Pallium-encoded valence-specific chemosensory amplification of eye-body coordination in larval zebrafish

Reviewer #1 (Public review):

Summary:

This study was designed to manipulate and analyze the effects of chemosensory cues on visuomotor control. They approach this by analyzing how eye-body coordination and brain-wide activity are altered with specific chemosensation in larval zebrafish. After analyzing the dynamics of coupled saccade-tail coordination sequences - directionally linked and typically coupled to body turns - the authors investigated the effects of sensory cues shown to be either aversive or appetitive on freely swimming zebrafish on the eye-body coordination. Aversive chemicals lead to an increase in saccade-tail sequences in both number and dynamics, seemingly facilitating behaviors like escape. Brain-wide imaging led the authors to neurons in the telencephalic pallium as a target to study eye-body coordination. Pallium neuron activity correlated with both aversive chemicals and coupled saccade-tail movements.

Recommendations for improvement are minimal. So much of the data is ultimately tabular, and the figures are an impenetrable wall of datapoints. 1c is an excellent example: three concentrations are presented, but it's rare for the three averages to trend appropriately. The key point, which is that aversive odors are repulsive and attractive odors (sometimes) attractive just gets lost in showing the three concentrations individually; it also makes direct comparisons impossible. There are similar challenges abound in the violin plots in 4e-4h, the error bars on the "fits" in 4i-4m, and so on. We recommend selecting an illustrative subset of data to present to permit interpretation and putting the rest in a supplemental table. (Presenting) less is more (effective).

Reviewer #2 (Public review):

Summary:

The manuscript by Sy SKH. et al. on pallium encoded chemosensory impact of eye-body coordination describes how the valence of chemosensory stimuli can affect the coordination of eye saccades with tail flips. They show that aversive valence stimuli can increase both the strength and frequency of tail flips through a pallium-mediated circuit.

Overall, the manuscript is well-written and easy to follow, although the figures are quite dense, the methodology is mostly sound, and the improvement to the fish on chips system is very interesting. The methods description is thorough and welcome, making the experiments clear. The limited number of animals, and the spread between 5 and 6dpf is a concern as most of the statistics seem to have been done on the individual events, and not the number of biological samples.

The initial behavioural experiments are very promising. However, the conclusions surrounding the role of the pallium are a lot more speculative and not supported by the results.

Comments:

(1) The fish on chips 2.0 methods show a lot of promise for future studies of chemosensory stimuli, combined with whole-brain imaging. This will provide new avenues of research for zebrafish neuroscientists.

(2) Chemosensory cues would have a very different timing than visual cues; timing is very important for multisensory integration. How do the authors suggest those are integrated? How would they differentiate between an integration of various cues or a different arousal state, as they describe in the introduction?

(3) Studies have looked at chemosensation in Drosophila, including multisensory integration, which should be discussed by the authors (see the work of Mark Frye, amongst others).

(4) In the brain imaging methods, there is a mention of robustly behaving larvae. Does that mean that an exclusion criterion was used to select only 5 larvae? If so, this should be stated clearly. The authors also do not mention how they avoid the switch to a passive state that one of the coauthors has observed in closed closed-loop setup. The authors should comment on this point.

(5) Were the statistics in Figure 2 done with an n of 5, or do they assume that each tail flip and saccade is an independent event? I would imagine the latter would have inflated p-values and should be avoided.

(7) Page 7: Why do the authors think that the cumulative effect of these minor differences could lead to very different behavioural goals? Especially when comparing to actual startle responses, which are extremely strong and stereotypical. How do their observations compare to the thermosensory navigation of larval zebrafish observed by Martin Haesemeyer, for example, or the work of the RoLi lab?

(8) Page 8: Figure 5, I am confused by the y-axis of g, in e and f, the values are capped at 2, whereas in g they go up to 6, with apparently a number of cells whose preference is out of the y-axis limit (especially in Q2). Having the number of cells in each quadrant would also help to assess if indeed there is some preference in the pallium towards Q1.

