Feedback inhibition by a descending GABAergic neuron regulates timing of escape behavior in Drosophila larvae

Reviewer #1 (Public Review):

Summary:
Zhu et al. set out to better understand the neural mechanisms underlying Drosophila larval escape behavior. The escape behavior is comprised of several sequenced movements, including a lateral roll motion followed by fast crawling. The authors specifically were looking to identify neurons important for the roll-to-crawl transition.

Strengths:
This paper is clearly written. The experiments are logical and complementary. They support the author's main claim that SeIN128 is a type of descending neuron that is both necessary and sufficient to modulate the termination of rolling.

Weaknesses:
-This manuscript is narrowly focused on Drosophila larval escape behavior. It would be more accessible to a broader audience if this work was put into a larger context of descending control.
-In general, the rigor is high. However, a few control experiments are missing.

Reviewer #2 (Public Review):

Summary:
This study discovered a neural mechanism that serves as a switch from rolling to fast crawling behaviors in Drosophila larvae. It addressed important open questions of how neural circuits determine the sequence of locomotor behaviors and how animals switch from one behavior to another. Overall, its results support the conclusions. The experimental approaches should be described more clearly.

The escape behavior of Drosophila larvae includes rolling followed by fast crawling, where the neural mechanism of this sequence is unclear. The authors identified SeIN128, a group of descending neurons that facilitates rolling termination and shortens crawling latency. By investigating the EM connectome of larval CNS, they found that SeIN128 receives inputs from Basin-2 and A00c neurons, which are reported to facilitate rolling. SeIN128 makes reciprocal inhibitory synapses onto Basin-2 and A00c. Gad staining indicates that SeIN128 neurons are GABAergic, and inhibition of SeIN128 caused increased rolling probability and prolonged rolling. RNAi knockdown of GABA receptors in Basins further validated that SeIN128 inhibits Basins via GABAergic inputs. Lastly, the authors found that SeIN128 inhibits rolling induced by two types of Basin neurons, Basin-2 and Basin-4. Overall, SeIN128 forms a feedback inhibition ensemble that terminates rolling and shifts the animal to crawling.

Strengths:
- The question (i.e., the neural circuitry of action selection) addressed by this study is important.
- Larval and adult Drosophila is a powerful model system in neuroscience study, with rich genetic tools, diverse behaviors, and well-studied nervous systems. This study makes good use of them.
- The experiments, analyses, and results are mostly rigorous and support the major claims. This study combined multiple innovative approaches, such as automated, machine-learning-based behavioral assays, EM reconstruction of larval CNS neurons, and genetic manipulation of specific neurons.

Weaknesses:
- The description of methods and quantification for certain analyses are not clear or detailed enough for a comprehensive judgment of rigorousness, or for other scientists to repeat the experiments. This especially applies to the algorithm.
- "Corkscrew-like rolling" is not an accurate term for larval rolling. The neuromuscular basis of rolling was recently studied by Cooney et. al., showing that rolling is the circumferential propagation of muscle activity where all segments contract similarly and synchronously.
- The readability of the manuscript (text and figures) needs improvement, especially in making it understandable for a general audience. The addition of visual representations, simplifying the complex names of neurons, avoiding overall long sentences, and providing sufficient background introduction may help.

Reviewer #3 (Public Review):

Summary: Drosophila larvae exhibit characteristic escape behavior in response to a noxious stimulus. The underlying nociceptive circuit that regulates the temporal dynamics of escape behavior - bending, rolling, and crawling remains unclear. Using behavioral prototypes with optical stimulation and imaging, the authors show the function of descending neurons (SeIN128) in the termination of the rolling and subsequent initiation of the crawling behavior. The study further establishes the functional connectome of SeIN128, Basin-2, and A00c neurons, forming an inhibitory feedback circuit that regulates the rolling-escape sequences.

Strength: The study provides anatomical and functional evidence for temporal dynamics of escape behaviors in Drosophila larvae. Authors convincingly show the function of bilaterally descending neurons (previously identified SeIN128 neurons) in the transition of escape sequences. Based on the previous studies and functional connectome analysis, the study shows that SeIN128 neurons form a GABAergic feedback circuit with Basin-2, a second-order interneuron, and A00c, an ascending neuron downstream of Basin-2. Activation of SeIN128 neurons terminates the rolling by suppressing Basin-2 activity, facilitating subsequent rapid escape crawling. Thus, it establishes the function of feedback inhibition in temporal dynamics of escape behavior and contributes to a mechanistic understanding of the nociceptive circuits.

Weakness: The manuscript is written clearly; however, the presentation of the data needs to be improved for readability. The data and discussion establish the function of SeIN128 and Basin-2 in escape behavior, but the role of A00c neurons needs to be clarified.