Author response:
The following is the authors’ response to the original reviews.
Public Reviews:
Reviewer #1 (Public review):
Summary:
This study presents convincing findings that oligodendrocytes play a regulatory role in spontaneous neural activity synchronisation during early postnatal development, with implications for adult brain function. Utilising targeted genetic approaches, the authors demonstrate how oligodendrocyte depletion impacts Purkinje cell activity and behaviours dependent on cerebellar function. Delayed myelination during critical developmental windows is linked to persistent alterations in neural circuit function, underscoring the lasting impact of oligodendrocyte activity.
Strengths:
(1) The research leverages the anatomically distinct olivocerebellar circuit, a well-characterized system with known developmental timelines and inputs, strengthening the link between oligodendrocyte function and neural synchronization.
(2) Functional assessments, supported by behavioral tests, validate the findings of in vivo calcium imaging, enhancing the study's credibility.
(3) Extending the study to assess the long-term effects of early-life myelination disruptions adds depth to the implications for both circuit function and behavior.
We appreciate these positive evaluation.
Weaknesses:
(1) The study would benefit from a closer analysis of myelination during the periods when synchrony is recorded. Direct correlations between myelination and synchronized activity would substantiate the mechanistic link and clarify if observed behavioral deficits stem from altered myelination timing.
We appreciate the reviewer’s thoughtful suggestion and have expanded the manuscript to clarify how oligodendrocyte maturation relates to the development of Purkinje-cell synchrony. The developmental trajectory of Purkinje-cell synchrony has already been comprehensively characterized by Good et al. (2017, Cell Reports 21: 2066–2073): synchrony drops from a high level at P3–P5 to adult-like values by P8. We found that the myelination in the cerebellum starts to appear from P5-P7 (Figure S1A, B), indicating that the timing of Purkinje cell desynchronization coincides with the initial appearance of oligodendrocytes and myelin in the cerebellum. To determine whether myelin growth could nevertheless modulate this process, we quantified ASPA-positive oligodendrocyte density and MBP-positive bundle thickness and area at P10, P14, P21 and adulthood (Fig. 1J, K, Fig. S1E). Both metrics increase monotonically and clearly lag behind the rapid drop in synchrony, indicating that myelination could be not the primary trigger for the desynchronization. When oligodendrocytes were ablated during the second postnatal week, the synchrony was reduced (new Fig. 2). Thus, once myelination is underway, oligodendrocytes become critical for maintaining the synchrony, acting not as the initiators but as the stabilizers and refiners of the mature network state.
We have added the new subsection in discussion (lines 451–467) now in which we propose a two-phase model. Phase I (P3–P8): High early synchrony is generated by non-myelin mechanisms (e.g. transient gap junctions, shared climbing-fiber input). Phase II (P8-). As oligodendrocytes proliferate and ensheath axons, they fine-tune conduction velocity and stabilize the mature, low-synchrony network state.
We believe these additions fully address the reviewer’s concerns.
(2) Although the study focuses on Purkinje cells in the cerebellum, neural synchrony typically involves cross-regional interactions. Expanding the discussion on how localized Purkinje synchrony affects broader behaviors - such as anxiety, motor function, and sociality - would enhance the findings' functional significance.
We appreciate the reviewer’s helpful suggestion and have expanded the Discussion (lines 543–564) to clarify how localized Purkinje-cell synchrony can influence broader behavioral domains. In the revised text we note that changes in PC synchrony propagate into thalamic, prefrontal, limbic, and parietal targets, thereby impacting distributed networks involved in motor coordination, affect, and social interaction. Our optogenetic rescue experiments further support this framework, as transient resynchronization of PCs normalized sociability and motor coordination while leaving anxiety-like behavior impaired. This dissociation highlights that different behavioral domains rely to varying degrees on precise cerebellar synchrony and underscores how even localized perturbations in Purkinje timing can acquire system-level significance.
