Regional heterogeneities of oligodendrocytes determine biased distribution pattern of Ranvier nodes along single axons in sound localization circuit

Reviewer #1 (Public review):

Summary:

The manuscript by Egawa and colleagues investigates differences in nodal spacing in an avian auditory brain stem circuit. The results are clearly presented and data are of very high quality. The authors make two main conclusions:

(1) Node spacing, i.e. internodal length, is intrinsically specified by the oligodendrocytes in the region they are found in, rather than axonal properties (branching or diameter).

(2) Activity is necessary (we don't know what kind of signaling) for normal numbers of oligodendrocytes and therefore the extent of myelination.

These are interesting observations, albeit phenomenon. I have only a few criticisms that should be addressed:

(1) The use of the term 'distribution' when describing the location of nodes is confusing. I think the authors mean rather than the patterns of nodal distribution, the pattern of nodal spacing. They have investigated spacing along the axon. I encourage the authors to substitute node spacing or internodal length for node distribution.

(2) In Seidl et al. (J Neurosci 2010) it was reported that axon diameter and internodal length (nodal spacing) were different for regions of the circuit. Can the authors help me better understand the difference between the Seidl results and those presented here?

(3) The authors looked only in very young animals - are the results reported here applicable only to development, or does additional refinement take place with aging?

(4) The fact that internodal length is specified by the oligodendrocyte suggests that activity may not modify the location of nodes of Ranvier - although again, the authors have only looked during early development. This is quite different than this reviewer's original thoughts - that activity altered internodal length and axon diameter. Thus, the results here argue against node plasticity. The authors may choose to highlight this point or argue for or against it based on results in adult birds?:

Significance:

This paper may argue against node plasticity as a mechanism for tuning of neural circuits. Myelin plasticity is a very hot topic right now and node plasticity reflects myelin plasticity. this seems to be a circuit where perhaps plasticity is NOT occurring. That would be interesting to test directly. One limitation is that this is limited to development.

Reviewer #2 (Public review):

Summary:

Egawa et al describe the developmental timeline of the assembly of nodes of Ranvier in the chick brainstem auditory circuit. In this unique system, the spacing between nodes varies significantly in different regions of the same axon from early stages, which the authors suggest is critical for accurate sound localization. Egawa et al set out to determine which factors regulate this differential node spacing. They do this by using immunohistological analyses to test the correlation of node spacing with morphological properties of the axons, and properties of oligodendrocytes, glial cells that wrap axons with the myelin sheaths that flank the nodes of Ranvier. They find that axonal structure does not vary significantly, but that oligodendrocyte density and morphology varies in the different regions traversed by these axons, which suggests this is a key determinant of the region-specific differences in node density and myelin sheath length. They also find that differential oligodendrocyte density is partly determined by secreted neuronal signals, as (presumed) blockage of vesicle fusion with tetanus toxin reduced oligodendrocyte density in the region where it is normally higher. Based on these findings, the authors propose that oligodendrocyte morphology, myelin sheath length, and consequently nodal distribution are primarily determined by intrinsic oligodendrocyte properties rather than neuronal factors such as activity.

Major comments:

(1) It is essential that the authors validate the efficiency of TeNT to prove that vesicular release is indeed inhibited, to be able to make any claims about the effect of vesicular release on oligodendrogenesis/myelination.

(2) Related to 1, can the authors clarify if their TeNT expression system results in the whole tract being silenced? It appears from Fig. 6 that their approach leads to sparse expression of TeNT in individual neurons, which enables them to measure myelination parameters. Can the authors discuss how silencing a single axon can lead to a regional effect in oligodendrocyte number?

