Spinal lumbar dI2 interneurons contribute to stability of bipedal stepping

  1. Baruch Haimson
  2. Yoav Hadas
  3. Nimrod Bernat
  4. Artur Kania
  5. Monica A Daley
  6. Yuval Cinnamon
  7. Aharon Lev-Tov
  8. Avihu Klar

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Abstract

Peripheral and intraspinal feedback is required to shape and update the output of spinal networks that execute motor behavior. We report that lumbar dI2 spinal interneurons in chicks receive synaptic input from afferents and premotor neurons. These interneurons innervate contralateral premotor networks in the lumbar and brachial spinal cord, and their ascending projections innervate the cerebellum. These findings suggest that dI2 neurons function as interneurons in local lumbar circuits, are involved in lumbo-brachial coupling, and that part of them deliver peripheral and intraspinal feedback to the cerebellum. Silencing of dI2 neurons leads to destabilized stepping in posthatching day 8 hatchlings, with occasional collapses, variable step profiles, and a wide-base walking gait, suggesting that dI2 neurons may contribute to the stabilization of the bipedal gait.

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  1. Version published to 10.7554/elife.62001 on eLife
  2. eLife

    ###Reviewer #2:

    This work addresses the possibility that developmentally-characterized di2 neurons contribute to the ventral spinocerebellar tract and regulate stepping in the chick. The work is sound considering that most information we have on spinal subtypes are for ventrally-born and local circuit interneurons (i.e. motor related), but less is known about the dorsally-born types and about long-range projecting neurons that link the spinal cord with higher integrative centers. Here, using a combination of cell-type specific manipulations, circuit tracing tools and kinematic analysis of gaits in the chick authors propose that spinal di2 interneurons contain multiple subgroups including a population that sends projection to the cerebellum. Silencing di2 neurons overall leads to impaired stepping.

    Overall, the strategy is sound and there is potential novelty, provided the weaknesses in the scientific demonstration listed below can be first addressed, experimentally and/or by additional analysis. Equally importantly also, the work suffers from a severe lack of clarity (writing, figures, results).

    I start with the scientific weaknesses:

    1. Synaptic connections rely mostly on the anatomical overlap between di2 cells and the synaptic field of their putative pre-synaptic partners. While this is indeed suggestive, it is not enough to ascertain actual synaptic connections, and even less so in a comparative manner between the different groups. Furthermore, some tracers (e.g PRVmCherry) do not seem to be under a synapse-specific promoter, so labelled elements might just as well be passing fibers. Clearer evidence of actual connections should be provided, functionally if possible or at the very least by showing clearer putative boutons onto neuronal somata/dendrites, quantifying them and quantifying differences between input cell types. Current figures (2F / 3B', C', D' / 4C, D', E', F') are not sufficiently convincing since we see only one cell and can barely detect boutons visually on some of them (not to mention that pseudo-colors keep changing, see other comment below). In addition, please consider using the term "putative" or "presumed" synapses, contacts and connections throughout the study.

    2. The loss of function and gait analysis is stronger and convincingly presented. However, unless I missed it, the strategy silences all di2 neurons but cannot discriminate the contributions of the pre-cerebellar ones. This poses problems for the interpretation of the data. Since this paper is about either subpopulations of di2, or the vSCT (see other comment about general scope of the work), it would be more robust if more specific silencing was included. It is currently assumed that one likely mechanism for the disturbed gait owes to the function of di2 as precerebellar neurons (line 385, 389) but the phenotype could also, or even entirely, be due to their proprio-spinal connectivity. This is a major caveat.

    On top of this, writing and data presentation MUST be substantially improved on multiple aspects:

    1. Please have the manuscript deeply proofread. In addition to numerous English mistakes (missing "the", "or", plural and singulars, lots of unnecessary commas, etc...) examples of confused writing include (non-exhaustive list):

    (a) Line 128: what does this phrase mean ("TF expression is redundant"...)

    (b) Line 159: I don't understand here, the Di2 ascend to the cerebellum, cross the midline to the targeted di2? To which Di2 do the authors refer to here, it sounds like they are in the cerebellum, or that the ascending Di2 redescend to the spinal cord...

    (c) The term targeted is in fact used alternatively and confusingly to refer to either "manipulated" cells, "synaptically-targeted" cells, there is also "targeted overground locomotion",....

