Serotonergic modulation of motor subspace dynamics drives a sleep-independent quiescent state

Reviewer #1 (Public review):

The wide-ranging serotonergic projections emerging from the Dorsal Raphe nucleus (DRN) are suggestive of a central role in regulating brain-wide activity and behavioural states. DRN activity has been associated with diverse functions, ranging from mood, motivation and pain regulation to sleep and cognitive flexibility. Its far-reaching connectivity made it challenging to assess the brain-wide effect of its activation, especially during behaviour.

The present study by Qi et al. addresses these challenges by combining state-of-the-art tracking microscopy with the whole-brain accessibility of the larval zebrafish model. To investigate the effect of DRN activation, the authors leveraged the Tg(tph2:ChrimsonR) line to optogenetically activate tph2-positive neurons in the DRN, while monitoring changes in brain-wide activity, locomotion and auditory-stimuli evoked responses.

Optogenetic activation had a suppressing effect on locomotion, which the authors distinguished from inducing sleep by the maintenance of posture and its sleep disturbing effect of nighttime stimulations. Further, the authors report a distinct effect of DRN activation on motor-related, but not auditory-related neuronal subspaces, identified by demixed principal component analysis.

In addition, rather than affecting all motor-correlated neurons similarly, tph2+ DRN-mediated suppression focused on neurons encoding high-amplitude or turning motion.

In summary, the work of Qi et al. provides solid evidence for a predominant role of the DRN in wake-state motor suppression by aptly combining the vast data-acquisition possibilities of the larval zebrafish model with computational methods to extract relevant information.

The brain-wide scope of the analysis is a key strength, reducing bias, confirming the involvement of known motor and auditory regions, and providing a valuable dataset for future analyses.

While the results well support the conclusion of the authors, certain biological and technical aspects demand discussion.

Reviewer #2 (Public review):

Summary:

The authors examine the effects of activating the dorsal raphe nucleus serotonergic system using a combination of calcium imaging and optogenetics in freely moving larval zebrafish. Their findings show that optogenetic stimulation induces a state of behavioral quiescence.

They further investigate whether this state corresponds to sleep or reduced motor activity. Analyses of posture and sleep-related paradigms indicate that serotonergic activation primarily suppresses motor output rather than promoting sleep. Notably, this suppression appears to be bout type-dependent, with stronger effects on neurons associated with larger tail amplitudes and turning angles.

In addition, auditory stimulation experiments reveal no significant impact of serotonin on sound encoding.

Strengths:

The study combines advanced experimental techniques with state-of-the-art analytical methods, enabling precise and compelling insights into the role of serotonergic modulation. The experiments and analyses are well aligned with the questions being addressed, and the results appear robust and reliable.

Moreover, the implementation of experiments that combine calcium imaging and optogenetics in freely moving animals is technically challenging and appears well justified in the context of the research questions.

Weaknesses:

While the analytical techniques employed are sophisticated and appear to be appropriately applied, their presentation makes the manuscript difficult to follow. Although the explanations are provided in the Methods section, including more guidance in the main text, such as how to interpret each analytical approach and what outcomes would be expected under different scenarios, would help readers who are less familiar with these techniques.

Providing this context would better guide the reader in navigating the figures, broaden the accessibility of the work, and ultimately increase its impact.

While the authors discuss different quiescent states mediated by serotonin reported in previous studies, their interpretation is limited to stating that "a common feature shared by these distinct behavioral states is a pronounced reduction in movement," and consequently proposing that activation of dorsal raphe nucleus is not sufficient to specify a particular behavioral state, but rather plays a primary role in driving motor suppression.

In my view, a more thorough attempt to determine whether the observed state corresponds to any of the previously described forms of quiescence, or represents a subset or variant of them, would strengthen the manuscript. This would help better integrate the findings with the existing literature.

For example, given that the authors have access to whole-brain activity data, it would be valuable to examine and discuss whether there are shared patterns of activation with previously reported quiescent states.

The manuscript largely avoids discussing the mechanisms underlying the observed motor suppression. For instance, is this effect driven directly by serotonin release onto target neurons? Is it mediated by glial activity, as suggested in other studies? Are additional neuromodulatory systems being recruited?

While addressing these questions may require substantial further work, potentially beyond the scope of the present study, the availability of whole-brain data provides an opportunity to at least explore or discuss these possibilities. In particular, it would be interesting to examine the recruitment of regions not directly stimulated but known to be associated with other neuromodulatory systems or promoting glial activation (e.g., the locus coeruleus).