(3) The authors need to fully revise their statistical analyses throughout and supply additional information that is needed to assess if their analyses are adequate:
(3.1) the authors use a variety of statistical tests and it is not always obvious why they chose a particular test. For example, in Fig. 2G they chose a Kruskal-Wallis test instead of a two-way ANOVA or Mann-Whitney U test, which are much more common in the field. What is the rationale for the test choice?
(3.2) in some cases, the choice of test appears wholly inappropriate. For example, in Fig. 3H-K, an unpaired t-test is inappropriate if the two regions were analysed in the same samples. In Fig. 5, was a t-test used for comparisons between multiple groups in the same dataset? If so, an ANOVA may be more appropriate.
(3.3) in some cases, the authors do not mention which test was used (Fig 3: E-G no test indicated, despite asterisks; G/L/M - which regression test that was used? What does r indicate?)
(3.4) more concerningly, throughout the results, data may have been pseudo-replicated. t-tests and ANOVAs assume that each observation in a dataset is independent of the other observations. In figures 1-4 and 6 there is a very large "n" number, but the authors do not indicate what this corresponds to. This leaves it open to interpretation, and the large values suggest that the number of nodes, internodal segments, or cells may have been used. These are not independent experimental units, and should be averaged per independent biological replicate - i.e. per animal (N).
(3.5) related to the pseudo-replication issue, can the authors include individual datapoints in graphs for full transparency, per biological replicates, in addition or in alternative to bar-graphs (e.g. Fig. 5 and 6).

(4) The main finding of the study is that the density of nodes differs between two regions of the chicken auditory circuit, probably due to morphological differences in the respective oligodendrocytes. Can the authors discuss if this finding is likely to be specific to the bird auditory circuit?

(5) Provided the authors amend their statistical analyses, and assuming significant differences remain as shown, the study shows a correlation (but not causation) between node spacing and oligodendrocyte density, but the authors did not manipulate oligodendrocyte density per se (i.e. cell-autonomously). Therefore, the authors should either include such experiments, or revise some of their phrasing to soften their claims and conclusions. For example, the word "determine" in the title could be replaced by "correlate with" for a more accurate representation of the work. Similar sentences throughout the main text should be amended.

(6) The authors fail to introduce, or discuss, very pertinent prior studies, in particular to contextualize their findings with:
(6.1) known neuron-autonomous modes of node formation prior to myelination, e.g. Zonta et al (PMID 18573915); Vagionitis et al (PMID 35172135); Freeman et al (PMID 25561543)
(6.2) known effects of vesicular fusion directly on myelinating capacity and oligodendrogenesis, e.g. Mensch et al (PMID 25849985)
(6.3) known correlation of myelin length and thickness with axonal diameter, e.g. Murray & Blakemore (PMID 7012280); Ibrahim et al (PMID 8583214); Hildebrand et al (PMID 8441812).
(6.4) regional heterogeneity in the oligodendrocyte transcriptome (page 9, studies summarized in PMID 36313617)

Significance:

In our view the study tackles a fundamental question likely to be of interest to a specialized audience of cellular neuroscientists. This descriptive study is suggestive that in the studied system, oligodendrocyte density determines the spacing between nodes of Ranvier, but further manipulations of oligodendrocyte density per se are needed to test this convincingly.

Reviewer #3 (Public review):

Summary:

The authors have investigated the myelination pattern along the axons of chick avian cochlear nucleus. It has already been shown that there are regional differences in the internodal length of axons in the nucleus magnocellularis. In the tract region across the midline, internodes are longer than in the nucleus laminaris region. Here the authors suggest that the difference in internodal length is attributed to heterogeneity of oligodendrocytes. In the tract region oligodendrocytes would contribute longer myelin internodes, while oligodendrocytes in the nucleus laminaris region would synthesize shorter myelin internodes. Not only length of myelin internodes differs, but also along the same axon unmyelinated areas between two internodes may vary. This is an interesting contribution since all these differences contribute to differential conduction velocity regulating ipsilateral and contralateral innervation of coincidence detector neurons. However, the demonstration falls rather short of being convincing.

Major comments:

(1) The authors neglect the possibility that nodal cluster may be formed prior to myelin deposition. They have investigated stages E12 (no nodal clusters) and E15 (nodal cluster plus MAG+ myelin). Fig. 1D is of dubious quality. It would be important to investigate stages between E12 and E15 to observe the formation of pre-nodes, i.e., clustering of nodal components prior to myelin deposition.