    (d) Stage HH18 is sometimes referred to as E3. Please be consistent throughout.

    (e) When describing inputs onto di2, add "neurons" (i.e. "onto di2 neurons").

    1. I would appreciate more background on di2 neurons in the introduction and why these have been investigated. Currently, most of this is given in the first paragraph of the results (lines 91-100 and also line 103). Also, it is stated first that "the role of di2 neurons is elusive due to the lack of genetic targeting means" (line 59). This contradicts the later statement that "the progenitor pdi2 expresses [various transcription factors]", and that the "post mitotic di2 are defined by..." (line 103). Please clarify what is known and not known about di2 already in the introduction.

    2. Related to the above, it is not sufficiently clear what is investigated here. The genetic identity of ventral spinocerebellar neurons? Or the diversity of di2 neurons? In the way the introduction is written, it gives the impression that it is the former, but then functional investigations are not specific enough (since they are targeted to the overall di2 population, see dedicated comment later). Authors should revise to make clearer what is the scope of the work.

    3. Histology Figures should be made more convincing, self-explanatory, and to a higher standard.

    (a) Anatomical landmarks must be placed on all figures, e.g: the midline and minimal nuclei of the cerebellum, the deep cerebellar nuclei should be indicated in Fig S4,... Also, please give the orientation axis on all figures (especially the ones illustrating large territories, like 2B, 4A).

    (b) Add the CTB or HSV tracer on Fig. 2A and check coherence: I believe for instance that HSP is wrongly stated instead of HSV in Fig 2D and PRV is wrongly stated instead of CTB in Fig 2F (and there might be other confusions throughout).

    (c) It is extremely confusing that histology pseudo-colors are sometimes changed from one related figure to the other, for unclear reasons (e.g. 2B, 2B', 2C, also 2C and S4A...). Consistency will help the reader go through all panels and figures comparatively.

    (d) Figures must be addressed in proper order. This also applies to supplemental figures. Otherwise, it gives the impression we have missed something.

    (e) What is the rationale for plotting the overlap in area versus volume (Figure 2H, I)? If overlap with area shows a higher percentage than with volume, does it mean that the overlap is only limited to a given A/P plane? I'm really confused about this representation and its meaning.

    1. Authors should avoid relying on subjective formulations like "that reside at the lateral dorsal aspect of lamina VII". Instead, they MUST demonstrate the positioning of Di2 neurons into the different spinal laminae with some form of quantitative measurements. This is currently just an "impression" that large, precerebellar Di2 are more ventral, in lamina VII and possibly VIII but without the representation of lamina borders on figures, this information cannot be appreciated by the reader. It is all essential that these borders are depicted in Figures and neurons be quantitatively allocated to each laminae. In addition/alternatively, authors should report the average D/V position of the different subtypes and test for significant differences to make the case of different spatially-confined populations stronger.

    2. FoxD3 expression on Supplemental Figure 2B is not convincing. It is also not reported in the statistics of Fig 1E. Do we have to assume that all di2 investigated here are FoxD3-positive? If so, one would need a better illustration and quantifications should be given. Otherwise, I would suggest simply relying on the literature and removing Figure S1B which is not helping. On other panels of that supplemental Figure 2, please add arrow/arrowheads on all neurons that are or are not co-labelled so we can appreciate co-labelling.

    3. The demonstration that di2 are excitatory is essential. It is the title of a paragraph (line 102), thus I think that the corresponding data with the neurotransmitters (Vglut2, GAD) would deserve to be in the main Figures. Also, the chosen illustration only shows ONE double-labelled cell with Vglut2. Authors should be able to show a field of view that more convincingly conveys the message with more cells.

  3. eLife

    ###Reviewer #1:

    This is a well-put-together manuscript describing carefully performed circuitry dissection and functional analysis of dl2 neurons in the chick. A genetic toolbox is used taking advantage of the electroporation technique applied to the embryos. The findings include a fairly convincing connectome for dl2 neurons and a functional phenotype that is, unfortunately, rather unsatisfying. The investigators conclude that dl2 interneurons regulate "stability" of bipedal stepping in the chick, which is fine, but the analysis misses an opportunity to more fully explore what the instability involves and thus to perhaps shed more light on the likely roles of this neuron population. The concerns/issues 3 and 4 below focus on this issue and the need for additional careful analysis of the behavior that will allow the phenotype to be more precisely described or ascribed to some aspect of stepping that might guide future studies in other models. For example, can the link between partial collapse and over-extensions be made more solid and thus argue that reduced extensor gain might be what results in the instability? What other analysis could be performed using the existing data/video to better describe the behavioral phenotype?

