1. Evaluation Summary:

    This manuscript uses viral tracing to identify interneurons, throughout the spinal cord, which synapse onto motoneurons innervating pairs of flexor and extensor hindlimb muscles. Importantly, the data identifies single premotor interneurons which travel to, and presumably regulate the activity of, multiple motor pools. It is possible that these premotor neurons are involved in regulating muscle stiffness across a joint.

    (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 #1 agreed to share their name with the authors.)

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  2. Reviewer #1 (Public Review):

    This is a well written manuscript in which the authors describe experiments in which they have used restricted viral tracing to identify premotor neurons which contact multiple motor pools. The finding that single premotor interneurons contact both flexor and extensor motor pools is interesting, and raises the possibility that these neurons may form the neural circuitry involved in regulating muscle stiffness around a joint. Generally speaking, the anatomical data could be improved by providing specific information regarding the rostra-caudal location of the various classes of premotor interneurons. While results of the experiments which investigate the neurotransmitter phenotype, and genetic background, of these divergent premotor interneurons can be used to devise testable hypotheses regarding the characteristics of the neurons, drawing conclusions based on the data presented seems premature. It is likely that this work will drive subsequent studies that attempt to manipulate the activity of these neurons, and definitively determine their function, however caution should be shown interpreting the results here, as the data is solely anatomical and does not shed light into the physiological role of this circuitry.

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  3. Reviewer #2 (Public Review):

    The study by Ranzano et al. set out to reveal if the spinal cord contain motor circuits that can support co-activation and co-inhibition of diverse flexor and extensor motor neuron pools in the mouse spinal cord. For this they use modified rabies virus in a mouse model and with a set-up that will allow selective mono-synaptically restricted labeling of premotor neurons projecting to functional synergist or antagonist ankle motor neuron pools along the entire spinal cord. They show that a minor percentage of premotor motor neurons projecting to either synergist or antagonist pair of motor neuron pools diverge. Divergent premotor neurons were seen both close and rostral to the target lumbar motor neuron pools, but with an increased proportion with distance from the lumbar cord. In the cervical spinal cord the largest proportion of divergent neurons where commissural excitatory neurons with molecular characteristics of the V0 class. The study provide important, new and convincing data on the spinal anatomical landscape of distributed motor networks that may coordinate synergistic activity as well as mediate co-contraction of antagonist muscles across multiple motor pools in the same limb or across limbs.

    Overall the claims are well supported by the data. Some aspects of methods could need clarification and some aspects of the claims are weakened by lack of identification of premotor neuron populations. The discussion of the data could perhaps be made stronger by linking the present data to functional studies.

    1. Transsynaptic method. The authors use a different model for trans-synaptic tracing than most previous studies in the spinal cord: namely the RGT mouse line crossed with ChAT-cre mice and combined with a retrograde labelling of motor neurons. The distribution of transsynaptic flexor and extensor related premotor neurons in this model is different from previously reported. Data for this are presented in (Ronzano et al. 2021 BioRxiv). But it will be useful to mention this here as well. The authors discuss why the labeling is not caused by a second jump from V0C neurons or motor neurons labelled by collaterals. Another course of contamination is at the level of the muscle. All injections are done in newborn mice with tiny muscle. It would be useful to know how the authors secured that there is no viral spread peripherally.

    2. Identity of neurons. A limitation of the study is that there is no transmitter phenotyping of the divergent premotor neurons in the lumbar and thoracic region. The divergent neurons can be either excitatory or inhibitory and cause coactivation or co-inhibition, respectively of synergist or antagonists. Except for the cervical CNs there is no evidence for transmitter-phenotype in the data. Perhaps the authors could just mention that this would have required in situ hybridization in the double injected animals or a third color RabV togheter with the GlyT2-GFP mice. The identification of V0 neruons interneurons is suboptimal. The use of specific AB (Evx) for the V0 population could have provided a better characterization (see Crone et al. 2008). Maybe also mention that the commissural neurons could be SIM1.

