Prevention of PKCi effect by the presence of a UAS-GFP construct

Description of the experiment and the figures Groups represent a cross between the tubGal80ts;UAS-PKCi line (which is effective in blocking self-learning when expressed pan neuronal) and 2 lines with specific combination of 3 construct: d42Gal4 or c380Gal4, as well as chaGal80 (inhibit Gal4 expression in cholinergic neutrons) and UAS-GFP (used to localize expression). In both cases, we have no effect on self learning. While we know that if the GFP construct is taken out, the prevention works (Colomb et al., in prep), we conclude that the presence of a second UAS element prevents the expression of the PKC inhibitor. <strong>Representation legend .</strong> The score during the final test is depicted in boxplots (bar is the median, boxes are quartiles, bars are 100% of the data not considering the outliers presented by points). <em>Drosophila </em>self-learning. The flies are tethered to a torque meter which measures the angular momentum around the fly's vertical body axis (yaw torque), caused by attempted turning maneuvers. Attempted left (or right) turning maneuvers are punished during training phases and no predictive stimuli are present. Thus, the only predictor of punishment is the behavior of the fly. If the fly continue to avoid the punished behavior when laser is absent during test phases, we talk about self-learning. Scoring and learning protocol. A score corresponding to the proportion of time the fly is doing the safe turning attempt is calculated for each 2 minutes period. Before training the score is around 0 (no preference). During training phases, the score rises since the fly avoid doing the laser punishment. After two rounds of 4 minutes training separated by a 2min test, a final 2 minutes test round is performed. The performance during these two minutes is used for statistical analysis. After the experiment, the torque force of the fly is tested again using its optomotor response and the accuracy of the laser position (the fly is sacrificed by letting the laser on for about 15s). If any of these two tests is not satisfactory, the data is not included in the analysis. <br><br> Detailed information. Only females were tested, flies were grown at 25° and did receive an heat shock (35° for 4 hours) 0.5 to 4 hours before being tested at 25°. NB: data where the pretest score was higher than 0.65 were excluded from the analysis. Full genotype of the flies tested: CS x T_PKCi : tubGal80ts/+;UASPKCi/+ . GFP, c380;chaG80 x T_PKCi : UAS-GFP,C380-GAL4/+;;cha-Gal80/+ . GFP; d42G4,chaG80 x T_PKCi : UAS-GFP;tubGal80ts/+;UASPKCi/d42Gal4,chaGal80 . GFP, c380G4;chaG80 x CS : UAS-GFP,C380-GAL4;tubGal80ts/+;UASPKCi/cha-Gal80 . GFP; d42G4,chaG80 x CS : UAS-GFP;;d42Gal4,chaGal80/+ . ------ flybase numbers: tubGal80^ts= P{tubP-GAL80ts} (FBtp0017264)[on the 2nd chromosome], d42GAl4= FBti0002759 c380Gal4 = FBti0016294 UAS-GFP = unknown UASPKCi = FBti0010565 cha-Gal80 = unknown

## 实验与图表说明
实验所用组群为tubGal80ts;UAS-PKCi品系（该品系在泛神经元表达时可有效阻断自学习）与两个携带三种特定构建体的品系的杂交后代：分别携带d42Gal4或c380Gal4，同时包含chaGal80（可抑制胆碱能神经元中的Gal4表达）以及UAS-GFP（用于定位基因表达位点）。两种杂交组合均未对果蝇自学习产生显著影响。已知移除GFP构建体后，该抑制效应可正常发挥（Colomb等，待发表），据此我们推断：第二个UAS元件的存在会阻断PKC抑制剂的表达。

**图例说明**
最终测试阶段的得分以箱线图呈现（图中横线代表中位数，箱体代表四分位数区间，须线覆盖除散点表示的异常值外的全部有效数据）。

*果蝇（Drosophila）*自学习实验范式：
果蝇被固定于扭矩仪上，该仪器可采集果蝇围绕身体垂直轴的角动量（即偏航扭矩），该扭矩由果蝇试图做出转向动作时产生。训练阶段中，果蝇的左侧（或右侧）转向动作会被施加惩罚，且训练全程无任何预测性刺激。因此，唯一可预示惩罚的因素仅为果蝇自身的行为。若在测试阶段无激光刺激时，果蝇仍能持续避免曾被惩罚的转向动作，则视为发生自学习。

评分与学习流程：
每2分钟为一个统计周期，计算该周期内果蝇做出安全转向尝试的时间占比，以此作为得分。训练前，果蝇的得分约为0（无转向偏好）。训练阶段中，由于果蝇会主动规避激光惩罚，得分会逐步升高。实验依次进行两次间隔2分钟测试的4分钟训练循环，随后开展为期2分钟的最终测试，以该阶段的表现得分作为统计分析的依据。

实验结束后，通过果蝇的视动反应再次测试其扭矩能力，并验证激光定位的准确性（通过持续照射激光约15秒处死果蝇）。若上述两项测试任意一项未达标，则该果蝇的相关数据将被排除出统计分析。

## 详细实验参数
本次实验仅选用雌性果蝇，果蝇于25℃环境下培养，且在25℃环境下开展测试前的0.5至4小时内，接受了4小时的35℃热激处理。注意：预测试得分高于0.65的数据将被排除出最终分析。

### 受试果蝇完整基因型
1. CS × T_PKCi：tubGal80ts/+;UASPKCi/+
2. GFP, c380;chaG80 × T_PKCi：UAS-GFP,C380-GAL4/+;;cha-Gal80/+
3. GFP; d42G4,chaG80 × T_PKCi：UAS-GFP;tubGal80ts/+;UASPKCi/d42Gal4,chaGal80
4. GFP, c380G4;chaG80 × CS：UAS-GFP,C380-GAL4;tubGal80ts/+;UASPKCi/cha-Gal80
5. GFP; d42G4,chaG80 × CS：UAS-GFP;;d42Gal4,chaGal80/+

### FlyBase数据库编号
tubGal80^ts = P{tubP-GAL80ts}（FBtp0017264，位于2号染色体）；
d42Gal4 = FBti0002759；
c380Gal4 = FBti0016294；
UAS-GFP 无对应已知编号；
UASPKCi = FBti0010565；
cha-Gal80 无对应已知编号。