Graham Stephanie, Rogers Ryan P, Alper Richard H
Department of Pharmaceutical Sciences, University of Saint Joseph School of Pharmacy, Hartford, CT, United States; Department of Biology, University of Saint Joseph, West Hartford, CT, United States.
Department of Biology, University of Saint Joseph, West Hartford, CT, United States; Department of Sciences, Wentworth Institute of Technology, Boston, MA, United States.
J Pharmacol Toxicol Methods. 2016 Jan-Feb;77:76-80. doi: 10.1016/j.vascn.2015.10.004. Epub 2015 Nov 7.
The purpose of these studies was to describe a novel application of an automated data acquisition/data reduction system, DanioVision™ by Noldus. DanioVision™ has the ability to detect changes in locomotor activity in third instar Drosophila melanogaster larvae. The noncompetitive GABAA receptor antagonist picrotoxin (PTX), was used as a pharmacologic agent to decrease locomotor activity.
Two strains of Drosophila were used in these studies; wild-type flies and flies with a mutation in the Rdl gene (Rdl(MD-RR)). Rdl(MD-RR)Drosophila are naturally occurring mutants that express an aberrant form of the GABAA receptor, which has a lower affinity for PTX, but not GABA itself. Larvae, extracted from food in 20% sucrose, were randomly placed into vials containing vehicle or PTX (0.03-3mM). After incubation of 2-24h, individual larvae were put in each well of a 6-well culture plate previously coated with 2% agar, the plate was then placed in the DanioVision™ apparatus. The activity of individual larva was recorded for 5 min, digitized and analyzed using Ethovision® XT software.
Incubation of third instar wild-type larvae in 1mM PTX for 4 or 24h decreased activity; whereas, a 2h incubation in PTX was without effect. PTX caused a concentration-dependent decrease in activity as demonstrated by consistently reduced locomotor activity with 1.0 and 3.0mM: 0.3mM resulted in variable decreases in locomotor activity and 0.03 mM yielded no effect. By contrast, PTX did not affect activity in Rdl(MD-RR) larvae even at the highest concentration, 3.0mM.
Using an automated data acquisition system, it was found that PTX decreases activity in third instar Drosophila larvae due to a selective blockade of the GABAA receptor. The method will reduce the likelihood of human error and bias, as well as increase the speed and ease of data collection and analysis.
这些研究的目的是描述一种由Noldus公司生产的自动化数据采集/数据简化系统DanioVision™的新应用。DanioVision™能够检测三龄黑腹果蝇幼虫运动活性的变化。非竞争性γ-氨基丁酸A型（GABAA）受体拮抗剂印防己毒素（PTX）被用作降低运动活性的药物。
在这些研究中使用了两种果蝇品系；野生型果蝇和Rdl基因发生突变的果蝇（Rdl(MD-RR)）。Rdl(MD-RR)果蝇是天然存在的突变体，其表达一种异常形式的GABAA受体，该受体对PTX的亲和力较低，但对GABA本身的亲和力正常。从含20%蔗糖的食物中取出的幼虫被随机放入含有赋形剂或PTX（0.03 - 3 mM）的小瓶中。在2 - 24小时的孵育后，将单个幼虫放入预先涂有2%琼脂的6孔培养板的每个孔中，然后将培养板放入DanioVision™仪器中。记录单个幼虫5分钟的活性，使用Ethovision® XT软件进行数字化和分析。
将三龄野生型幼虫在1 mM PTX中孵育4或24小时会降低活性；而在PTX中孵育2小时则没有效果。PTX导致活性呈浓度依赖性降低，如1.0和3.0 mM时运动活性持续降低所示：0.3 mM导致运动活性有变化的降低，0.03 mM则没有效果。相比之下，即使在最高浓度3.0 mM时，PTX也不影响Rdl(MD-RR)幼虫的活性。
使用自动化数据采集系统发现，PTX由于对GABAA受体的选择性阻断而降低三龄果蝇幼虫的活性。该方法将降低人为误差和偏差的可能性，同时提高数据收集和分析的速度及便利性。
