxed-NMDA receptor R1 (NR1) strain, significant reduction of NR1 mRNA and NR1-IR was found in the dentate hilus of the mutants at 12 weeks of age, using in situ hybridization and immunohistochemistry. Surprisingly, reduction of NR1 mRNA was not evident in area CA3, cerebellar nuclei, and preoptic nuclei, most likely due to the relative inefficiency of Cre-mediated recombination of the floxed NR1 loci. Therefore, our hippocampal mossy cell-restricted NR knockout line could provide an ideal animal model for the study of mossy cell function. Using these mice, Seiichiro Jinde and others are now developing behavioral research strategies to elucidate in vivo NR function of mossy cells in the acquisition and retrieval of context specificity in an associative memory, such as the ability to distinguish two similar input patterns in a context (pattern separation). We are also generating another mutant line with transgenic expression of Cre recombinase-mediated inducible diphtheria toxin receptor (DTR) under control of the alphaCaMKII promoter, which limits expression to neurons of the forebrain. After crossed with the Cre-expressing mouse strain, Cre#4688, the resulting double-transgenic mutant will express DTR in the surface of Cre-expressing cells only in the forebrain. Since expression of DTR allows binding of the B subunit of DT and subsequent receptor-mediated endocytosis, application of diphtheria toxin (DT) induces the selective ablation of Cre-expressing cells. We plan to evaluate the role of mossy cells in dentate excitability and epileptogenesis using these inducible-mossy cell ablation mice.