Calcium Imaging of Neuronal Activity in Drosophila Can Identify Anticonvulsive Compounds.
Calcium Imaging of Neuronal Activity in Drosophila Can Identify Anticonvulsive Compounds.
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DOI：
10.1371/journal.pone.0148461
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Baines RA
中科院分区：
文献类型：
--
作者：
Streit AK;Fan YN;Masullo L;Baines RA
虽然现在有许多抗癫痫药物（aed）可用，但大约三分之一的癫痫患者对药物干预反应不佳。造成这种情况的原因很复杂，但可能反映了越来越多的已确定的使个体易患这种疾病的突变。因此，开发新的治疗方法来解决这一未满足的临床需求是一个明确的要求。果蝇（Drosophila melanogaster）中存在的类似癫痫发作行为的基因突变，为利用这种昆虫强大的遗传学来识别新的细胞靶标以促进设计更有效的抗癫痫药提供了可能性。在这项研究中，我们使用GCaMP（一种有效的钙报告因子）的神经元表达，使用非侵入性和快速的方法来成像神经元活动。三龄幼虫离体中枢神经系统中运动神经元的表达表现出在腹侧神经索各节段之间传递的钙活性波。在同一神经元中，相邻节段之间的钙峰间隔时间通常大于200毫秒。暴露于抽搐前药（微毒素或4-氨基吡啶）可将分离降低至200毫秒以下，显示相邻节段之间活动的同步性增加。增加的同步性是癫痫的特征，在癫痫基因突变体：bangsenseless1 （bss1）和麻痹k1270t （paraK1270T）中也同样观察到。bss1暴露于临床使用的抗癫痫药物（苯妥英或加巴喷丁）可显著降低同步性。在这项研究中，我们使用同向性测量来评估已知和新型抗惊厥化合物（抗疼痛、异硫酸盐、依托哌啶、雷帕霉素和双锥体多）降低癫痫样中枢神经系统活性的有效性。我们进一步表明，这些化合物还可以降低果蝇在一个确定的运动神经元（aCC）中的电压门控持续Na+电流（INaP）。我们的联合检测提供了一种快速可靠的方法来筛选未知化合物作为抗惊厥药的潜力。
Although there are now a number of antiepileptic drugs (AEDs) available, approximately one-third of epilepsy patients respond poorly to drug intervention. The reasons for this are complex, but are probably reflective of the increasing number of identified mutations that predispose individuals to this disease. Thus, there is a clear requirement for the development of novel treatments to address this unmet clinical need. The existence of gene mutations that mimic a seizure-like behaviour in the fruit fly, Drosophila melanogaster, offers the possibility to exploit the powerful genetics of this insect to identify novel cellular targets to facilitate design of more effective AEDs. In this study we use neuronal expression of GCaMP, a potent calcium reporter, to image neuronal activity using a non-invasive and rapid method. Expression in motoneurons in the isolated CNS of third instar larvae shows waves of calcium-activity that pass between segments of the ventral nerve cord. Time between calcium peaks, in the same neurons, between adjacent segments usually show a temporal separation of greater than 200 ms. Exposure to proconvulsants (picrotoxin or 4-aminopyridine) reduces separation to below 200 ms showing increased synchrony of activity across adjacent segments. Increased synchrony, characteristic of epilepsy, is similarly observed in genetic seizure mutants: bangsenseless1 (bss1) and paralyticK1270T (paraK1270T). Exposure of bss1 to clinically-used antiepileptic drugs (phenytoin or gabapentin) significantly reduces synchrony. In this study we use the measure of synchronicity to evaluate the effectiveness of known and novel anticonvulsive compounds (antipain, isethionate, etopiside rapamycin and dipyramidole) to reduce seizure-like CNS activity. We further show that such compounds also reduce the Drosophila voltage-gated persistent Na+ current (INaP) in an identified motoneuron (aCC). Our combined assays provide a rapid and reliable method to screen unknown compounds for potential to function as anticonvulsants.