Somatotopic organization among parallel sensory pathways that promote a grooming sequence in Drosophila

Reviewer #1 (Public Review):

Sensory neurons of the mechanosensory bristles on the head of the fly project to the sub esophageal ganglion (SEZ). In this manuscript, the authors have built on a large body of previous work to comprehensively classify and quantify the head bristles. They broadly identify the nerves that various bristles use to project to the SEZ and describe their region-specific innervation in the SEZ. They use dye-fills, clonal labelling, and electron microscopic reconstructions to describe in detail the phenomenon of somatotopy - conserved peripheral representations within the central brain - within the innervation of these neurons. In the process they develop novel tools to access subsets of these neurons. They use these to demostrate that groups of bristles in different parts of the head control different aspects of the grooming sequence.

Reviewer #2 (Public Review):

The authors combine genetic tools, dye fills and connectome analysis techniques to generate a "first-of-its-kind", near complete, synaptic resolution map of the head bristle neurons of Drosophila. While some of the BMN anatomy was already known based on previous work by the authors and other researchers, this is the first time a near complete map has been created for the head BMNs at electron microscopy resolution.

Strengths:
1. The authors cleverly use techniques that allow moving back and forth between periphery (head bristle location) and brain, as well as moving between light microscopy and electron microscopy data. This allows them to first characterize the pathways taken by different head BMNs to project to the brain and also characterize anatomical differences among individual neurons at the level of morphology and connectivity.
2. The work is very comprehensive and results in a near complete map of all head BMNs.
3. Authors also complement this anatomical characterization with a first-level functional analysis using optogenetic activation of BMNs that results in expected directed grooming behavior.

Weaknesses:
1. The clustering analysis is compelling but cluster numbers seem to be arbitrarily chosen instead of by using some informed metrics.
2. It could help provide context if authors revealed some of the important downstream pathways that could explain optogenetics behavioral phenotypes and previously shown hierarchical organization of grooming sequences.
3. In contrast to the rigorous quantitative analysis of the anatomical data, the behavioral data is analyzed using much more subjective methods. While I do not think it is necessary to perform a rigorous analysis of behaviors in this anatomy focused manuscript, the conclusions based on behavioral analysis should be treated as speculative in the current form e.g. calling "nodding + backward walking" as an avoidance response is not justified as it currently stands. Strong optogenetic activation could lead to sudden postural changes that due to purely biomechanical constraints could lead to a couple of backward steps as seen in the example videos. Moreover since the quantification is manual, it is not clear what the analyst interprets as backward walking or nodding. Interpretation is also concerning because controls show backward walking (although in fewer instances based on subjective quantification).

Summary:
The authors end up generating a near-complete map of head BMNs that will serve as a long-standing resource to the Drosophila research community. This will directly shape future experiments aimed at modeling or functionally analyzing the head grooming circuit to understand how somatotopy guides behaviors.

Reviewer #3 (Public Review):

Eichler et al. set out to map the locations of the mechanosensory bristles on the fly head, examine the axonal morphology of the bristle mechanosensory neurons (BMNs) that innervate them, and match these to electron microscopy reconstructions of the same BMNs in a previously published EM volume of the female adult fly brain. They used BMN synaptic connectivity information to create clusters of BMNs that they show occupy different regions of the subesophageal zone brain region and use optogenetic activation of subsets of BMNs to support the claim that the morphological projections and connectivity of defined groups of BMNs are consistent with the parallel model for behavioral sequence generation.

The authors have beautifully cataloged the mechanosensory bristles and the projection paths and patterns of the corresponding BMN axons in the brain using detailed and painstaking methods. The result is a neuroanatomy resource that will be an important community resource. To match BMNs reconstructed in an electron microscopy volume of the adult fly brain, the authors matched clustered reconstructed BMNs with light-level BMN classes using a variety of methods, but evidence for matching is only summarized and not demonstrated in a way that allows the reader to evaluate the strength of the evidence. The authors then switch from morphology-based categorization to non-BMN connectivity as a clustering method, which they claim demonstrates that BMNs form a somatotopic map in the brain. This map is not easily appreciated, and although contralateral projections in some populations are clear, the distinct projection zones that are mentioned by the authors are not readily apparent. Because of the extensive morphological overlap between connectivity-based clusters, it is not clear that small projection differences at the projection level are what determines the post-synaptic connectivity of a given BMN cluster or their functional role during behavior. The claim the somatotopic organization of BMN projections is preserved among their postsynaptic partners to form parallel sensory pathways is not supported by the result that different connectivity clusters still have high cosine similarity in a number of cases (i.e. Clusters 1 and 3, or Clusters 1 and 2). Finally, the authors use tools that were generated during the light-level characterization of BMN projections to show that specifically activating BMNs that innervate different areas of the head triggers different grooming behaviors. In one case, activation of a single population of sensory bristles (lnOm) triggers two different behaviors, both eye and dorsal head grooming. This result does not seem consistent with the parallel model, which suggests that these behaviors should be mutually exclusive and rely on parallel downstream circuitry.

This work will have a positive impact on the field by contributing a complete accounting of the mechanosensory bristles of the fruit fly head, describing the brain projection patterns of the BMNs that innervate them, and linking them to BMN sensory projections in an electron microscopy volume of the adult fly brain. It will also have a positive impact on the field by providing genetic tools to help functionally subdivide the contributions of different BMN populations to circuit computations and behavior. This contribution will pave the way for further mechanistic study of central circuits that subserve grooming circuits.