Mapping Serotonergic Dynamics using Drug-Modulated Molecular Connectivity

Reviewer #1 (Public Review):

This paper by Ionescu et al. applies novel brain connectivity measures based on fMRI and serotonin PET both at baseline and following ecstasy use in rats. There are multiple strengths to this manuscript. First, the use of connectivity measures using temporal correlations of 11C-DASB PET, especially when combined with resting state fMRI, is highly novel and powerful. The effects of ecstasy on molecular connectivity of the serotonin network and salience network are also quite intriguing.

I would like the authors to discuss and justify their use of high-dose (1.3%) isolfurane. A recent consensus paper on rat fMRI (Grandjean et al., "A Consensus Protocol for Functional Connectivity Analysis in the Rat Brain.") found that medetomidine combined with low dose isoflurane provided optimal control of physiology and fMRI signal. To overcome any doubts about the effects of the high-dose anaesthetic I'd encourage the authors to show the results of their functional connectivity specificity using the same or similar image processing protocol as described in that consensus paper. This is especially true since the fMRI ICs in Figure 2A appear fairly restricted.

I'd also be interested to read more about why the cerebellum was chosen as a reference region, given that serotonin is highly expressed in the cerebellum, and what effects the choice of reference region has on their quantification.

The PET ICs appear less bilateral than the fMRI ICs. Is that simply a thresholding artefact or is it a real signal?

"The data will be made available upon reasonable request" is not sufficient - please deposit the data in an open repository and link to its location.

Reviewer #2 (Public Review):

Summary:

The article aims to describe a novel methodology for the study of brain organization, in comparison to fMRI functional connectivity, under rest vs. controlled pharmacological stimulation.

Strengths:

Solid study design with pharmacological stimulation applied to assess the biological significance of functional and (novel) molecular connectivity estimates.

Provides relevant information on the multivariate organization of serotoninergic system in the brain.

Provides relevant information on the sensitivity of traditional (univariate PET analysis, fMRI functional connectivity) and novel (molecular connectivity) methods in measuring pharmacological effects on brain function.

Weaknesses:

While the study protocol is referenced in the paper, it would be useful to at least report whether the study uses bolus, constant infusion, or a combination of the two and the duration of the frames chosen for reconstruction. Minimal details on anesthesia should also be reported, clarifying whether an interaction between the pharmacological agent for anesthesia and MDMA can be expected (whole-brain or in specific regions).

Some terminology is used in a bit unclear way. E.g. "seed-based" usually refers to seed-to-voxel and not ROI-to-ROI analysis, or e.g. it is a bit confusing to have IC1 called SERT network when in fact all ICs derived from DASB data are SERT networks. Perhaps a different wording could be used (IC1 = SERT xxxxx network; IC2= SERT salience network) .

The limited sample size for the rats undergoing pharmacological stimulation which might make the study (potentially) not particularly powerful. This could not be a problem if the MDMA effect observed is particularly consistent across rats. Information on inter-individual variability of FC, MC, and BPND could be provided in this regard.

Author Response:

We thank the reviewers for their insightful feedback. In our revised version of the manuscript, we will address all points raised.

Regarding the preprocessing (Reviewer 1), we agree that the StandardRat pipeline is optimal for newly acquired datasets. However, since this study involves reanalyzing an already published dataset (Ionescu et al., JNM, 2023), which was preprocessed, analyzed, and published before the StandardRat paper, we aimed to maintain the same preprocessing. This approach allows for consistent interpretation of the readout regarding functional and molecular connectivity in the context of our previously published findings. Nonetheless, we agree that providing full access to the data will enable other researchers to reproduce our results using the StandardRat preprocessing pipeline and perform additional analyses on this rich dataset. Therefore, we will provide full access to the data via an open repository, as the reviewer suggested.

Regarding anesthesia, we acknowledge that this is a limitation of our study, as more recent studies have indicated superior protocols. However, we and others have shown that, while not ideal, isoflurane at the used dose maintains stable physiology and does not cause burst suppression in rats. We will amend our discussion to reflect these points.

Regarding the other points, we will amend the manuscript to provide more detail on the experimental design, including the tracer application as suggested by Reviewer 2, and clarify parts of the analysis that are unclear in the current version. Additionally, we agree with Reviewer 2 that our current terminology may cause confusion, and we will amend it accordingly. We will also discuss the other points raised by the reviewers, such as the reduced sample size for the pharmacological cohort as limitations in our discussion.

Thank you for your understanding and the opportunity to improve our manuscript.