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  1. Evaluation Summary:

    This paper seeks to expand our understanding of how spinally-projecting serotonergic neurons either inhibit or facilitate nociception depending on physiological context. Capitalizing on differential susceptibility to AAVretro transduction, the authors suggest identification of functional serotonergic subunits within the medullary raphe - one that includes innervation of the superficial dorsal horn and may modulate sensitivity to peripheral thermal stimuli, and another that includes innervation of a deeper lamina of the dorsal horn and may modulate sensitivity to mechanical von Frey stimulation. As well, the viral techniques and findings may inform the design and interpretation of work in the field.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 and Reviewer #3 agreed to share their name with the authors.)

  2. Reviewer #3 (Public Review):

    Ganley et al have investigated the anatomical organisation and function of descending serotoninergic (5-HT) pathways that originate in the medulla and project to the spinal cord. It has long been known that descending 5-HT pathways had powerful modulatory effects. However, previous studies have shown that this system can either facilitate or inhibit pain, although relatively little was known about the underlying mechanisms. The authors initially confirm previous findings that 5-HT neurons in the lateral pargigantocellularis (LPGi) nucleus project to the superficial laminae of the dorsal horn, while those in the nucleus raphe magnus (NRM) innervate more ventral regions, including the deep dorsal horn. In the course of this work, they made an apparently serendipitous discovery, that retrograde tracing from the spinal cord with the AAV2Retro serotype preferentially captures projections from LPGi, while largely sparing those from the NRM. They then exploit this finding, together with selective targeting of NRM by means of small midline injections into the medulla, to examine the effects of chemogenetically activating either the NRM or LPGi components of the descending serotoninergic system. Their behavioural results show a clear and extremely interesting distinction: activating the LPGi 5-HT cells decreases responses to hot or cold stimuli (with no apparent effect on those to mechanical stimuli), while activating the NRM 5-HT cells increases mechanical sensitivity, with no effect on responses to thermal stimuli. This suggests that both sensory modality affected, and the polarity of the effect, depend on the source of the 5-HT cells and that these are mediated through different regions of the dorsal horn.

    In the course of the study, the authors also provide interesting insights into the vagaries of two widely used viral tracing techniques: monosynaptic rabies tracing and the use of AAV2Retro for retrograde tracing. For example, they show that medullary 5-HT neurons are capable of being directly infected with rabies virus, but are not labelled through transsynaptic transport, probably reflecting the lack of synapses formed by many of these cells. Their findings show the importance of caution when interpreting negative results with these techniques.

    The article is generally well written and illustrated. The work has been conducted carefully, and the interpretation is suitably cautious.

  3. Reviewer #2 (Public Review):

    This paper will be of interest to scientists within the fields of sensory processing and neuromodulation, as it deepens our understanding of descending serotonergic neuron subsystems and their differential innervation of dorsoventral spinal cord regions and thus likely differential roles in sensory tasks. Sophisticated viral strategies coupled with transgenic approaches are deployed and exploited in novel ways, which will also be of interest. However, in its current form, the paper would benefit from additional controls, more complete data presentation and analyses, and broader inclusion of the literature and its implications for interpreting the present work.

    The identification of a functional subdivision within the medullary serotonergic raphe as relates to sensitivity to peripheral thermal versus mechanical stimuli adds to the ongoing discussion on pain control circuitry. By uncovering and appreciating a differential susceptibility to AAV2retro infection, the authors identified a novel means by which to classify descending, spinal-cord-projecting serotonergic neurons. The analyses of various viral methods for the study of serotonergic neurons also add to ongoing discussions in the field.

    The interpretation of some experiments is not fully supported by the data presented. Additional data is needed, genetic tools require validation for the intended use, and in some cases control experiments need expansion (as relates to the use of DREADDs). As well, the manuscript narrative might be better served if some figures/panels were presented as supplemental, and if the context and discussion included a broader swath of the work published in this area.

  4. Reviewer #1 (Public Review):

    The authors thoroughly investigated four different standard methods for circuit tracing, and show that these methods differentially label lateral versus midline hindbrain serotonergic neurons and their processes. The authors then made use of these differences to individually target the two neuronal populations. In particular, the lateral paragigantocellularis (LPGi) were successfully labeled with an intersectional methodology owing to their distinctive susceptibility to AAV2retro transduction. While the differences are useful in this study, the authors do not address the biological underpinnings of these differences in labeling. Instead, the initial component of the study describes these labeling differences.

    Based on these identified differences in labeling, the authors demonstrate that medial and lateral hindbrain serotonergic neuron axon projections innervate different regions of the spinal cord. Interestingly, nucleus raphe magnus (NRM) neurons project to most of the spinal cord but are absent from the superficial lamina. Considering the superficial lamina corresponds to the site of C-fiber and A-LTRM dorsal root ganglion sensory neuron innervation, it implies that serotonergic neurons in the NRM may not regulate pain circuits directly involving these nociceptors. Indeed, the authors demonstrate that activation of NRM neurons increases mechanical sensitivity. In contrast, LPGi axons were projected to the superficial lamina, and activation of these neurons decreased thermal sensitivity. This study does not elucidate how descending serotonergic projections are involved in the modulation of these pain circuits; i.e. through synaptic transmission with primary afferents or interneurons. Moreover, it is not clear whether these projections are different between lamina I and lamina II, or whether these vary along the rostrocaudal axis. Since distinct spinal cord lamina is innervated differentially by specific subtypes of peripheral neurons - the authors could consider co-labeling descending neuronal projections with known laminar and DRG subtype markers.

    To address the functional implications of the innervation patterns seen, the authors investigated the involvement of lateral and medial hindbrain neurons in thermal and mechanical pain circuits using three behavioral assays: Hargreaves, Cold plantar, and von Frey. This is the major point of the study - namely that the lateral paragigantocellularis serotonergic neurons modulate thermal sensation, while the medial serotonergic neurons modulate responses to mechanical stimuli. This provides a good start to understanding the functional differences. To fully understand the role of hindbrain serotonergic neurons in descending pain modulation, these studies should be expanded to include chemical, pruriceptive, proprioceptive, and high and low-threshold mechanical stimuli, as well as chronic pain.

    It is also clear that a shortcoming of this study is that the authors did not study sex differences although such differences are well-documented in serotonin signaling and pain perception.