High-resolution awake mouse fMRI at 14 Tesla

Reviewer #1 (Public Review):

Summary:

The authors bring together implanted radiofrequency coils, high-field MRI imaging, awake animal imaging, and sensory stimulation methods in a technological demonstration. The results are very detailed descriptions of the sensory systems under investigation.

Strengths:

- The maps are qualitatively excellent for rodent whole-brain imaging.
- The design of the holder and the coil is pretty clever.

Weaknesses:

- Some unexpected regions appear on the whole brain maps, and the discussion of these regions is succinct.
- The authors do not make the work and effort to train the animals and average the data from several hundred trials apparent enough. This is important for any reader who would like to consider implementing this technology.
- The data is not available. This does not let the readers make their own assessment of the results.

Reviewer #2 (Public Review):

Summary:

The manuscript by Hike et al. entitled 'High-resolution awake mouse fMRI at 14 Tesla' describes the implementation of awake mouse BOLD-fMRI at high field. This work is timely as the field of mouse fMRI is working toward collecting high-quality data from awake animals. Imaging awake subjects offers opportunities to study brain function that are otherwise not possible under the more common anesthetized conditions. Not to mention the confounding effects that anesthesia has on neurovascular coupling. What has made progress in this area slow (relative to other imaging approaches like optical imaging) is the environment within the MRI scanner (high acoustic noise) - as well as the intolerance of head and body motion. This work adds to a relatively small, but quickly growing literature on awake mouse fMRI. The findings in the study include testing of an implanted head-coil (for MRI data reception). Two designs are described and the SNR of these units at 9.4T and 14T are reported. Further, responses to visual as well as whisker stimulation recorded in acclimated awake mice are shown. The most interesting finding, and most novel, is the observation that mice seem to learn to anticipate the presentation of the stimulus - as demonstrated by activations evident ~6 seconds prior to the presentation of the stimulus when stimuli are delivered at regular intervals (but not when stimuli are presented at random intervals). These kinds of studies are very challenging to do. The surgical preparation and length of time invested into training animals are grueling. I also see this work as a step in the right direction and evidence of the foundations for lots of interesting future work. However, I also found a few shortcomings listed below.

Weaknesses:

(1) The surface coil, although offering a great SNR boost at the surface, ultimately comes at a cost of lower SNR in deeper more removed brain regions in comparison to commercially available Bruker coils (at room temperature). This should be quantified. A rough comparison in SNR is drawn between the implanted coils and the Bruker Cryoprobe - this should be a quantitative comparison (if possible) - including any differences in SNR in deeper brain structures. There are drawbacks to the Cryoprobe, which can be discussed, but a more thorough comparison between the implanted coils, and other existing options should be provided (the Cryoprobe has been used previously in awake mouse experiments). Further, the details of how to build the implanted coils should be provided (shared) - this should include a parts list as well as detailed instructions on how to build the units. Also, how expensive are they? And can they be reused?

(2) In the introduction, the authors state that "Awake mouse fMRI has been well investigated". I disagree with this statement and others in the manuscript that give the reader the impression that awake experiments are not a challenging and unresolved approach to fMRI experiments in mice (or rodents). Although there are multiple labs (maybe 15 worldwide) that have conducted awake mouse experiments (with varying degrees of success/thoroughness), we are far from a standardized approach. This is a strength of the current work and should be highlighted as such. I encourage the authors to read the recent systematic review that was published on this topic in Cerebral Cortex by Mandino et al. There are several elements in there that should influence the tone of this piece including awake mouse implementations with the Bruker Cryoprobe, prevalence of surgical preparations, and evaluations of stress.

(3) The authors also comment on implanted coils reducing animal stress - I don't know where this comment is coming from, as this has not been reported in the literature (to my knowledge) and the authors don't appear to have evaluated stress in their mice.

(4) Following on the above point, measures of motion, stress, and more details on the acclimation procedure that was implemented in this study should be included.

(5) It wasn't clear to me at what times the loop versus "Figure 8" coil was being used, nor how many mice (or how much data) were included in each experiment/plot. There is also no mention of biological sex.

(6) Building on the points above, the manuscript overall lacks experimental detail (especially since the format has the results prior to the methods).

(7) An observation is made in the manuscript that there is an appreciable amount of negative BOLD signal. The authors speculate that this may come from astrocyte-mediated BOLD during brain state changes (and cite anesthetized rat and non-human primate experiments). This is very strange to me. First, the negative BOLD signal is not plotted (please do this), further, there are studies in awake mice that measure astrocyte activation eliciting positive BOLD responses (see Takata et al. in Glia, 2017).