(3) The authors discuss the possibility of oligodendrocyte-mediated synapse elimination as a possible mechanism behind their findings, drawing from relevant recent literature on oligodendrocyte precursor cells. However, there are no data presented supporting this assumption. The authors should explain why they think the mechanism behind their observation extends beyond the contribution of myelination or remove this point from the discussion entirely.
We thank the reviewer for pointing out that our original discussion of oligodendrocyte-mediated synapse elimination was not directly supported by data in the present manuscript. Because we are actively analyzing this question in a separate, follow-up study, we have deleted the speculative passage to keep the current paper focused on the demonstrated, myelination-dependent effects. We believe this change sharpens the mechanistic narrative and fully addresses the reviewer’s concern.
(4) It would be valuable to investigate the secondary effects of oligodendrocyte depletion on other glial cells, particularly astrocytes or microglia, which could influence long-term behavioral outcomes. Identifying whether the lasting effects stem from developmental oligodendrocyte function alone or also involve myelination could deepen the study's insights.
We thank the reviewer for raising this point and have performed the requested analyses. Using IBA1 immunostaining for microglia and S100b for Bergmann glia, we quantified cell density and these marker signal intensity at P14 and P21. Neither microglial or Bergmann-glial differed between control and oligodendrocyte-ablated mice at either time‐point (new Figure S2). These results indicate that the behavioral phenotypes we report are unlikely to arise from secondary activation or loss of other glial populations.
We now added results (lines 275–286) and also discuss myelination and other oligodendrocyte function (lines 443–450). It remains difficult to disentangle conduction-related effects from myelination-independent trophic roles of oligodendrocytes. We therefore note explicitly that future work employing stage-specific genetic tools or acute metabolic manipulations will be required to parse these contributions more definitively.
(5) The authors should explore the use of different methods to disturb myelin production for a longer time, in order to further determine if the observed effects are transient or if they could have longer-lasting effects.
We agree that distinguishing transient from enduring effects is critical. Importantly, our original submission already included data demonstrating a persistent deficit of PC population synchrony (Fig. 4, previous Fig. 3): (i) at P14—the early age after oligodendrocyte ablation—population synchrony is reduced, and (ii) the same deficit is still present in adults (P60–P70) despite full recovery of ASPA-positive cell density and MBP-area and -thickness (Fig. 2H-K, Fig. S1E, and Fig. 4). We also performed the ablation of oligodendrocytes after the third postnatal week. Despite a similar acute drop in ASPA-positive cells, neither population synchrony nor anxiety-, motor-, or social behaviors differed from littermate controls. Thus, extending myelin disruption beyond the developmental window does not exacerbate or prolong the phenotype, whereas a short perturbation within that window leaves a permanent timing defect. These findings strengthen our conclusion that it is the developmental oligodendrocyte/myelination program itself—rather than ongoing adult myelin production—that is essential for establishing stable network synchrony. We now highlight this point explicitly in the revised Discussion (lines 507–522).
(6) Throughout the paper, there are concerns about statistical analyses, particularly on the use of the Mann-Whitney test or using fields of view as biological replicates.
We appreciate the reviewer’s guidance on appropriate statistical treatment. To address these concerns we have re-analyzed all datasets that contained multiple measurements per animal (e.g., fields of view, lobules, or trials) using nested statistics with animal as the higher-order unit. Specifically, we applied a two-level nested ANOVA when more than two groups were compared and a nested t-test when two conditions were present. The re-analysis confirmed all original conclusions. Because the nested models yielded comparable effect sizes to the Mann–Whitney tests, we have retained the mean ± SEM for ease of comparison with prior literature but now also report all values for each mouse in Table 1. In cases where a single measurement per mouse was compared between two groups, we used the Mann–Whitney test and present the results in the graphs as median values.