(9) Figure 6: How is the onset of neuronal activity determined compared to the motor stimulus? Looking at Supplementary Figure 8, it is quite unclear how the pallium is different from the OB or subpallium. The label of onset delay is also confusing in this figure.

(10) Page 9: I do not think that the small differences observed in the pallium are as clear-cut as the authors make them out to be, or that they provide such strong evidence of their importance. As there are no interventions showing any causality in the presence of these pallium responses and the sensorimotor responses, these could represent different arousal states rather than any integration of sensory information.

Reviewer #3 (Public review):

The manuscript investigates the coupling of saccadic eye movements (S) with directed tail flips (T). The remarkable discovery is that tail flips that are preceded by a conjugate sacced (S-T) can be credibly classified as specific "volitional" turns that are distinguished from the standard tail movements that seem to be more of a spontaneous and "impulsive" nature.

They show that 'turning intent', as indicated by a small increase in S, is elevated by aversive odors, while 'gliding intent', as indicated by a decrease in S and an increase in undulation cycles, is elevated by appetitive odors.

This is a very important finding, which is backed up by a thorough behavioral analysis, and the identification of neural populations in the pallium and sub-pallium that clearly distinguish between these kinds of turns is very promising. Here they identify neuronal populations that are preferentially active during - and predictive of - coupled (S-T) versus isolated (T) tail flips.

Especially the fact that S-T turns (but not T turns) can be predicted already by pre-event, ramping, pallial activity is intriguing.

The authors then go on and demonstrate that the frequency of (S-T) turns is modulated in fish exposed to appetitive or aversive odors.
Specifically, they quantify the aversiveness and appetitive-ness of several odors in a free swimming assay. They select a couple of these odors based on their valence, and they demonstrate that these odors induce moderate modulation in the frequency of eye saccades (S) and tail flips (T) and (S-T) turns.

The study is rigorous and thorough, and the findings are informative and novel.

In important controls, they confirm that brain-wide imaging can distinguish between appetitive and aversive contexts, and they show that pallial activation by aversive odors is consistent with neural activity in the rhombencephalon that correlates with turning activity, whereas sub-pallial activation by appetitive odors correlates with rhombencephalic activity related to gliding.

Overall, this manuscript is very good.

Author response:

We thank the editors and all reviewers for the detailed evaluation of the work and the overall positive remarks, as well as the constructive feedback to improve our manuscript. Based on the integrated comments of the reviewers and advice of the reviewing editor, we will suitably address all comments raised by the reviewers, and we outline our revision plan below:

Interpretation of findings

● We will carefully reframe our interpretation of the data regarding the role of the pallium in the coupled saccade-tail turning events, and clearly state that we have not established a causal role, which requires additional perturbation experiments.

● We will also acknowledge the confounding interpretation that the pallial activities recorded may also represent or include arousal state signals.

Streamlining the presentation

● In the introduction, we will better contextualize our study with additional discussions on (i) the advantageous use of zebrafish to study chemosensation, factoring in differences in the spread of chemical cues in water vs. air, and (ii) the disruption of eye-body coordination and underlying neural circuits.

● We will streamline the presentation of data in Fig. 1 by keeping the overall responses of the larvae to each chemical across concentrations in the main figure, while moving suitable additional details to a supplementary figure.

● Similarly, for each of the subsequent main figures, wherever suitable we will select an illustrative, core set of panels to retain in the main figure, and move other more detailed plots to supplementary figures.

● We will incorporate additional references and discussions of the past literature, including relating our findings to (i) chemosensation/multisensory integration in Drosophila, (ii) thermosensation-driven and navigational behavior in larval zebrafish, and (iii) fleeing or escape behavior in zebrafish and other species.

● We will clarify our animal subject inclusion criteria, that all larval subjects with sufficiently high-quality, stable imaging were included (i.e., we only excluded larvae because of insufficient quality of imaging, but not other factors).

● For applicable plots, adding suitable additional details to the plots or legends (e.g., clarification of measures, specifying numbers of cells).

Data analysis and statistics

● We will perform additional data analysis, by making comparisons with statistics performedon fish subject-level, and include confident intervals wherever applicable.