Altered reactivity to threatening stimuli in Drosophila models of Parkinson’s disease, revealed by a trial-based assay

Reviewer #1 (Public Review):

Summary: Translating discoveries from model organisms to humans is often challenging, especially in neuropsychiatric diseases, due to the vast gaps in the circuit complexities and cognitive capabilities. Kajtor et al. propose to bridge this gap in the fly models of Parkinson's disease (PD) by developing a new behavioral assay where flies respond to a moving shadow by modifying their locomotor activities. The authors believe the flies' response to the shadow approximates their escape response to an approaching predator. To validate this argument, they tested several PD-relevant transgenic fly lines and showed that some of them indeed have altered responses in their assay.

Strengths: This single-fly-based assay is easy and inexpensive to set up, scalable, and provides sensitive, quantitative estimates to probe flies' optomotor acuity. The behavioral data is detailed, and the analysis parameters are well-explained.

Weaknesses: While the abstract promises to give us an assay to accelerate fly-to-human translation, the authors need to provide evidence to show that this is indeed the case. They have used PD lines extensively characterized by other groups, often with cheaper and easier-to-setup assays like negative geotaxis, and do not offer any new insights into them. The conceptual leap from a low-level behavioral phenotype, e.g. changes in walking speed, to recapitulating human PD progression is enormous, and the paper does not make any attempt to bridge it. It needs to be clarified how this assay provides a new understanding of the fly PD models, as the authors do not explore the cellular/circuit basis of the phenotypes. Similarly, they have assumed that the behavior they are looking at is an escape-from-predator response modulated by the central complex- is there any evidence to support these assumptions? Because of their rather superficial approach, the paper does not go beyond providing us with a collection of interesting but preliminary observations.

Reviewer #2 (Public Review):

In this study, Kajtor et al investigated the use of a single-animal trial-based behavioral assay for the assessment of subtle changes in the locomotor behavior of different genetic models of Parkinson's disease of Drosophila. Different genotypes used in this study were Ddc-GAL4>UAS-Parkin-275W and UAS- α-Syn-A53T. The authors measured Drosophila's response to predator-mimicking passing shadow as a threatening stimulus. Along with these, various dopamine (DA) receptor mutants, Dop1R1, Dop1R2 and DopEcR were also tested.
The behavior was measured in a custom-designed apparatus that allows simultaneous testing of 13 individual flies in a plexiglass arena. The inter-trial intervals were randomized for 40 trials within 40 minutes duration and fly responses were defined into freezing, slowing down, and running by hierarchical clustering. Most of the mutant flies showed decreased reactivity to threatening stimuli, but the speed-response behavior was genotype invariant.
These data nicely show that measuring responses to the predator-mimicking passing shadows could be used to assess the subtle differences in the locomotion parameters in various genetic models of Drosophila.

The understanding of the manifestation of various neuronal disorders is a topic of active research. Many of the neuronal disorders start by presenting subtle changes in neuronal circuits and quantification and measurement of these subtle behavior responses could help one delineate the mechanisms involved. The data from the present study nicely uses the behavioral response to predator-mimicking passing shadows to measure subtle changes in behavior. However, there are a few important points that would help establish the robustness of this study.

1. The visual threat stimulus for measuring response behavior in Drosophila is previously established for both single and multiple flies in an arena. A comparative analysis of data and the pros and cons of the previously established techniques (for example, Gibson et al., 2015) with the technique presented in this study would be important to establish the current assay as an important advancement.
2. Parkinson's disease mutants should be validated with other GAL-4 drivers along with Ddc-GAL4, such as NP6510-Gal4 (Riemensperger et al., 2013). This would be important to delineate the behavioral differences due to dopaminergic neurons and serotonergic neurons and establish the Parkinson's disease phenotype robustly.
3. The DopEcR mutant genotype used for behavior analysis is w1118; PBac{PB}DopEcRc02142/TM6B, Tb1. Balancer chromosomes, such as TM6B,Tb can have undesirable and uncharacterised behavioral effects. This could be addressed by removing the balancer and testing the DopEcR mutant in homozygous (if viable) or heterozygous conditions.
4. The height of the arena is restricted to 1mm. However, for the wild-type flies (Canton-S) and many other mutants, the height is usually more than 1mm. Also, a 1 mm height could restrict the fly movement. For example, it might not allow the flies to flip upside down in the arena easily. This could introduce some unwanted behavioral changes. A simple experiment with an arena of height at least 2.5mm could be used to verify the effect of 1mm height.
5. The detailed model for Monte Carlo simulation for speed-response simulation is not described. The simulation model and its hyperparameters need to be described in more depth and with proper justification.
6. The statistical analysis in different experiments needs revisiting. It wasn't clear to me if the authors checked if the data is normally distributed. A simple remedy to this would be to check the normality of data using the Shapiro-Wilk test or Kolmogorov-Smirnov test. Based on the normality check, data should be further analyzed using either parametric or non-parametric statistical tests. Further, the statistical test for the age-dependent behavior response needs revisiting as well. Using two-way ANOVA is not justified given the complexity of the experimental design. Again, after checking for the normality of data, a more rigorous statistical test, such as split-plot ANOVA or a generalized linear model could be used.
7. The dopamine receptor mutants used in this study are well characterized for learning and memory deficits. In the Parkinson's disease model of Drosophila, there is a loss of DA neurons in specific pockets in the central brain. Hence, it would be apt to use whole animal DA receptor mutants as general DA mutants rather than the Parkinson's disease model. The authors may want to rework the title to reflect the same.