Preclinical neuroscientist interested in awake fMRI in rodentsBrain fMRI in rodent animal models is a fundamental tool for exploring brain functions. However in rodents one requires anesthetics to control stress level and reduce motion. While anesthesia does maximize experimental control, it affects brain activity and restricts the type of experiments one can perform. Several have shown how it is possible to image awake animals but in the majority of cases this involves using a combination of initial anesthesia followed by forced restraint. These studies have shown that anesthesia has implications for BOLD signals. We describe a strategy for awake rat fMRI which relies on habituating the animal to a gentle restraint ("snuggle sleeve") and a head restraining system (see Figure 1). The goal was to show that any level of anesthesia during the process perturbs the study under investigation and that the head posting method combined with extensive training and habituation can provide better results for fMRI.

The setup used for is shown in Figure 1. Rats were slowly acclimated using short but systematic training procedure in a mock scanner setup. Rats were trained eight days prior to fMRI scans. Each training day lasted half hour with animal in mock scanner, head posted and with gradient sounds (~140dB). Custom made “snuggle sleeves” were adjustable to enhance comfort and guarantee restraint while still allowing access to head-nut and paws. After several sessions rats entered voluntarily into the “snuggle sleeve”; no initial anesthesia was required. Following a session blood was drawn and corticosteroid levels (stress hormone) was measured.

10 male Sprague Dawley rats (325-400g) were used in this study. Two fMRI sessions consisting of a 1) resting state fMRI, and 2) stimulus-evoked fMRI were conducted sequentially on the awake rats. The same experiments were then repeated under anesthesia ( ~1% isoflurane/O2). Resting-statefMRI lasted 8 minutes (no external stimuli). Stimulus-evoked fMRI was comprised of 6 repetitive stimulus ON/OFF blocks (2sec-off/12sec-on/36sec-off). The air-puff stimuli used consisted in a 5 g force innocuous stimuli on right hind paw. MR experiments were carried out on a Bruker Clinscan 7T. We used an EPI sequence with TR= 2000ms, TE=18ms, in-plane resolution= 0.38mmx0.38mm, slice thickness=0.5 mm, number of repetitions=240 for resting-state fMRI and 150 for stimulus-evoked fMRI. Post-hoc analysis on head motion was estimated using FSL’s MCFLIRT tool. The motion timeseries was calculated to measure the root-mean-square displacement from one time point relative to the preceding time point.

After few training-days stress was greatly reduced, as observed behaviorally (e.g. little or no struggling, eye secretions indicative of stress in rat, or excessive vocalizations ) and through stress hormone levels (data not shown here ). Head motion for each rat was recorded from imaging session (Figure2) for both experiments. Directional displacements are color encoded with the stimulation pattern shown in black (Fig 2A). Except in one case, awake rats did not exhibit excessive head motion ( < 70um). No significant difference between awake and anesthetized. For stimulus-evoked fMRI, the head motion did not appear to be associated with the pattern of the stimulation, (i.e. it did not interfere with our ability to detect the relevant signal changes). In awake condition, unlilateral air-puff stimulation (Figure 3) resulted in significant increase in fMRI signal in bilateral S1, bilateral thalamus, and prefrontal regions including ACC and mPFC (P < 0.05, FWE corrected); the greatest signal change was on the hemisphere contralateral to the stimulation. No significant change was found in anesthetized condition (P < 0.05, FWE corrected). Given that contralateral S1 hind limb region showed the most prominent response to the air-puff stimulation, we examined how S1 hind limb region was functionally coupled to other brain areas using resting state (Figure 4). We found that when rats are awake, S1 hind limb region showed significant correlations with the same region in the other hemisphere. Anesthesia reduces this connection between hemispheres.In the present study we demonstrate that rats trained over the course of 8 days can be restrained in a comfortable “snuggle sleeve” and head posted while undergoing fMRI. This setup not only reduces motion but does not require any initial anesthesia. Habituation with this instrumental setup allows rats to be awake, conscious and be relatively still during fMRI experiments. In addition rats had more fMRI activation in response to air-puff stimulation and showed more widespread functional connectivity in awake condition, compared to when the rats were anesthetized. These results suggest that the described methodology can enhance performance of fMRI experiments in awake condition rodents.