Major
(1) The authors present compelling evidence that early loss of myelination disrupts synchronous firing prematurely. However, synchronous neuronal firing does not equate to circuit synchronization. It is improbable that myelination directly generates synchronous firing in Purkinje cells (PCs). For instance, Foran et al. (1992) identified that cerebellar myelination begins around postnatal day 6 (P6), while Good et al. (2017) recorded a developmental decline in PC activity correlation from P5-P11. To clarify myelin's role, we recommend detailed myelin imaging through light microscopy (MBP staining at higher magnification) to assess the extent of myelin removal accurately. Myelin sheaths, as shown by Snaidero et al. (2020), can persist after oligodendrocyte (OL) death, particularly following DTA induction (Pohl et al. 2011). Quantification of MBP+ area, rather than mean MBP intensity, is necessary to accurately measure myelin coverage.
We appreciate the reviewer’s concern that residual sheaths might remain after oligodendrocyte ablation and have therefore re-examined myelin at higher spatial resolution. Then, two independent metrics were extracted: MBP⁺ area fraction in the white matter and MBP⁺ bundle thickness (new Figure 1J, K, and Fig. S1E). We confirm a robust, transient loss of myelin at P10 and P14 as shown by the reduction of MBP⁺ area and MBP⁺ bundle thickness. Both parameters recovered to control values by P21 and adulthood, indicating effective remyelination. These data demonstrate that, in our paradigm, oligodendrocyte ablation is accompanied by substantial sheath loss rather than the persistent myelin reported after acute toxin exposure. We have added them in Result (lines 266–271).
The results reinforce the view that myelin removal and/or loss of trophic support during a narrow developmental window drive the long-term hyposynchrony and behavioral phenotypes we report. We have added the new subsection in discussion (lines 443–450) now in which we propose a two-phase model. Phase I (P3–P8): High early synchrony is generated by non-myelin mechanisms (e.g. transient gap junctions, shared climbing-fiber input). Phase II (P8-). As oligodendrocytes proliferate and ensheath axons, they fine-tune conduction velocity and stabilize the mature, low-synchrony network state. We believe these additions fully address the reviewer’s concerns.
(2) Surprisingly, the authors speculate about oligodendrocyte-mediated synaptic pruning without supportive data, shifting the focus away from the potential impact of myelination. Even if OLs perform synaptic pruning, OL depletion would likely maintain synchrony, yet the opposite was observed. Further characterisation of the model and the potential source of the effect is needed.
We thank the reviewer for pointing out that our original discussion of oligodendrocyte-mediated synapse elimination was not directly supported by data in the present manuscript. Because we are actively analyzing this question in a separate, follow-up study, we have deleted the speculative passage to keep the current paper focused on the demonstrated, myelination-dependent effects. We believe this change sharpens the mechanistic narrative and fully addresses the reviewer’s concern.
(3) Improved characterization of the DTA model would add clarity. Although almost all infected cells are reported as OLs, quantification of infected OL-lineage cells (e.g., via Olig2 staining) would verify this. It remains possible that observed activity changes are driven by OL-independent demyelination effects. We suggest cross-staining with Iba1 and GFAP to rule out inflammation or gliosis.
We thank the reviewer for this important suggestion and have expanded our histological characterization accordingly. First, to verify that DTA expression is confined to mature oligodendrocytes, we co-stained cerebellar sections collected 7 days after AAV-hMAG-mCherry injection with Olig2 (pan-OL lineage) and ASPA (mature OL marker) as shown in Figure S1C-D. Quantitative analysis revealed that 100 % of mCherry⁺ cells were Olig2⁺/ASPA⁺, whereas mCherry signal was virtually absent in Olig2⁺/ASPA⁻ immature oligodendrocytes. These data confirm that our DTA manipulation targets mature myelinating OLs rather than earlier lineage stages. We have added them in Result (lines 260–262).
Second, to examine indirect effects mediated by other glia, we performed cross-staining with IBA1 (microglia) and S100β (Bergmann). Cell density and fluorescence intensity for each marker were indistinguishable between control and DTA groups at P14 and P21 (Figure S2A-H). Thus, neither inflammation nor astro-/microgliosis accompanies OL ablation. We have added them in Result (lines 275–286).
Collectively, these results demonstrate that the observed desynchronization and behavioral phenotypes arise from specific loss of mature oligodendrocytes and their myelin, rather than from off-target viral expression or secondary glial responses.
