Impact of neuronal chloride transport on treatment of seizures

描述（由申请人提供）：本研究针对两种癫痫发作对抗惊厥治疗反应不佳的情况：新生儿癫痫发作和难治性颞叶癫痫（TLE）。这两种情况都有一个不寻常的功能：：在这两种情况下，神经元都被主要的抑制性神经递质GABA兴奋而不是抑制。GABA是兴奋性的，因为在这些条件下的神经元积累细胞内氯，这逆转了GABAA受体介导的电流。NKCC 1是一种将氯离子输入神经元的氯离子转运蛋白，KCC 2是一种输出氯离子的氯离子转运蛋白。在拟议的研究中，我们将测试NKCC1活性是否超过KCC2活性在新生儿和红藻氨酸模型的TLE。将在新生儿脑、TLE红藻氨酸盐模型和成年对照动物中评价转运蛋白功能和表达。我们将使用海马切片的全细胞和短杆菌肽穿孔贴片记录来测量这两种转运蛋白的动力学。我们将使用蛋白质印迹和免疫细胞化学来确定这两种转运蛋白的表达水平。NKCC 1氯转运蛋白对利尿剂布美他尼非常敏感。使用大鼠幼仔中急性新生癫痫发作的红藻氨酸盐模型和成年大鼠中慢性TLE的红藻氨酸盐模型，我们将测试用布美他尼阻断NKCC 1介导的氯化物积累是否会恢复GABAA受体介导的抑制，从而改善这两种类型的癫痫发作.为了量化对癫痫发作的影响，我们将使用急性和慢性，无线电遥测数字脑电图记录和计算机化癫痫发作分析。布美他尼已经在抑制NKCC 1的剂量下作为利尿剂在人类新生儿中进行了测试，因此布美他尼治疗新生儿癫痫发作是一种新的可行的治疗方法，对于这种疾病没有有效的治疗方法，并且终身发病的风险非常高。同样，布美他尼诱导的GABAA受体介导的抑制作用在难治性TLE中的恢复可以为由于其发作区的位置而不适合癫痫手术的患者提供非手术治疗替代方案。

DESCRIPTION (provided by applicant): This research is directed at two conditions in which seizures respond poorly to anticonvulsant therapy: neonatal seizures and intractable temporal lobe epilepsy (TLE). These two conditions share an unusual feature: in both conditions, neurons are excited rather than inhibited by GABA, the principal inhibitory neurotransmitter. GABA is excitatory because neurons in these conditions accumulate intracellular chloride, which reverses GABAA receptor-mediated current flow. NKCC1 is a chloride transporter that imports chloride into neurons, and KCC2 is a chloride transporter that exports chloride. In the proposed research we will test whether NKCC1 activity exceeds KCC2 activity in the neonate and in the kainate model of TLE. Transporter function and expression will be evaluated in the neonatal brain, in the kainate model of TLE, and in adult control animals. We will use whole-cell and gramicidin perforated patch recordings in hippocampal slices to measure the kinetics of these two transporters. We will use western blots and immunocytochemistry to determine the level of expression of the two transporters. The NKCC1 chloride transporter is exquisitely sensitive to the diuretic bumetanide. Using a kainate model of acute neonatal seizures in the rat pup, and the kainate model of chronic TLE in the adult rat, we will test whether blocking NKCC1-mediated chloride accumulation with bumetanide will restore GABAA receptor- mediated inhibition and thereby ameliorate these two types of seizures. To quantify the effects on seizures we will use acute and chronic, radiotelemetric digital EEG recordings and computerized seizure analysis. Bumetanide has already been tested as a diuretic in human neonates at doses that inhibit NKCC1, so bumetanide treatment of neonatal seizures is a new and feasible treatment of a disorder for which there is no effective therapy and a very high risk of lifelong morbidity. Similarly, bumetanide-induced restoration of GABAA receptor-mediated inhibition in intractable TLE could provide a non-surgical therapeutic alternative to patients who are not candidates for epilepsy surgery due to the location of their ictal onset zones.