(2) The claim that axonal diameter is constant along the axonal length need to be demonstrated at the EM level. This would also allow to measure possible regional differences in the thickness of the myelin sheath and number of myelin wraps.

(3) The observation that internodal length differs is explain by heterogeneity of sources of oligodendrocyte is not convincing. Oligodendrocytes a priori from the same origin remyelinate shorter internode after a demyelination event.

Significance:

The authors suggest that the difference in internodal length is attributed to heterogeneity of oligodendrocytes. In the tract region oligodendrocytes would contribute longer myelin internodes, while oligodendrocytes in the nucleus laminaris region would synthesize shorter myelin internodes. Not only length of myelin internodes differs, but also along the same axon unmyelinated areas between two internodes may vary. This is an interesting contribution since all these differences contribute to differential conduction velocity regulating ipsilateral and contralateral innervation of coincidence detector neurons.

Author response:

General Statements

We sincerely appreciate the constructive comments from the reviewers, which have significantly enhanced the clarity and rigor of our manuscript. Most of their suggestions have already been incorporated into the revised version. Additionally, we are conducting an additional experiment to further substantiate our conclusions, and preliminary data seem to support our findings.

As pointed out by Reviewer #1, the regulation of neural circuit function by oligodendrocytes is currently a highly significant and actively studied topic. Our study demonstrates that regional heterogeneity in oligodendrocytes underlies the microsecond-level computational processes in the sound localization circuit. We believe this work represents a substantial contribution to the field.

Description of the planned revisions

• Evaluation of node formation along axons sparsely expressing eTeNT (related to Reviewer #2: comment 1)

Based on the approximately 90% expression efficiency of A3V-eTeNT in NM neurons, we interpreted that vesicular release from NM axons was largely inhibited in the NL region, leading to the suppression of oligodendrogenesis and the subsequent emergence of unmyelinated segments. However, the effects of eTeNT on myelination are likely diverse, and a possibility remains that eTeNT directly disrupted axon-oligodendrocyte interactions, preventing oligodendrocytes from myelinating the axons expressing eTeNT.

To test this possibility, we have initiated an additional experiment to evaluate formation of nodes along axons, while expressing eTeNT sparsely by electroporation. Preliminary results indicated that unmyelinated segments did not increase, supporting our original conclusion. After completion of the experiment, we will include the findings as a Supplementary Figure associated with Figure 6, which will provide a clearer understanding of how eTeNT influences myelination.

Description of the revisions that have already been incorporated in the transferred manuscript

• Revised terminology from "nodal distribution" to "nodal spacing" throughout the manuscript. (Reviewer #1: comment 1)

• Emphasized that our analyses were focused on the main trunk of NM axons (Reviewer #1: comment 2) We explicitly stated throughout the manuscript that we analyzed the main trunk of NM axons and made it clear that our findings do not contradict those by Seidl et al. (J Neurosci 2010), showing the similar axon diameter between midline and ventral NL regions (page 7, line 7).

• Added an explanation on the maturation of sound localization circuit (Reviewer #1: comment 3) We explained that chickens have high ability of sound localization at hatch, emphasizing that the sound localization circuit is almost fully developed by E21 (page 4, line 12).

• Emphasized the diverse effects of neuronal activity on oligodendrocytes (page 10, line 18) (Reviewer #1: comment 4)

• Added details on the efficiency of A3V-eTeNT expression in NM neurons to the Results section (page 8, line 5) (Reviewer #2: comment 1)

• Made it clear in Figure Legend for Figure 6D that the analysis was conducted under the condition, where most of the axons were labeled by A3V-eTeNT (page 31, line 9) (Reviewer #2: comment 2)

• Clarified the rationale for statistical test selection (Reviewer #2: comment 3.1)

• Reanalyzed all statistical data with appropriate methods using R (Reviewer #2: comment 3.2)

