Hippocampal subfield CA1-3 shows differential structural and functional network plasticity after stress-reducing socio-affective mental training

Reviewer #1 (Public Review):

Valk and Engert et al. examined the potential relations between three different mental training modules, hippocampal structure and functional connectivity, and cortisol levels over a 9-month period. They found that among the three types of mental training: Presence (attention and introspective awareness), Affect (socio-emotional - compassion and prosocial motivation), and Perspective (socio-cognitive - metacognition and perspective taking) modules; Affect training most consistently related to changes in hippocampal structure and function - specifically, CA1-3 subfields of the hippocampus. Moreover, decreases in diurnal cortisol correlated to bilateral increases in volume, and decreases in diurnal and chronic cortisol left CA1-3 functional connectivity. Chronic cortisol levels also related to right CA4/DG volume and left subiculum function. The authors demonstrate that mindfulness training programs impact hippocampus and are a potential avenue for stress interventions, a potential avenue to improve health. The data contribute to the literature on plasticity of hippocampal subfields during adulthood, the impact of mental training interventions on the brain, and the link between CA1-3 and both short- and long-term stress changes. Additional clarification and extension of the methods is needed to strengthen the authors' conclusions.

The authors thoughtfully approached the study of hippocampal subfields, utilizing a method designed for T1w images that outperformed Freesurfer 5.3 and that produced comparable results to an earlier version of ASHS. However, given the use of normalized T1-weighted images to delineate hippocampal subfield volume, some caution may be warranted (Wisse et al. 2020). While the authors note the assessment of quality control processes, the difficulty in ensuring valid measurement is an ongoing conversation in the literature. This also extends to the impact of functional co-registration using segmentations. I appreciate the inclusion of Table 5 in documenting reasons for missing data across subjects. Providing additional details on the distribution of quality ratings across subfields would help contextualize the results and ensure there is equal quality of segmentations across subfields.

Given the consistent pattern of finding results with CA1-3, in contrast to other subfields, it would help to know if the effects of the different training modules on subfields differed from each other statistically (i.e., not just that one is significant, and one is not) to provide an additional context of the strength of results focused on Affect training and CA1-3 (for example, those shown in Figure 3).

Reviewer #2 (Public Review):

In this study, Valk, Engert et al. investigated effects of stress-reducing behavioral intervention on hippocampal structure and function across different conditions of mental training and in relation to diurnal and chronic cortisol levels. The authors provide convincing multimodal evidence of a link between hippocampal integrity and stress regulation, showing changes in both volume and intrinsic functional connectivity, as measured by resting-state fMRI, in hippocampal subfield CA1-3 after socio-affective training as compared to training in a socio-cognitive module. In particular, increased CA1-3 volume following socio-affective training overlapped with increased functional connectivity to medial prefrontal cortex, and reductions in cortisol. The conclusions of this paper are well supported by the data, although some aspects of the data analysis would benefit from being clarified and extended.

A main strength of the study is the rigorous design of the behavioral intervention, including test-retest cohorts, an active control group, and a previously established training paradigm, contributing to an overall high quality of included data. Similarly, systematic quality checking of hippocampal subfield segmentations contributes to a reliable foundation for structural and functional investigations.

Another strength of the study is the multimodal data, including both structural and functional markers of hippocampal integrity as well as both diurnal and chronic estimates of cortisol levels. However, the included analyses are not optimally suited for elucidating multivariate interrelationships between these measures. Instead, effects of training on structure and function, and their links to cortisol, are largely characterized separately from each other. This results in the overall interpretation of results, and conclusions, being dependent on a large number of separate associations. Adopting multivariate approaches would better target the question of whether there is cortisol-related structural and functional plasticity in the hippocampus after mental training aimed at reducing stress.

The authors emphasize a link between hippocampal subfield CA1-3 and stress regulation, and indeed, multiple lines of evidence converge to highlight a most consistent role of CA1-3. There are, however, some aspects of the results that limit the robustness of this conclusion. First, formal comparisons between subfields are incomplete, making it difficult to judge whether the CA1-3, to a greater degree than other subfields, display effects of training. Relatedly, it would be of interest to assess whether changes in CA1-3 make a significant contribution to explaining the link between hippocampal integrity and cortisol, as compared to structure and functional connectivity of the whole hippocampus. Second, both structural and functional effects (although functional to a greater degree), were most pronounced in the specific comparison of "Affect" and "Perspective" training conditions, possibly limiting the study's ability to inform general principles of hippocampal stress-regulation.

Author Response:

We would like to thank the Editors and Reviewers for their positive evaluations, constructive comments, and for the opportunity to revise our manuscript. We feel that the comments and suggestions will further improve our manuscript.

In the updated manuscript we aim to incorporate all suggested changes and considerations provided by the Reviewers. In particular, we will provide further information on the quality-control ratings per subfield, as suggested by Reviewer 1. Moreover, we will evaluate whether the training-related changes were specific to CA1-3, rather than just showing significant alterations in CA1-3 and not in the other subfields. Last, as suggested by Reviewer 2, we will additionally test for multivariate associations between hippocampal subfield structure and function, to further evaluate the specificity of hippocampal subfield change as a function of training and cortisol.