Gradual Restraint Habituation for Awake Functional Magnetic Resonance Imaging Combined With a Sparse Imaging Paradigm Reduces Motion Artifacts and Stress Levels in Rodents

Abstract

Functional magnetic resonance imaging, as a non-invasive technique, offers unique opportunities to assess brain function and connectivity under a broad range of applications, ranging from passive sensory stimulation to high-level cognitive abilities, in awake animals. This approach is confounded, however, by the fact that physical restraint and loud unpredictable acoustic noise must inevitably accompany fMRI recordings. These factors induce marked stress in rodents, and stress-related elevations of corticosterone levels are known to alter information processing and cognition in the rodent. Here, we propose a habituation strategy that spans specific stages of adaptation to restraint, MRI noise, and confinement stress in awake rats and circumvents the need for surgical head restraint. This habituation protocol results in stress levels during awake fMRI that do not differ from pre-handling levels and enables stable image acquisition with very low motion artifacts. For this, rats were gradually trained over a period of three weeks and eighteen training sessions. Stress levels were assessed by analysis of fecal corticosterone metabolite levels and breathing rates. We observed significant drops in stress levels to below pre-handling levels at the end of the habituation procedure. During fMRI in awake rats, after the conclusion of habituation and using a non-invasive head-fixation device, breathing was stable and head motion artifacts were minimal. A task-based fMRI experiment, using acoustic stimulation, conducted 2 days after the end of habituation, resulted in precise whole brain mapping of BOLD signals in the brain, with clear delineation of the expected auditory-related structures. The active discrimination by the animals of the acoustic stimuli from the backdrop of scanner noise was corroborated by significant increases in BOLD signals in the thalamus and reticular formation. Taken together, these data show that effective habituation to awake fMRI can be achieved by gradual and incremental acclimatization to the experimental conditions. Subsequent BOLD recordings, even during superimposed acoustic stimulation, reflect low stress-levels, low motion and a corresponding high-quality image acquisition. Furthermore, BOLD signals obtained during fMRI indicate that effective habituation facilitates selective attention to sensory stimuli that can in turn support the discrimination of cognitive processes in the absence of stress confounds.

Male and Female C57BL/6 Mice Respond Differently to Awake Magnetic Resonance Imaging Habituation

“…Furthermore, mean FCM concentrations for both male and female on the last day of MRI habituation appear below that of pre handling levels (Figure 7A). This is consistent with other groups who reported decreased FCM concentration levels after several days of awake MRI training (King et al, 2005;Tsurugizawa et al, 2020;Russo et al, 2021). Lastly, during our MRI habituation period, both male and female mice were able to recover, maintain and increase their body weight.…”

“…For most labs, however, scanner resources are too scarce to include an actual MRI scanner in the habituation so we opted for a habituation protocol using a simulated scanner environment only to ensure our study is relevant to as many labs as possible. For future work it may be beneficial to include resting days as implemented in similar studies (Russo et al, 2021). However, our FCM data shows that if habituation is not maintained stress of handling increases again (Post time point in Figure 7A, both sexes).…”

Modeling Brain Dysconnectivity in Rodents

“…Efforts are presently underway to create protocols aimed at optimizing habituation to the MRI environment and mitigating the stressful effects of head fixation, body restraint and noise via a gradual and incremental habituation protocols (63)(64)(65)(66). Some of these studies have shown that these procedure can result in stress-related corticosterone levels comparable to pre-handling levels in both mice (57) and rats (67). In the latter species, thanks to a sparse MRI sequence tuned to reduce acoustic stress, animals could even discriminate auditory stimulation from the background scanner noise (67).…”

Translational Magnetic Resonance Imaging in Autism Spectrum Disorder From the Mouse Model to Human

“…Despite common functional connectivity between awaked and lightly anesthetized state ( Grandjean et al, 2020 ; Tsurugizawa and Yoshimaru, 2021 ), fMRI signal changes to the physiological stimuli are significantly disturbed by the anesthetic ( Peeters et al, 2001 ; Tsurugizawa et al, 2010 , 2016 ; Aksenov et al, 2015 ; Tokunaga et al, 2021 ; You et al, 2021 ). As an alternative, awake mouse fMRI is developed using habituation training to reduce stress during MRI experiments ( Tsurugizawa et al, 2013 , 2020 , 2021 ; Bergmann et al, 2016 ; Yoshida et al, 2016 ; Madularu et al, 2017 ; Chen et al, 2020 ; Russo et al, 2021 ). Although the habituation protocol needs refinement to reduce stress efficiently ( Low et al, 2016 ), awake fMRI can be an essential tool to bridge the human MRI and rodent MRI.…”