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对抗惊厥药物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特点：在这两种情况下，神经元都被GABA（主要的抑制性神经递质）兴奋而不是抑制。GABA是兴奋性的，因为在这些条件下神经元在细胞内积累氯化物，这逆转了GABAA受体介导的电流。NKCC1是一种氯离子转运体，将氯离子输入神经元，而KCC2是一种氯离子转运体，将氯离子输出神经元。在我们提出的研究中，我们将测试NKCC1活性是否超过KCC2活性在新生儿和盐酸盐模型TLE。转运蛋白的功能和表达将在新生儿脑、TLE的盐酸盐模型和成年对照动物中进行评估。我们将在海马切片中使用全细胞和革兰西丁穿孔贴片记录来测量这两种转运体的动力学。我们将使用免疫印迹和免疫细胞化学来确定这两种转运蛋白的表达水平。NKCC1氯离子转运体对利尿剂布美他胺非常敏感。利用大鼠幼鼠急性新生儿癫痫发作的盐酸盐模型和成年大鼠慢性TLE的盐酸盐模型，我们将测试布美他尼阻断nkcc1介导的氯积累是否会恢复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癫痫发作分析。布美他尼已经作为一种利尿剂在人类新生儿中进行了测试，其剂量可以抑制NKCC1，因此布美他尼治疗新生儿癫痫是一种新的可行的治疗方法，这种疾病没有有效的治疗方法，而且终生发病率很高。同样，布美他尼诱导的GABAA受体介导的顽固性TLE抑制的恢复可以为由于其起病区位置而不适合癫痫手术的患者提供非手术治疗选择。

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.