Diffusion MRS tracks distinct trajectories of neuronal development in the cerebellum and thalamus of rat neonates

Reviewer #1 (Public Review):

In this work, Ligneul and coauthors implemented diffusion-weighted MRS in young rats to follow longitudinally and in vivo the microstructural changes occurring during brain development. Diffusion-weighted MRS is here instrumental in assessing microstructure in a cell-specific manner, as opposed to the claimed gold-standard (manganese-enhanced MRI) that can only probe changes in brain volume. Differential microstructure and complexification of the cerebellum and the thalamus during rat brain development were observed non-invasively. In particular, lower metabolite ADC with increasing age were measured in both brain regions, reflecting increasing cellular restriction with brain maturation. Higher sphere (representing cell bodies) fraction for neuronal metabolites (total NAA, glutamate) and total creatine and taurine in the cerebellum compared to the thalamus were estimated, reflecting the unique structure of the cerebellar granular layer with a high density of cell bodies. Decreasing sphere fraction with age was observed in the cerebellum, reflecting the development of the dendritic tree of Purkinje cells and Bergmann glia. From morphometric analyses, the authors could probe non-monotonic branching evolution in the cerebellum, matching 3D representations of Purkinje cells expansion and complexification with age. Finally, the authors highlighted taurine as a potential new marker of cerebellar development.

From a technical standpoint, this work clearly demonstrates the potential of diffusion-weighted MRS at probing microstructure changes of the developing brain non-invasively, paving the way for its application in pathological cases. Ligneul and coauthors also show that diffusion-weighted MRS acquisitions in neonates are feasible, despite the known technical challenges of such measurements, even in adult rats. They also provide all necessary resources to reproduce and build upon their work, which is highly valuable for the community.

From a biological standpoint, claims are well supported by the microstructure parameters derived from advanced biophysical modelling of the diffusion MRS data. The assumption of metabolite compartmentation, forming the basis of cell-specific microstructure interpretation of dMRS data, remains debated and should be considered with care (Rae, Neurochem Res, 2014, https://doi.org/10.1007/s11064-013-1199-5). External cross-validation of some of the authors' claims, in particular taurine in the thalamus switching from neurons to astrocytes during brain development, would be a highly valuable addition to this study.

Specific strengths:

(1) The interpretation of dMRS data in terms of cell-specific microstructure through advanced biophysical modelling (e.g. the sphere fraction, modelling the fraction of cell bodies versus neuronal or astrocytic processes) is a strong asset of the study, going beyond the more commonly used signal representation metrics such as the apparent diffusion coefficient, which lacks specificity to biological phenomena.
(2) The fairly good data quality despite the complexity of the experimental framework should be praised: diffusion-weighted MRS was acquired in two brain regions (although not in the same animals) and longitudinally, in neonates, including data at high b-values and multiple diffusion times, which altogether constitutes a large-scale dataset of high value for the diffusion-weighted MRS community.
(3) The authors have shared publicly data and codes used for processing and fitting, which will allow one to reproduce or extend the scope of this work to disease populations, and which goes in line with the current effort of the MR(S) community for data sharing.

Specific weaknesses:

(1) This work lacks an introduction and a discussion about diffusion MRI, which is already a validated technique to assess brain development non-invasively. Although water lacks cell-specificity compared to metabolites, several studies have reported a decrease in water ADC and increased fractional anisotropy with brain maturation, associated with the myelination process and decreased water content (overview in Hüppi, Chapt. 30 of "Diffusion MRI: Theory, Methods, and Applications", Oxford University Press, 2010). Interestingly, the same observations are found in this work (decreased ADC with age for most metabolites in both brain regions), which should have been commented on. Moreover, the authors could have reported water diffusion properties in addition to metabolites', as I believe the water signal, used for coil combination and/or Eddy currents corrections, is usually naturally acquired during diffusion-weighted MRS scans.
(2) It is unclear why the authors have normalized metabolite concentrations (measured from low b-values diffusion-weighted MRS spectra) to the macromolecule concentrations. First, it is not specified whether in vivo macromolecules were acquired at each age or just at one time point. Second, such ratios are not standard practice in the MRS community so this choice should have been explained. Third, the macromolecule content was reported to change with age (Tkac et al., Magn Reson Med, 2003), therefore a change in metabolite to macromolecule ratio with age cannot be interpreted unequivocally.
(3) Some discussion is missing about the choice of the analytical biophysical model (although a few are compared in Supplementary Materials), in particular: is a model of macroscopic anisotropy relevant in cerebellum, made of a large fraction of oriented white matter tracks, and does the model remain valid at different ages given white matter maturation and the ongoing myelination process?

Reviewer #2 (Public Review):

Summary:

The authors set out to non-invasively track neuronal development in rat neonates, which they achieved with notable success. However, the direct relationship between the results and broader conclusions regarding developmental biology and potential human implications is somewhat overstretched without further validation.

Strengths:

If adequately revised and validated, this work could have a significant impact on the field, providing a non-invasive tool for longitudinal studies of brain development and neurodevelopmental disorders in preclinical settings.

Weaknesses:

(1) Consistency and Logical Flow:

- The manuscript suffers from a lack of strategic flow in some sections. Specifically, transitions between major findings and methodological discussions need refinement to ensure a logical progression of ideas. For example, the jump from the introduction of developmental trajectories and the technicalities of MRS (Magnetic Resonance Spectroscopy) processing on page 3 could benefit from a bridging paragraph that explicitly states the study's hypotheses based on existing literature gaps.

(2) Scientific Rigour:

- While the novel application of diffusion-weighted MRS is commendable, there's a notable gap in the rigorous validation of this approach against gold-standard histological or molecular techniques. Particularly, the assertions regarding the sphere fraction and morphological changes inferred from biophysical modelling mandates direct validation to solidify the claims made. A study comparing these in vivo findings with ex vivo confirmation in at least a subset of samples would significantly enhance the reliability of these conclusions.

(3) Clarity and Novelty:

- The manuscript often delves deeply into technical specifics at the expense of accessibility to readers not deeply familiar with MRS technology. The introduction and discussions would benefit from a clearer elucidation of why these specific metabolite markers were chosen and their known relevance to neuronal and glial cells, placing this in the context of what is novel compared to existing literature.
- The novelty aspect could be reinforced by a more structured discussion on how this method could change the current understanding or practices within neurodevelopmental research, compared to the current state of the art.

(4) Completeness:

- The Discussion section requires expansion to offer a more comprehensive interpretation of how these findings impact the broader field of neurodevelopment and psychiatric disorders. Specifically, the implications for human studies or clinical translation are touched upon but not fully explored.
- Further, while supplementary material provides necessary detail on methodology, key findings from these analyses should be summarized and discussed in the main text to ensure the manuscript stands complete on its own.

(5) Grammar, Style, Orthography:

- There are sporadic grammatical and typographical errors throughout the text which, while minor, detract from the overall readability. For example, inconsistencies in metabolite abbreviations (e.g., tCr vs Cr+PCr) should be standardized.

(6) References and Additional Context:

- The current reference list is extensive but lacks integration into the narrative. Direct comparisons with existing studies, especially those with conflicting or supportive findings, are scant. More dedicated effort to contextualize this work within the existing body of knowledge would be beneficial.