A comprehensive mechanosensory connectome reveals a somatotopically organized neural circuit architecture controlling stimulus-aimed grooming of the Drosophila head

Reviewer #1 (Public review):

Summary:

Calle-Schuler et. al. reconstruct all the pre- and post-synaptic neurons to the bristle mechanosensory neurons on the adult fly head to understand how neural circuits determine the sequential motor patterns during fly grooming. They find that most presynaptic neurons, interneurons, and excitatory postsynaptic neurons are also somatotopically organized, such that each neuron is more connected to bristles mechanosensory neurons that are closer on the head and less connected to bristles mechanosensory neurons that are further away. These include the direct BMN-BMN circuits, excitatory interneurons, as well as the inhibitory networks. They also identify that the entire hemi-lineage 23b forms excitatory postsynaptic circuits with BMNs, highlighting how these circuits and hence their function could be developmentally determined.

Strengths:

This is a complete map of all the neurons that make 5 or more pre- and post-synaptic connections of the fly head BMNs. Using this, the authors have identified various trends, such as ascending neurons providing most of the GABAergic inhibitory input, which could provide the presynaptic inhibition essential for the parallel model for sequential grooming generation. Moreover, they identified that the entire cholinergic hemilineage 23b is postsynaptic to BMNs.

Weaknesses:

Although the somatotropic organization is an elegant mechanism to generate sequential motor sequences during grooming, none of the analyses in the paper directly demonstrate that this somatotropic connectivity is sufficient to generate hierarchical suppression and reconstruct the grooming sequence. If somatotropic organization is sufficient, then hierarchical clustering should recover the grooming sequence. Their detailed connectome enables the authors to test if some networks are more crucial for grooming sequence than others: to what extent can each network individually (ascending neurons-BMN alone) or a combination (BMN-BMN, ascending-BMN, BMN-descending, etc.) recover the sequence observed during grooming. If all the pre- and post-synaptic neurons put together cannot explain the sequence, then the sequence is probably determined by individual synaptic strengths or other key downstream neurons.

Reviewer #2 (Public review):

Summary:

Schuler et al. present an extensive analysis of the synaptic connectivity of mechanosensory head bristles in the brain of Drosophila melanogaster. Based on the previously described set of bristle afferent neurons, (BMNs), located on the head, the study aims to provide a complete, quantitative assessment of all synaptic partners in the ventral brain. Activation of head bristles induces grooming behavior, which is hierarchically organized, and hypothesized to be grounded in a parallel cellular architecture in the central brain. The authors found evidence that, at the synaptic level, neurons downstream of the BMN afferents, namely the postsynaptic LB23 interneurons and recurrent GABAergic neurons (involved in sensory gain control), are organized in parallel, following the somatotopic organization described for the BMN afferents. This study, therefore, represents an important step towards a better understanding of the cellular circuits that govern the hierarchical order of sequentially organized grooming behavior in Drosophila melanogaster.

The study is well done, the images are well designed and extensive in number, but the account is challenging to read and digest for the reader outside the Drosophila /connectome community. It is amazing what can be done with the connectome nowadays using the up-to-date FAFB dataset, the analytical and visual tools (as in FlyWire), in combination with known anatomy/physiology/behavior in DM. I suggest that the authors provide more detail on hemilineages, their relationship to the FAB connectome, the predicted neurotransmitter identity, and the use of statistical CatMAID tools used in some of the Figures.

A graphical summary at the end of the study would be very useful to highlight the important findings focusing on neuron populations identified in this study and their position in the hypothesized parallel central circuitry of BMNs.

Reviewer #3 (Public review):

Summary:

The authors set out to extend their previous mapping of Drosophila head mechanosensory neurons (Eichler et al., 2024) by reconstructing their full second-order connectome. Their aim is to reveal how bristle mechanosensory neurons (BMNs) interface with excitatory and inhibitory partners to generate location-specific grooming movements, and to identify the circuit motifs and developmental lineages that support this transformation.

Strengths:

The strengths of this work are clear. The authors present a comprehensive synaptic-resolution connectome for BMNs, identifying nearly all of their pre- and postsynaptic partners. This dataset reveals important circuit motifs:

(1) BMNs provide feedforward excitation to descending neurons, feedforward inhibition to interneurons, and are themselves strongly regulated by GABAergic presynaptic inhibition.

(2) These motifs together support the idea that BMN activity is locally gated and hierarchically suppressed, fitting well with known behavioural sequences of grooming.

(3) The study also shows that connectivity preserves somatotopy, such that BMNs from neighbouring bristle populations converge onto shared partners, while distant BMNs remain segregated.

(4) A developmental analysis reveals both primary and secondary partners, suggesting a layered scaffold plus adult-specific elaborations.

(5) Finally, the identification of hemilineage 23b (LB23) as a core postsynaptic pathway - incorporating previously described antennal grooming neurons (aBN2) - provides a striking link between developmental lineage, anatomical connectivity, and behavioral output.

(6) Together, the dataset represents a valuable resource for the neuroscience community and a foundation for future functional studies.

Weaknesses:

There are also some weaknesses that mostly only limit clarity.

(1) The writing is dense, with results often presented in a cryptic fashion and the functional implications deferred to the discussion. As a result, the significance of circuit motifs such as BMN→motor or reciprocal inhibitory loops is sometimes buried, rather than highlighted when first described.

(2) Some assumptions require more explanation for non-specialist readers - for example, how bristle identity is inferred in EM in the absence of cuticular structures, or what is meant by "ascending" and "descending" in a dataset that does not include the ventral nerve cord. While some of this comes from the earlier paper, it would help readers of this one to explain this.

(3) Visualization choices also sometimes obscure key conclusions: network graphs can be visually appealing but do not clearly convey somatotopy or BMN-type differences; heatmaps or region-level matrices would make the parallel, block-like organization of the circuit more evident.

(4) The data might also speak to roles beyond grooming (e.g., mechanosensory modulation of posture or feeding), and a brief acknowledgement of this would broaden the impact.

(5) The restriction to one hemisphere should be explicitly acknowledged as a limitation when framing this as a 'comprehensive' connectome.

Overall, the authors achieve their main goal: they convincingly show that BMNs connect into parallel, somatotopically organized pathways, with LB23 providing a key lineage-based link from sensory input to grooming output. The dataset is carefully analyzed, and while the presentation could be streamlined, the connectome will be a valuable resource for researchers studying sensory processing, motor control, and the logic of circuit organization.