    3. The discussion is insightful but should perhaps link the data more directly to functional studies for example by considering how synergies are bound together across limb during locomotion which could both involve co-activation of synergies or co-inhibition of antagonists from commissural neurons (see Bellardita and Kiehn 2015; Butt and Kiehn 2003) or ipsilateral neurons (Levine et al. 2014; among others). It would also be useful to discuss how co-inhibition of synergists leads to functional movements.

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  4. Reviewer #3 (Public Review):

    The study by Ronzano et al uses monosynaptically-restricted transsynaptic labeling to reveal populations of "divergent" premotor interneurons, neurons that are presynaptic to multiple motor neuron pools. Various pairings of muscle injections are analyzed, including hindlimb synergists and antagonists, and hindlimb-forelimb combinations. They show that divergent neurons innervating both synergist and antagonist motor pools are similarly located and found in similar numbers. These neurons exist throughout the cord, in decreasing number but higher proportions of the total labeled neurons with more rostral locations (lumbar to thoracic to cervical cord). A subset of the population of the long descending divergent neurons is identified as part of the V0 class, with some similarly located (and possibly overlapping) neurons projecting the forelimb muscles in addition to hindlimb muscles. Other studies have shown distributions of premotor interneurons from single motor neuron pools. The novelty here is the focus on interneurons with divergent innervations of multiple motor pools.

    One of the major differences (and advantages) of this study from prior muscle injections of transsynaptic virus is that rather than co-injecting the AAV containing the G glycoprotein, necessary for transsynaptic transfer, into the muscle with the modified Rabies virus, they genetically specify it to motor neurons (and other cholinergic neurons) using Chat-Cre. The advantage of this is that the potential confounds of transfer via the sensory neuron and/or selective AAV transfection/G expression by a particular subpopulation of motor neurons is removed. Although the possibility of 'double jumps' (MNs to ChAT interneurons to upstream neurons) is possible, it is less likely to occur in sufficient numbers in the time of the experiment.

    The data presented are comprehensive for the motor pools examined. The location analyses are extensive. Divergent neurons demonstrated by the dual virus strategy are further supported by the demonstration of terminals of hindlimb premotor interneurons synapsing on thoracic and cervical motor neurons. The manuscript is well written. Overall, data are clearly presented and limitations are fully discussed. This study can form the basis for future studies regarding specific identity and function of the divergent populations.

    Main limitations of the study are related to the tracing technique used. The authors are fully transparent about these limitations in the Discussion. As pointed out, this technique is not optimal for quantifying double labeled neurons. Conclusions regarding the existence and location of neurons projecting to at least two motor pools can be made. However, these are likely to be severely underestimated and it is not possible to determine if these neurons are more broadly presynaptic to other motor pools in the limb (or beyond). The reduced efficiency of infection by two viruses due to viral interference is mentioned and relevant sources demonstrating the limitation are cited. Therefore, the data are solid but the functionally-related interpretations and conclusions are somewhat limited and speculative.

    The other related limitation inherent in the technique is the efficiency of transfer of even a single virus. Analysis is presented regarding a comparison with a more efficient virus, suggesting transsynaptic efficiency may be ~25%, but this does not fully get at the issue. The efficiency of the starter or seed cell labeling is not mentioned. Quantification of motor neurons taking up the RabV would be helpful as this will be directly related to the potential number of presynaptic neurons. This is especially crucial with the forelimb injections, in which 0-6 muscles were injected.

    The percentage of divergent interneurons is also underestimated in that the denominator is single labeled neurons presynaptic to either motor neuron pool. Information may be gained by determining whether different portions of premotor neurons to one over another are more likely to be divergent. This is particularly the case with antagonist injections but also for comparisons of relative proportions of dual labeled neurons premotor to synergists and antagonists. This would likely need to be combined or controlled by the percent (or number) of motor neurons from each pool that are labeled to indicate potential differences in starter cells as mentioned above. Counterbalancing is mentioned in the Methods but it is not clear that is fully possible with an n=2 or 3.

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