    Major Concerns:

    1. The connectome part of the work appears solid and supports the concept that a subpopulation of the population are likely VSCT neurons, that the non VSCT neurons receive the bulk of the afferent input and that these neurons project to contralateral dl2 neurons (some which may be VSCT) and other premotor neurons. Anatomically, the only concern is that no distinctions were made between the lumbar and brachial populations, and if differences in these populations exist, it would be important and interesting to describe them.

    2. Figure 2 Characterization of dl2/VSCT neurons as being primarily large dl2 neurons is quite convincing, and the observation that the dl2 neurons account for 10% of the VSCT axons is also of interest and quite compelling. A question arises, however, about the source, rostrocaudally, of the VSCT neurons and tract. Is the 10% for the total or for a specific level or levels? Can more be said/quantified about differences in these populations at different spinal levels?

    3. Whole-body collapses and subsequent over-extensions are important and speak to changes in reflex arc and motor output. The statement "usually followed by" over-extension should be followed-up. Can this be further quantified? Are the two events linked or distinct, and did over-extensions happen in the absence of collapses?

    4. These issues mesh with the lower knee height and angle of the TMP joint, even when collapses are excluded. It appears as though the control system to maintain muscle shortening (force output of extensors) is altered. I agree that stability is compromised, but could we go further to state that the compromise is due to extensor gain control?

  4. eLife

    ###Reviewer #3:

    This study by Haimson et al. aims at examining the diversity of dI2 interneurons and their role in coordinating activity across different regions of the spinal cord and in reporting back activity to the brain. The results show that dI2 interneurons comprise different sub-classes based on their axonal projections, soma diameter and transmitter identity. They also show that some dI2 interneurons project rostrally from the lumbar spinal cord and make putative synaptic contacts with other dI2 interneurons in the brachial spinal cord on their way to the cerebellum. Finally, it is shown that some dI2 interneurons receive putative inputs from DRG neurons and may serve to transmit movement-related feedback. An indiscriminate silencing of dI2 interneurons results in instability of locomotion. Overall, this study reports some interesting observations by showing the heterogeneity of dI2 interneurons and their potential function. I have the following concerns:

    1. 12% express Pax2 and are considered inhibitory. However, Gad is expressed in only 25% of dI2 interneurons while vGlut is expressed in 88%. These proportions suggest that there are dI2 neurons that co-express vGlut and Gad. Is this the case? Are there additional inhibitory dI2 neurons in addition to those expressing Pax2 which could explain the fact that Gad labels 25% of dI2 neurons. These points need some clarifications and discussion.

    2. Of all dI2 interneurons, 91% are small diameter and 9% are large diameter neurons - large diameter neurons are mostly apparent in the lumbar spinal cord. The small and large diameter dI2 neurons cannot be differentiated by their expression of TFs, but can be distinguished by their transmitter identity? Is the proportion of small and large diameter neurons the same along the spinal cord?

    3. Do all dI2 neurons receive putative synaptic contacts from DRG neurons? Unless I have missed it, it would be helpful to provide quantification of the number of small vs large diameter dI2 neurons with regard to the different putative synaptic contacts they receive from DRG neurons, dI2 and V1 interneurons.

    4. Lines 218-220: It is stated that DRG putative contacts are mainly targeting dorsal dI2 neurons while ventral ones receive virtually no contacts. Since large diameter VSCT dI2 neurons are located ventrally, they do not seem to receive direct sensory information. However, the authors conclude that VSCT dI2 neurons receive sensory input (lines 227-228) and also in the Discussion. There seem to be a mismatch between the results and the conclusion drawn by the authors (lines 374-377). Unless I am missing something here, this is not consistent with the conclusions of this study. Please clarify.

    5. The silencing experiments are interesting, however it is unclear which sub-class of dI2 neurons and at what level (lumbar vs brachial spinal cord or cerebellum) the observed behavioral perturbations take place. It is possible to selectively silence excitatory vs inhibitory or only VSCT neurons to provide some link between dI2 sub-classes and behavioral perturbations.

  5. Version published to 10.1101/2020.01.07.898072 on bioRxiv