(4) The use of an independent model of myelin loss, such as the inducible Myrf knockout mouse with a MAG promoter, to assess if oligodendrocyte loss causes temporary or sustained impacts, employing an extended knockout model like Myrf cKO with MAG-Cre viral methods would be advantageous.
We agree that distinguishing transient from enduring effects is critical. Importantly, our original submission already included data demonstrating a persistent deficit of PC population synchrony (Fig. 4, previous Fig. 3): (i) at P13-15—the early age after oligodendrocyte ablation—population synchrony is reduced, and (ii) the same deficit is still present in adults (P60–P70) despite full recovery of ASPA-positive cell density and MBP-area and -thickness (Fig. 2H-K, Fig. S1E, and Fig. 4). We also performed the ablation of oligodendrocytes after the third postnatal week. Despite a similar acute drop in ASPA-positive cells, neither population synchrony nor anxiety-, motor-, or social behaviors differed from littermate controls. Thus, extending myelin disruption beyond the developmental window does not exacerbate or prolong the phenotype, whereas a short perturbation within that window leaves a permanent timing defect. These findings strengthen our conclusion that it is the developmental oligodendrocyte/myelination program itself—rather than ongoing adult myelin production—that is essential for establishing stable network synchrony. We now highlight this point explicitly in the revised Discussion (lines 507–522).
(5) For statistical robustness, the use of non-parametric tests (Mann-Whitney) necessitates reporting the median instead of the mean as the authors do. Furthermore, as repeated measurements within the same animal are not independent, the authors should ideally use nested ANOVA (or nested t-test comparing two conditions) to validate their findings (Aarts et al., Nat. Neuroscience 2014). Alternatively use one-way ANOVA with each animal as a biological replicate, although this means that the distribution in the data sets per animal is lost.
We appreciate the reviewer’s guidance on appropriate statistical treatment. To address these concerns we have re-analyzed all datasets that contained multiple measurements per animal (e.g., fields of view, lobules, or trials) using nested statistics with animal as the higher-order unit. Specifically, we applied a two-level nested ANOVA when more than two groups were compared and a nested t-test when two conditions were present. The re-analysis confirmed all original conclusions. Because the nested models yielded comparable effect sizes to the Mann–Whitney tests, we have retained the mean ± SEM for ease of comparison with prior literature but now also report all values for each mouse in Table 1. In cases where a single measurement per mouse was compared between two groups, we used the Mann–Whitney test and present the results in the graphs as median values.
Minor Points
(1) In all figures, please specify the ages at which each procedure was conducted, as demonstrated in Figure 2A.
All main and supplementary figures now specify the exact postnatal age.
(2) Clarify the selection criteria for regions of interest (ROI) in calcium imaging, and provide representative ROIs.
We appreciate the reviewer’s guidance. We have clarified that our ROI detection followed the protocol reported by our previous paper (Tanigawa et al., 2024, Communications Biology) (lines 177-178) and representative Purkinje cell ROIs are now shown in Fig. 2B.
(3) Include data on the proportion of climbing fiber or inferior olive neurons expressing Kir and the total number of neurons transfected, which would help contextualize the observed effects on PC synchronization and its broader implications for cerebellar circuit function.
We appreciate the reviewer’s guidance. New Fig. 7C summarizes the efficiency of AAV-GFP and AAV-Kir2.1-GFP injections into the inferior olive. Across 4 mice PCs with GFP-labeled CFs was detected in 19.3 ± 11.9 (mean ± S.D.) % for control and 26.2 ± 11.8 (mean ± S.D.) % for Kir2.1 of PCs. These numbers are reported in the Results (lines 373–375).
(4) Higher magnification images in Figures 1 and S3 would improve visual clarity.
We have addressed the request for higher-magnification images in two ways. First, all panels in Figure S3 were placed on a larger canvas. Second, in Figure 1 we adjusted panel sizes to emphasize fine structure: panel 1C already represents an enlargement of the RFP positive cells shown in 1B, and panel 1H and 1J now occupies a wider span so that every ASPA-positive cell body can be distinguished. Should the reviewer still require an even closer view, we have additional ready for upload.
