Decision letter | Norepinephrine is required to promote wakefulness and for hypocretin-induced arousal in zebrafish

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Norepinephrine is required to promote wakefulness and for hypocretin-induced arousal in zebrafish

Decision letter

Affiliation details

California Institute of Technology, United States
Louis Ptáček, Reviewing editor, University of California, San Francisco, United States

eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.

Thank you for sending your work entitled “Norepinephrine is Required to Promote Wakefulness and for Hypocretin-Induced Arousal in Zebrafish” for consideration at eLife. Your article has been favorably evaluated by Eve Marder (Senior editor) and three reviewers, one of whom, Louis Ptáček, is a member of our Board of Reviewing Editors.

The Reviewing editor and the other reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.

The manuscript by Singh and colleagues is a nice piece of work that is logical in its progression. One very novel and powerful technical advance is the in vivo optogenetics in a 96 well format. It would be better to not refer to this as “high-throughput” as many think of HTP in the context of biochemical/fluorescent assays that allow easy assessment of many thousand or more of assays. This is clearly HTP for a behavioral analysis though, and the authors could simply tone down the term. This approach takes advantage of the transparency of fish and the ability to excite proteins without surgical implantation that is necessary in mice. This study follows logically from examination of HCRT->HCRTR->NE signaling on “wakefulness”. The bottom line is that hypocretin promotes arousal through its receptor in the locus coeruleus (LC) and specifically on NE-expressing cells in the LC. Since the investigators are not technically looking at sleep, more careful language (rest-activity rhythms, “sleep-like behavior”) would be more appropriate. Many of us believe rest activity as described here will be conserved (although not identical) to mammalian sleep. Of course, there will be some periods of inactivity when an animal is awake, thus, the need for greater care with the semantics.

Essential revisions:

1) Prazosin is an alpha1 specific antagonist. Its use leads to decreased activity in fish, increased number of bouts of sleep-like behavior, and decreased latency. However, there is no discussion of other receptors, namely alpha2, beta1, and beta2. The dbh-/- experiment looks at the effect of decreasing NE which would have effects on all receptors.

2) Related to #1, the authors don't discuss the discrepancy in Prazosin and dbh experiments in panels H of Figures 1 and 2. Prazosin has no effect on bout length at night while it is significantly increased in the dbh experiment. How many (if any) paralogous genes exist in zebrafish? Is there redundancy? It is possible that this may relate to alpha1-specific affects vs. global NE affects. One additional experiment is to use clonidine (alpha2 agonist) (or the IV form, Dexmedetomidine), or beta1 antagonists (beta-blockers) to help dissect differential effects on different receptors.

3) The result that adult dbh-/- fish have higher mortality related to normal doses of the anesthetic tricaine is interesting, yet out of place and unsupported by the data provided. You may consider omitting this finding, or explaining better its relevance.

4) With respect to the prazosin experiments, the investigators overstate the case for the effect not being dopamine-mediated. This would require measuring dopamine levels directly. It would be worthwhile to review (and to reference) the work from Ken Solt's lab showing that DA actions in VTA are important for arousal from anesthesia and to tone down the conclusion to “suggests the affect in not dopamine-mediated”, this may also be useful in the discussion surrounding Figure 3.

5) The paper claims in several places that “NE is required for Hcrt-induced arousal” (this quote is from the Abstract). This is not true. There is a major quantitative effect in the mutant (and see below) but as the arousal enhancement is not eliminated, this bold statement is incorrect.

6) It would have been good had they used the drug prazosin in combination with the Hcrt-induced arousal rather than just the mutant fish alone. NE elimination need not be the only effect of the KO, especially as it is systemic and all through development. For example, there could be compensatory changes in the endocrine system. Replication with the drug would increase confidence in the conclusion.

7) How long was the pretreatment in Figure 1 (how fast-acting is the drug), was a dose-response curve done (where is 100 μM on this curve?), and was the drug used in the dbh KO fish? Was it without effect as one might predict if it is specific for alpha-adrenergic receptors?

8) The arousal threshold effects (Figure 3) are rather modest, and enigmatic. First in Figure 3A, the “increased response rate” (not a response rate, just an enhanced response) is probably just an independent effect as the authors suggest. However, this means that they should replot their data subtracting a fixed value from each mutant response, and then compare with the WT. There may be a relatively small residual effect. Even in Figure 3B, it is difficult to interpret the magnitude of the effect because it is not clear what the X-axis is (log base 2, base 10?). The fact that the drug and the mutant effects are similar (even if modest) appears to underscore the conclusion but still… And if the drug result (3B) is really more impressive than the mutant (1A), what does this do to the dopamine hypothesis? Perhaps the authors could consider that the excess sleep itself lowers the arousal threshold. (Too much sleep; sleep more lightly?). And the more sleep itself is no surprise. Is this possible?

9) The role of the LC is also no surprise given what has been done in rodents (e.g., Carter et al., 2012). It is however of value to pinpoint the role of NE within the LC. Given that the effect is far from complete, it means that there are other arousal systems that probably lie downstream of Hcrt – as indicated in the literature. It would be good if the authors discussed some of these and the likelihood that they also play a role in fish.

10) The authors spend considerable effort emphasizing the controversy in the sleep phenotype of mouse dbh knockouts. While it is true that two groups reported different sleep results for those mice, it is unclear whether this fish study truly resolves the controversy – it only provides supporting evidence that NA is wake-promoting in a phylogenetic sense and is consistent with analysis of the role of octopamine in Drosophila sleep. The evidence they provide for hypocretin promoting arousal partially via NA is a nice extension of a prior study done in mice by Carter et al. 2012, which implicated LC neurons in the arousing effects of hypocretin.