• Clearly indicated which statistical tests were used in each figure (Reviewer #2: comment 3.3)

• Clarified what n represents and N used in each experiment (Reviewer #2: comment 3.4)

• Added individual data points to bar graphs in Figure 5 and 6 (Reviewer #2: comment 3.5)

• Emphasized the importance of comparing the ITD circuit with that of rodents (page 11, line 32) (Reviewer #2: comment 4)

• Softened the expressions related to "determine" (Reviewer #2: comment 5)

Our study demonstrates that regional differences in the intrinsic properties of oligodendrocytes are the prominent determinant of nodal spacing patterns. However, we acknowledge that this does not establish a direct causation. Accordingly, relevant expressions have been revised throughout the manuscript.

• Added references (Reviewer #2: comment 6)

• Corrected units in Figure 1G (Reviewer #2: comment 7)

• Added discussion about the involvement of pre-nodal clusters in the regional differences in nodal spacing (page 9, line 35) (Reviewer #3: comment 1).

Related to this issue, we have added new data to Figure 6I.

• Discussed the possibility that the developmental origin and/or the pericellular microenvironment of OPCs contributed to the regional heterogeneity of oligodendrocytes (page 9, line 21) (Reviewer #3: comment 3).

• Added references used in the response to reviewers into the main text.

• Corrected the data error in Figure 6G, H

• Corrected the dataset in Figure 3E

We limited the data in Figure 3E–G to those measuring both myelin length and diameter simultaneously.

Description of analyses that authors prefer not to carry out

• Analysis in adult chickens (Reviewer #1: comment 3,4)

The chick brainstem auditory circuit is nearly fully developed by E21, and we have also demonstrated that nodal spacing increases by approximately 20% while maintaining regional differences up to P9. Therefore, our study covers the period from pre-myelination to postfunctional maturation, and we think that the necessity of analyzing aged animals is small.

• Functional evaluation of the efficiency of eTeNT suppression (Reviewer #2: comment 1)

It is technically challenging to quantitatively assess the inhibition of vesicular release by eTeNT in NM axons given that multiple synapses from different NM axons converge onto postsynaptic neurons. In addition, previous studies have already validated the efficacy of this construct in multiple species. Therefore, we will not evaluate electrophysiologically the extent of vesicular release inhibition by eTeNT in this study. Instead, we have provided clear evidence that A3V-eTeNT is expressed efficiently and leads to notable phenotypic changes, such as the inhibition of oligodendrogenesis. (page 8, line 5).

• Replacing figures with data averaged per animal (Reviewer #2: comment 3.4)

Our study focuses on the distribution of morphological characteristics at the single-cell level rather than solely on group means. Averaging measurements per animal could obscure this cellular heterogeneity and potentially misrepresent our findings. Given that data distributions in our plots show clear distinctions, we believe that averaging per biological replicate is not essential in this case. If requested, we will be happy to provide the outputs of PlotsOfDifferences as supplementary source data files, similar to those used in eLife publications, for each figure.

• Additional experiments to manipulate oligodendrocyte density (Reviewer #2: comment 5)

We have already demonstrated that A3V-eTeNT reduces oligodendrocyte density in the NL region, and some of the arguments in our study are based on this result. Therefore, we think that further experiments are not necessary.

• Verification of the presence of pre-nodal clusters (Reviewer #3: comment 1)

We investigated the presence of pre-nodal clusters on NM axons, but we could not identify them in the immunohistochemistry of AnkG. As the occurrence of pre-nodal clusters varies depending on neuronal type, we consider that pre-nodal clusters are not prominent in the NM axons and that further experimental validation would not be necessary. Instead, we have added a discussion on the possibility that pre-nodal clusters contribute to regional differences in nodal spacing along NM axons (page 9, line 35).

• Axon diameter measurements using EM (Reviewer #3: comment 2)

This experiment was already done by Seidl et al. (2010), and hence, we do not think it necessary to repeat it. We believe that the relative differences in axon diameter between the regions could be adequately assessed using the optical approach with membrane-targeted GFP.