(5) Consider language editing to enhance overall clarity and readability.
The entire manuscript was edited to improve flow, consistency, and readability.
(6) Refine the discussion to align with the presented data.
We have refined the discussion.
Thank you once again for your constructive suggestions and comments. We believe these changes have improved the clarity and readability of our manuscript.
Reviewer #2 (Public review):
We appreciate Reviewer #2’s positive evaluation of our work and thank him/her for the constructive suggestions and comments. We followed these suggestions and comments and have conducted additional experiments. We have rewritten the manuscript and revised the figures according to the points Reviewer #1 mentioned. Our point-by-point responses to the comments are as follows.
Summary:
In this manuscript, the authors use genetic tools to ablate oligodendrocytes in the cerebellum during postnatal development. They show that the oligodendrocyte numbers return to normal post-weaning. Yet, the loss of oligodendrocytes during development seems to result in decreased synchrony of calcium transients in Purkinje neurons across the cerebellum. Further, there were deficits in social behaviors and motor coordination. Finally, they suppress activity in a subset of climbing fibers to show that it results in similar phenotypes in the calcium signaling and behavioral assays. They conclude that the behavioral deficits in the oligodendrocyte ablation experiments must result from loss of synchrony.
Strengths:
Use of genetic tools to induce perturbations in a spatiotemporally specific manner.
We appreciate these positive evaluation.
Weaknesses:
The main weakness in this manuscript is the lack of a cohesive causal connection between the experimental manipulation performed and the phenotypes observed. Though they have taken great care to induce oligodendrocyte loss specifically in the cerebellum and at specific time windows, the subsequent experiments do not address specific questions regarding the effect of this manipulation.
Calcium transients in Purkinje neurons are caused to a large extent by climbing fibers, but there is evidence for simple spikes to also underlie the dF/F signatures (Ramirez and Stell, Cell Reports, 2016).
We thank the reviewer for drawing attention to the work of Ramirez & Stell (2016), which showed that simple-spike bursts can elicit Ca²⁺ rises, but only in the soma and proximal dendrites of adult Purkinje cells. In our study, Regions of Interest were restricted to the dendritic arbor, where SS-evoked signals are essentially undetectable (Ramirez & Stell, 2016), whereas climbing-fiber complex spikes generate large, all-or-none transients (Good et al., 2017). Accordingly, even if a rare SS-driven event reached threshold it would likely fall outside our ROIs.
In addition, we directly imaged CF population activity by expressing GCaMP7f in inferior-olive neurons. Correlation analysis of CF boutons revealed that DTA ablation lowers CF–CF synchrony at P14 (new Fig. 3A–D). Because CF synchrony is a principal driver of Purkinje-cell co-activation, this reduction provides a mechanistic link between oligodendrocyte loss and the hyposynchrony we observe among Purkinje cells. Consistent with this interpretation, electrophysiological recordings showed that parallel-fiber EPSCs and inhibitory synaptic inputs onto Purkinje cells were unchanged by DTA treatment (Fig. 3E-H) , which makes it less likely that the reduced synchrony simply reflects changes in other excitatory or inhibitory synaptic drive.
That said, SS-dependent somatic Ca²⁺ signals could still influence downstream plasticity and long-term cerebellar function. In future work we therefore plan to combine somatic imaging with stage-specific oligodendrocyte manipulations to test whether SS-evoked Ca²⁺ dynamics are themselves modulated by oligodendrocyte support. We have added these descriptions in the Results (lines 288–294) and Discussion (lines 423–434).
Also, it is erroneous to categorize these calcium signals as signatures of "spontaneous activity" of Purkinje neurons as they can have dual origins.
Thank you for pointing out the potential ambiguity. In the revised manuscript we have clarified how we use the term “spontaneous activity” in the context of our measurements (lines 289-290). Our calcium imaging was restricted to the dendritic arbor of Purkinje cells, where calcium transients are dominated by climbing-fiber (CF) inputs (Ramirez & Stell, 2016; Good et al., 2017). Thus, the synchrony values reported here primarily reflect CF-driven complex spikes rather than mixed signals of dual origin. We have revised the Results section accordingly (lines 289–293) to make this measurement-specific limitation explicit.
