Effect of vigabatrin on gene expression in Drosophila head

We recently developed a fly model of pentylenetetrazol (PTZ)-induced long-term behavioral plasticity. Pharmacological validation suggests this model to be kindling-like. Hence, our model is of relevance in understanding the molecular pathogenesis of epileptogenesis as well as in screening potential antiepileptogenic agents. Vigabatrin (VGB) is an antiepileptic drug, and in the process of developing our fly model we generated gene expression profile of flies’ head secondary to treatment of the insects with VGB. Our results provide novel insights in to the drug’s mode of action. Keywords: Dose response Oregon-R wild type D. melanogaster was grown in standard fly medium consisting of agar-agar, maize powder, brown sugar, dried yeast, and nipagin. Flies were cultured at 24 +/- 1ºC, 60% RH, and 12 hrs light (9 AM to 9 PM) and 12 hours dark cycle. Ten to eleven days old unmated adult males were grown in either normal food (NF) or food containing 24 mg/ml of VGB for three days. Each culture vials contained 30 flies. Flies frozen in liquid nitrogen were agitated and the heads collected using cooled sieves. Total RNA was isolated from eight pools of frozen heads, every two of which represented a single parallel set of treatment in which four vials contained NF treated control flies, and four VGB treated individuals, using TRI REAGENT (Sigma) according to the manufacturer’s protocol. Double stranded cDNA was synthesized from 10 µg of total RNA using Microarray cDNA Synthesis Kit (Roche), and the cDNA purified using Micorarray Target Purification Kit (Roche), according to the manufacturer’s protocol. Each of the four sets of NF control and VGB treated cDNA samples, belonging to the four biological replicates, was used for labeling with either Cy3 or Cy5 dyes (Amersham Biosciences) using Microarray RNA Target Synthesis Kit T7 (Roche). The labeled products were purified by Microarray Target Purification Kit (Roche). The Cy3 and Cy5 labeled two cRNA samples of each biological replicate were pooled together, precipitated, washed, air-dried, and dissolved in 18MΩ RNAase free water (Sigma). Dye swapping was accomplished by hybridizing two arrays with NF control as Cy3- and VGB treated as Cy5- labeled sample, and the rest two as the opposite, NF as Cy5- and drug treated as Cy3- labeled sample. The labeled product was mixed with hybridization solution containing hybridization buffer (DIG Easy Hyb; Roche), 10mg/ml salmon testis DNA (0.05 mg/ml final concentration, Sigma) and 10mg/ml yeast tRNA (0.05 mg/ml final concentration, Sigma). The hybridization mixture was denatured at 65ºC and applied onto cDNA microarray slides (D12Kv1, CDMC, Toronto). The slides were covered by a coverslip (ESCO, Portsmouth, USA) and hybridization was allowed to take place in hybridization chamber (Corning) at 37ºC for 16 hrs. Following hybridization, the coverslips were removed in a solution containing 1X SSC and 0.1% SDS at 50ºC, and the slides washed in 1X SSC and 0.1% SDS (three times for 15 minutes each) in a coplin jar at 50ºC with occasional swirling and then transferred to 1X SSC and washed with gentle swirling at room temperature (twice for 15 minutes each). Slides were given a final wash in 0.1X SSC for 15 minutes and then liquid was quickly removed from the slide surface by spinning at 600 rpm for 5 minutes. Slides were scanned at 10µm resolution in GenePix 4000A Microarray Scanner (Molecular Devices). The 16 bit TIFF images were preprocessed and quantified using Gene Pix Pro 6.0 software (Molecular Devices). Ratio based normalization was performed using Acuity 4.0 software (Molecular Devices). All Spots with raw intensity less then 100U and less then twice the average background was ignored during normalization. Normalized data was filtered for the selection of features before further analysis. Only those spot were selected which contained only a small percentage (<3) of saturated pixels, were not flagged bad or found absent (flags >= 0), had relatively uniform intensity and uniform background (Rgn R2 (635/532) >= 0.6) and were detectable above background (SNR >= 3). Analyzable spots in at least three of the four biological replicates performed were retrieved for downstream analysis using Significance Analysis of Microarrays (SAM 2.21, Excel Add-In, Stanford) under the conditions of one class response and 100 permutations.

