Axonal Sprouting and Epilepsy after Traumatic CNS Injury

问题：癫痫是创伤性头部损伤的常见后果。其原因尚不清楚。假设：1)创伤性中枢神经系统损伤后神经营养因子的释放触发锥体细胞轴突萌发。2)突触后靶细胞的谷氨酸敏感性和兴奋性增加是部分去神经支配的结果。3)损伤引起的突触前轴突萌发和突触后兴奋性增加共同引起创伤后癫痫。模型：经Schaffer侧支横断后，海马切片培养CA3细胞萌发新的轴突侧支，CA1细胞对谷氨酸产生超敏感。这些现象可以解释病变引起的高兴奋性。该模型为研究创伤后癫痫的突触前和突触后机制提供了一种实验上易于处理和信息丰富的方法。因此，我们将利用神经解剖学、细胞生物学和电生理学技术来研究该模型中轴突损伤后高兴奋性的成因。目的1：确定损伤性高兴奋性的突触前机制。TrkB免疫粘附素，全长和显性负神经营养因子受体构建的生物转染，以及trk受体敲除小鼠的培养物，将用于验证trk受体的激活是损伤诱导的轴突生长所必需的假设。该假说预测，在trkB免疫粘附素存在的情况下，在转染了显性阴性irk受体的细胞中，或者在trk受体敲除小鼠的培养物中，病变诱导的芽不会发生。预计缺乏轴突发芽可以消除损伤引起的高兴奋性。目的2：确定损伤性高兴奋性的突触后机制。我们将使用全细胞电压钳和激光微光解针对单个远端树突的笼状神经递质，测试谷氨酸超敏感和内在高兴奋性在去神经支配后发生在CA1细胞中的假设。该假说预测，神经递质受体表达水平的变化和/或固有电压依赖性离子电导率的变化是先前在Schaffer侧枝横断后观察到的树突谷氨酸反应增强的基础。目标：更好地了解创伤后癫痫的病因，并最终提供新的和改进的预防性治疗策略来治愈这种疾病。

The Problem: Epilepsy is a common consequence of traumatic head injury. Its cause is unknown. The Hypotheses: 1) The release of neurotrophins after traumatic CNS injury triggers axonal sprouting by pyramidal cells. 2) The glutamate sensitivity and excitability of postsynaptic target cells increase as a consequence of partial denervation. 3) Injury-induced presynaptic axonal sprouting and increased postsynaptic excitability combine synergistically to cause posttraumatic epilepsy. The Model: After Schaffer collateral transection, CA3 cells in hippocampal slice cultures sprout new axon collaterals and CA1 cells become supersensitive to glutamate. These phenomena may account for the lesion-induced hyperexcitability. This model provides an experimentally tractable and informative approach for studying pre- and postsynaptic mechanisms of posttraumatic epilepsy. The genesis of hyperexcitability after axonal injury in this model will therefore be investigated using neuroanatomical, cell biological, and electrophysiological techniques. AIM 1: Determine the presynaptic mechanisms underlying injury- induced hyperexcitability. TrkB immunoadhesins, biolistic transfection with full length and dominant negative neurotrophin receptor constructs, and cultures derived from trk receptor knockout mice will be used to test the hypothesis that activation of trk receptors is required for injury-induced axonal sprouting. The hypothesis predicts that lesion-induced sprouting will not occur in the presence of trkB immunoadhesin, in cells transfected with dominant negative irk receptors or in cultures made from trk receptor knock-out mice. Lack of axonal sprouting is predicted to eliminate injury-induced hyperexcitability. AIM 2: Determine the postsynaptic mechanisms underlying injury- induced hyperexcitability. Using whole-cell voltage-clamp and laser microphotolysis of caged neurotransmitters targeted to individual distal dendrites, we will test the hypotheses that glutamate supersensitivity and intrinsic hyperexcitability occur in CA1 cells after denervation. The hypothesis predicts that changes in the levels of expression of neurotransmitter receptors and/or changes in intrinsic voltage-dependent ionic conductances underlie the potentiation of dendritic glutamate responses observed previously after Schaffer collateral transection. The Goal: to better understand the causes of posttraumatic epilepsy and, ultimately, to offer new and improved prophylactic therapeutic strategies to cure this disease.