Further, the effect of developmental oligodendrocyte ablation on the cerebellum has been previously reported by Mathis et al., Development, 2003. They report very severe effects such as the loss of molecular layer interneurons, stunted Purkinje neuron dendritic arbors, abnormal foliations, etc. In this context, it is hardly surprising that one would observe a reduction of synchrony in Purkinje neurons (perhaps due to loss of synaptic contacts, not only from CFs but also from granule cells).
We appreciate the reviewer’s comparison to Mathis et al. (2003). Mathis et al. used MBP–HSV-TK transgenic mice in which systemic FIAU treatment eliminates oligodendrocytes. When ablation began at P1, they observed severe dysmorphology—loss of molecular-layer interneurons, Purkinje-cell (PC) dendritic stunting, and abnormal foliation. Crucially, however, the same study reports that starting the ablation later (FIAU from P6-P20) left cerebellar cyto-architecture entirely normal.
Our AAV MAG-DTA paradigm resembles this later window. Our temporally restricted DTA protocol produces the same ‘late-onset’ profile—robust yet reversible hypomyelination with no loss of Purkinje cells, interneurons, dendritic length, or synaptic input (new Fig. S1–S2, Fig. 3E-H). The enduring hyposynchrony we report therefore cannot be attributed to the dramatic anatomical defects seen after prenatal ablation, but instead reveals a specific requirement for early-postnatal myelin in stabilizing PC synchrony, especially affecting CF-CF synchrony.
This clarification shows that we have carefully considered the Mathis model and that our findings not only replicate, but also extend the earlier work. We have added these description in Result (lines 273-286)
The last experiment with the expression of Kir2.1 in the inferior olive is hardly convincing.
We appreciate the reviewer’s concern and have reinforced the causal link between Purkinje-cell synchrony and behavior. To test whether restoring PC synchrony is sufficient to rescue behavior, we introduced a red-shifted opsin (AAV-L7-rsChrimine) into PCs of DTA mice raised to adulthood. During testing we delivered 590-nm light pulses (10 ms, 1 Hz) to the vermis, driving brief, population-wide spiking (new Fig. 8). This periodic re-synchronization left anxiety measures unchanged (open-field center time remained low) but rescued both motor coordination (rotarod latency normalized to control levels) and sociability (time spent with a novel mouse restored). The dissociation implies that distinct behavioral domains differ in their sensitivity to PC timing precision and confirms that reduced synchrony—not cell loss or gross circuit damage (Fig. S1F, S2)—is the primary driver of the motor and social deficits. Together, the optogenetic rescue establishes a bidirectional, mechanistic link between PC synchrony and behavior, addressing the reviewer’s reservations about the original experiment. We have added these descriptions in Result (lines 394-415)
In summary, while the authors used a specific tool to probe the role of developmental oligodendrocytes in cerebellar physiology and function, they failed to answer specific questions regarding this role, which they could have done with more fine-grained experimental analysis.
Thank you once again for your constructive suggestions and comments. We believe these changes have improved the clarity and readability of our manuscript.
Recommendations for the authors:
Reviewer #2 (Recommendations for the authors):
(1) Show that ODC loss is specific to the cerebellum.
We thank the reviewer for requesting additional evidence. To verify that oligodendrocyte ablation was confined to the cerebellum, we injected an AAV carrying mCherry under the human MAG promoter (AAV-hMAG-mCherry) into the cerebellum, and screened the whole brain one week later. As shown in the new Figure 1E–G, mCherry positive cells were present throughout the injected cerebellar cortex (Fig. 1E), but no fluorescent cells were detected in extracerebellar regions—including cerebral cortex, medulla, pons, midbrain. These data demonstrate that our viral approach are specific to the cerebellum, ruling out off-target demyelination elsewhere in the CNS as a contributor to the behavioral and synchrony phenotypes. We have added these descriptions in Result (lines 262-264)
(2) Characterize the gross morphology of the cerebellum at different developmental stages. Are major cell types all present? Major pathways preserved?