我们近期构建了戊四唑（pentylenetetrazol, PTZ）诱导的果蝇长期行为可塑性模型。药理学验证结果表明，该模型具有点燃样（kindling-like）特征，可用于解析癫痫发生的分子发病机制，同时可用于筛选潜在的抗癫痫发生剂。氨己烯酸（vigabatrin, VGB）是一种抗癫痫药物，在本果蝇模型的构建过程中，我们获取了经VGB处理的果蝇头部的基因表达谱，研究结果为阐明该药物的作用机制提供了新的见解。
关键词：剂量反应
Oregon-R野生型黑腹果蝇（Drosophila melanogaster, D. melanogaster）饲养于标准果蝇培养基中，该培养基由琼脂、玉米粉、红糖、干酵母和尼泊金组成。果蝇培养条件为：温度24±1℃，相对湿度60%，光暗周期为12小时光照（早9点至晚9点）、12小时黑暗。选取10~11日龄的未交配成年雄蝇，随机分为两组：正常食物（normal food, NF）组与含24mg/ml VGB的食物组，处理时长为3天。每个培养瓶中放置30只果蝇。将果蝇经液氮速冻后，通过震荡分离并使用冷筛收集头部。参照制造商说明书，使用TRI试剂（TRI REAGENT, Sigma）从8份冰冻头部混合样本中提取总RNA，每两份样本构成一组平行处理，其中4瓶为NF处理的对照果蝇，4瓶为VGB处理的果蝇。
取10μg总RNA，参照制造商说明书，使用微阵列cDNA合成试剂盒（Microarray cDNA Synthesis Kit, Roche）合成双链cDNA，并通过微阵列靶点纯化试剂盒（Microarray Target Purification Kit, Roche）纯化cDNA。针对4个生物学重复的NF对照与VGB处理的cDNA样本，分别使用微阵列RNA靶点合成试剂盒T7（Microarray RNA Target Synthesis Kit T7, Roche）搭配Cy3、Cy5荧光染料（Amersham Biosciences）进行标记，标记产物经微阵列靶点纯化试剂盒（Roche）纯化。将每个生物学重复的Cy3与Cy5标记的互补RNA（cRNA）样本混合，经沉淀、洗涤、风干后，溶于18MΩ无RNA酶水（Sigma）。染料互换实验设置为：两组阵列以NF对照为Cy3标记、VGB处理样本为Cy5标记，剩余两组则反之，即NF对照为Cy5标记、药物处理样本为Cy3标记。
将标记产物与杂交液混合，该杂交液包含杂交缓冲液（DIG Easy Hyb, Roche）、10mg/ml鲑鱼精DNA（终浓度0.05mg/ml, Sigma）与10mg/ml酵母tRNA（终浓度0.05mg/ml, Sigma）。杂交混合物于65℃变性后，滴加至cDNA微阵列玻片（D12Kv1, CDMC, 加拿大多伦多）上，使用盖玻片（ESCO, 美国朴次茅斯）覆盖，于杂交箱（Corning）中37℃孵育16小时完成杂交。杂交结束后，在50℃的1×SSC与0.1%SDS溶液中移除盖玻片，随后将玻片置于Coplin染色缸中，于50℃用1×SSC与0.1%SDS溶液洗涤3次，每次15分钟，期间轻微摇晃；再转移至室温下的1×SSC溶液中洗涤2次，每次15分钟，同样轻微摇晃。最后用0.1×SSC溶液进行终末洗涤15分钟，随后以600rpm转速离心5分钟，快速去除玻片表面残留液体。
使用GenePix 4000A微阵列扫描仪（Molecular Devices）以10μm分辨率扫描玻片，获取16位TIFF图像，再通过GenePix Pro 6.0软件（Molecular Devices）进行预处理与定量分析。使用Acuity 4.0软件（Molecular Devices）进行基于比值的标准化处理。在标准化过程中，剔除原始强度低于100U且低于平均背景强度两倍的斑点。对标准化后的数据进行筛选，以选取符合要求的特征：仅包含少量饱和像素（占比<3%）、未被标记为不良或缺失（标记值≥0）、强度与背景均一性良好（区域R平方值(635/532)≥0.6），且信噪比（SNR）≥3的斑点。将至少在4个生物学重复中的3个中可检测到的可分析斑点，使用微阵列显著性分析软件（Significance Analysis of Microarrays, SAM 2.21, Excel插件, 斯坦福大学）进行下游分析，设置条件为单类响应与100次置换。