Effect of sodium valproate on gene expression in Drosophila head. Drosophila melanogaster

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. Sodium valproate (NaVP) 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 NaVP. Our results provide novel insights in to the drug’s mode of action. Keywords: Dose response Overall design: 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 0.33 mg/ml of NaVP 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 NaVP 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 NaVP 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 18MO RNAase free water (Sigma). Dye swapping was accomplished by hybridizing two arrays with NF control as Cy3- and NaVP 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 (= 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氮（pentylenetetrazol, PTZ）诱导的果蝇长期行为可塑性模型。药理学验证表明该模型具有点燃样特征，因此其既可用于解析癫痫发生的分子发病机制，也可用于潜在抗癫痫药物的筛选。丙戊酸钠（sodium valproate, NaVP）是一种抗癫痫药物，在本模型开发过程中，我们获取了经NaVP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模式提供了全新视角。

关键词：剂量反应
整体实验设计：将Oregon-R野生型黑腹果蝇（Drosophila melanogaster, D. melanogaster）接种于含琼脂、玉米粉、红糖、干酵母以及尼泊金的标准果蝇培养基中，于24±1℃、相对湿度60%、12 h光照（早9点至晚9点）/12 h黑暗的循环条件下培养。选取10~11日龄未交配的成年雄果蝇，分为两组：一组饲喂正常培养基（NF），另一组饲喂含0.33 mg/ml NaVP的培养基，持续处理3天。每支培养管接种30只果蝇。

将果蝇置于液氮中速冻后，通过震荡分离头部并使用冷却筛收集。使用TRI REAGENT (Sigma)按照制造商说明书，从8组冻存头部样本中提取总RNA，每2组为一个平行处理组：其中4组为NF处理的对照组果蝇，4组为NaVP处理组果蝇。

以10 μg总RNA为模板，使用Microarray cDNA Synthesis Kit (Roche)合成双链cDNA，并按照制造商说明书通过Micorarray Target Purification Kit (Roche)对cDNA进行纯化。将4组生物学重复的NF对照组与NaVP处理组cDNA样本，分别使用Microarray RNA Target Synthesis Kit T7 (Roche)结合Cy3或Cy5染料(Amersham Biosciences)进行标记，标记产物通过Microarray Target Purification Kit (Roche)纯化。将每个生物学重复的Cy3与Cy5标记的cRNA样本混合，经沉淀、洗涤、风干后，溶于18MO无RNA酶水(Sigma)中。为完成染料互换，将两组阵列分别以NF对照组为Cy3标记、NaVP处理组为Cy5标记，剩余两组则采用相反的标记方式，即NF对照组为Cy5标记、药物处理组为Cy3标记。

将标记产物与杂交混合液混匀，该混合液包含杂交缓冲液(DIG Easy Hyb; Roche)、10mg/ml鲑鱼精DNA(终浓度0.05 mg/ml, Sigma)以及10mg/ml酵母tRNA(终浓度0.05 mg/ml, Sigma)。将杂交混合物于65℃变性后，点样至cDNA微阵列玻片(D12Kv1, CDMC, 多伦多)。使用盖玻片(ESCO, 美国朴次茅斯)覆盖玻片，于37℃杂交舱(Corning)中杂交16 h。杂交完成后，将玻片置于含1×SSC与0.1% SDS的50℃溶液中去除盖玻片，随后在Coplin染色缸中于50℃用1×SSC与0.1% SDS洗涤3次，每次15 min，期间轻微摇晃；再转移至1×SSC中室温轻柔洗涤2次，每次15 min。最后用0.1×SSC进行终末洗涤15 min，随后以600 rpm离心5 min快速去除玻片表面液体。

使用GenePix 4000A微阵列扫描仪(Molecular Devices)以10 μm分辨率扫描玻片。通过Gene Pix Pro 6.0软件(Molecular Devices)对16位TIFF图像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，剔除原始信号强度低于100U且低于平均背景强度两倍的所有点。进一步分析前，对筛选得到的特征点进行过滤：仅保留信号占比为0、强度与背景均一性良好(Rgn R2 (635/532) ≥ 0.6)且信噪比(SNR ≥ 3)的点。使用微阵列显著性分析工具(SAM 2.21, Excel插件, 斯坦福大学)，在单类别响应、100次置换检验的条件下，提取至少3次生物学重复中可分析的点用于后续分析。