We thank the reviewer for requesting additional evidence. To ensure that the developmental loss of oligodendrocytes did not globally disturb cerebellar architecture, we performed a comprehensive histological and electrophysiological survey during development. New data are presented (new Fig. S1–S2, Fig. 3E-H).
(1) Overall morphology. Low-magnification parvalbumin counterstaining revealed similar cerebellar area in DTA versus control mice at every age (Fig. S1F, G).
(2) Major neuronal classes. Quantification of parvalbumin-positive Purkinje cells and interneurons showed no differences in density between control and DTA (Fig. S2E, H, M, N, P). Purkinje dendritic arbors were not different between control and DTA (Fig. S2G, O).
(3) Excitatory and inhibitory synapse inputs. Miniature IPSCs and Parallel-fiber-EPSCs onto Purkinje cells were quantified; neither was differed between groups (Fig. 3E-G).
(4) Glial populations. IBA1-positive microglia and S100β-positive astrocytes exhibited normal density and marker intensity (Fig. S2).
Taken together, these analyses show that all major cell types are present at normal density, synaptic inputs from excitatory and inhibitory neurons are preserved, and gross cerebellar morphology is intact after DTA-mediated oligodendrocyte ablation.
(3) Recording of PNs to see whether the lack of synchrony is due to CFs or simple spikes.
We thank the reviewer for drawing attention to the work of Ramirez & Stell (2016), which showed that simple-spike bursts can elicit Ca<sup>2+</sup> rises, but only in the soma and proximal dendrites of adult Purkinje cells. In our study, Regions of Interest were restricted to the dendritic arbor, where SS-evoked signals are essentially undetectable (Ramirez & Stell, 2016), whereas climbing-fiber complex spikes generate large, all-or-none transients (Good et al., 2017). Accordingly, even if a rare SS-driven event reached threshold it would likely fall outside our ROIs.
In addition, we directly imaged CF population activity by expressing GCaMP7f in inferior-olive neurons. Correlation analysis of CF boutons revealed that DTA ablation lowers CF–CF synchrony at P14 (new Fig. 3A–D). Because CF synchrony is a principal driver of Purkinje-cell co-activation, this reduction provides a mechanistic link between oligodendrocyte loss and the hyposynchrony we observe among Purkinje cells. Consistent with this interpretation, electrophysiological recordings showed that parallel-fiber EPSCs and inhibitory synaptic inputs onto Purkinje cells were unchanged by DTA treatment (Fig. 3E-H) , which makes it less likely that the reduced synchrony simply reflects changes in other excitatory or inhibitory synaptic drive.
That said, SS-dependent somatic Ca<sup>2+</sup> signals could still influence downstream plasticity and long-term cerebellar function. In future work we therefore plan to combine somatic imaging with stage-specific oligodendrocyte manipulations to test whether SS-evoked Ca²⁺ dynamics are themselves modulated by oligodendrocyte support. We have added these descriptions in the Results (lines 301–312) and Discussion (lines 423–434).
(4) Is CF synapse elimination altered? Test using evoked EPSCs or staining methods.
We agree that directly testing whether oligodendrocyte loss disturbs climbing-fiber synapse elimination would provide a full mechanistic picture. We are already quantifying climbing fiber terminal number with vGluT2 immunostaining and recording evoked CF-EPSCs in the same DTA model; these data, together with an analysis of how population synchrony is involved in synapse elimination, will form the basis of a separate manuscript now in preparation. To keep the present paper focused on the phenomena we have rigorously documented—transient oligodendrocyte loss and the resulting long-lasting hyposynchrony and abnormal behaviors—we have removed the speculative sentence on oligodendrocyte-mediated synapse elimination. We believe this revision meets the reviewer’s request without over-extending the current dataset.
Thank you once again for your constructive suggestions and comments. We believe these changes have improved the clarity and readability of our manuscript.
