Disruption of thalamic functional connectivity is a neural correlate of dexmedetomidine-induced unconsciousness

1Understanding the neural mechanisms of consciousness is a fundamental challenge of current neuroscience research. We note that precise definitions of the words ‘consciousness’ and ‘unconsciousness’ remain ambiguous. However, loss of voluntary response in a task is an effective and clinically relevant definition of unconsciousness. Over the past decade, important insights have been gained by using functional magnetic resonance imaging (fMRI) to characterize the brain during sleep, and disorders of consciousness (DOC) such as coma, vegetative states, and minimally conscious states. In addition to being critical for patients to safely and humanely undergo traumatic surgical or diagnostic procedures, anesthesia has long been recognized as a tool for studying the neural mechanisms of loss and recovery of consciousness.

2Disruption of cortico-cortical connectivity has been proposed as a central mechanism to explain unconsciousness associated with general anesthesia, sleep, and DOC. However, recent evidence suggests that cortico-cortical functional connectivity may be maintained during unconsciousness. To develop more precise neuroanatomic and neurophysiological characterizations of this and other putative mechanisms, studies of anesthetic-induced unconsciousness could benefit from the use of multimodal imaging approaches, combined with neurophysiological understanding of the brain state induced by the anesthetic drug being studied.

3A challenge in using anesthetics to study unconsciousness is stating precise, possible mechanisms of anesthetic-induced brain states, and using an anesthetic which acts at single rather than multiple brain targets to test those mechanism. Among the several anesthetics in current use today, dexmedetomidine is an appealing choice for testing a specific mechanism of how an anesthetic alters the level of consciousness. Dexmedetomidine selectively targets pre-synaptic α2-adrenergic receptors on neurons projecting from the locus ceruleus to the pre-optic area. This leads to activation of inhibitory outputs to the major arousal centers in the midbrain, pons, and hypothalamus producing a neurophysiological and behavioral state that closely resembles NREM II sleep (Figure 1). Dexmedetomidine also acts at the locus ceruleus projections to the intralaminar nucleus of the thalamus, the basal forebrain and the cortex.

4Therefore, the aim of this study was to use a novel integrated positron emission tomography and magnetic resonance imaging (PET/MR) approach to characterize brain resting-state network activity and metabolism during dexmedetomidine-induced unconsciousness. This strategy offers several appealing features. First, PET scanning using fludeoxyglucose (18F-FDG) provides a sensitive estimate of the brain's metabolic state in terms of the cerebral metabolic rate of glucose (CMRglc). Second, comparison of CMRglc results with regional cerebral blood flow (rCBF) estimates obtained from pulsed Arterial Spin Labeling (pASL) fMRI signals allows a direct assessment of whether, as previously reported, flow-metabolism coupling is maintained during dexmedetomidine-induced unconsciousness. Third, we use the CMRglc results to inform the brain functional connectivity analysis derived from the Blood Oxygen Level Dependent (BOLD) fMRI signals.

5In 10 healthy volunteers, we used EEG recordings, and an auditory task to confirm that dexmedetomidine induced a loss of voluntary responsiveness, and exhibited a neurophysiological profile that was similar to non rapid eye movement (NREM) II sleep. We then studied 18F-FDG uptake during baseline and dexmedetomidine-induced unconsciousness in these 10 healthy volunteers in a separate experiment where we defined loss of consciousness as the onset of sustained eye closure and lack of response to a verbal request to open the eyes (Figure 2A). During both the baseline and dexmedetomidine-induced unconsciousness 18F-FDG study visits (two per subject), fMRI data were also recorded. Consistent with a global decrease in glucose metabolism, we found that during the unconscious state, there was a broad reduction in 18F-FDG standardized uptake values in the brain (Figure 2—figure supplement 1A). To quantify this result, we calculated CMRglc during the two 18F-FDG visits (Figure 2—figure supplement 1B). We found reduced CMRglc in thalamic, frontal, and parietal brain regions during the unconscious state (Figure 2B, Table 1). The difference in CMRglc exhibited a spatial distribution consistent with the previously described DMN and both left and right FPNs (Table 1). We found these network-specific CMRglc changes interesting because the DMN has been linked to stimulus-independent thought and self-consciousness, while the FPNs have been linked to conscious perception of the external environment and executive control initiation.

6To further understand the effects of dexmedetomidine-induced unconsciousness on rCBF and brain functional connectivity in these networks, we increased our statistical power for fMRI data analysis by studying an additional seven volunteers using fMRI only (Figure 2C). Analysis of the rCBF difference maps between the unconscious and baseline states showed decreases in rCBF during unconsciousness that overlapped with the same DMN and FPN regions that demonstrated reductions in CMRglc (Figure 2D, Figure 2—figure supplement 1, Table 2). This is consistent with maintenance of blood flow and metabolism coupling during dexmedetomidine-induced unconsciousness.

7Given that we observed reduced flow and metabolism in regions known to correspond to the DMN and FPNs, we next asked whether intrinsic functional connectivity within these networks was altered with dexmedetomidine-induced unconsciousness. First, we confirmed that during both wakefulness and unconsciousness, the DMN and bilateral FPNs were consistently identified in our volunteers (Figure 3, Table 3). Next, we performed a comparison of changes in these networks between the unconscious and awake states. When we compared the DMN between these two states, we found no difference in functional connectivity between the cortical regions of the DMN and FPNs, suggesting that cortico–cortico functional connectivity may be maintained during unconsciousness (Figure 3A–B, Table 3). However, we observed a reduction in functional connectivity between the thalamus and the DMN in the unconscious state (Figure 3A–B, Table 3). This loss of thalamic functional connectivity was observed in a region consistent with intralaminar, midline, mediodorsal, and ventral anterior nuclei. However, our ability to precisely resolve the thalamic nuclei implicated in loss of thalamo-cortical functional connectivity is limited because our methods are not sensitive to small focal differences. We also found that functional connectivity of the left cerebellar representation of the DMN was significantly reduced during unconsciousness (Figure 3B, Table 3). When we compared the FPNs in these two states, we found that the left and right FPNs showed opposite changes in functional connectivity with a cerebellar cluster, likely indicating a switch in hemispheric dominance for cortico-cerebellar functional connectivity to this region during unconsciousness (Figure 3C–F, Table 4, Table 5). Functional connectivity of the cerebellar representations of the FPNs was also significantly reduced (Figure 3D,F, Table 4, Table 5). Since cortico-cerebellar connections are mediated by the thalamus, the changes we observed in cortico-cerebellar functional connectivity are likely related to impaired thalamic functioning. Taken together, these results show that unconsciousness is associated with decreased thalamic CMRglc, rCBF, and functional connectivity to the DMN and support the notion that the thalamus plays a critical role in mediating unconsciousness.

8In order to further examine the role of the thalamus in unconsciousness, we next performed a seed-based functional connectivity analysis using the thalamic region with decreased DMN functional connectivity as the seed region. This thalamic seed also overlapped with thalamic regions showing reduced rCBF and CMRglc. Our seed-based analysis allowed us to more thoroughly examine changes in thalamic functional connectivity to all brain regions. We found that the thalamic seed was significantly functionally disconnected from posterior regions of the DMN (posterior cingulate cortex, precuneus, and inferior parietal lobules) during unconsciousness (Figure 4A, Table 6). Notably, the cortical regions showing reduced thalamic functional connectivity also overlapped with those demonstrating reduced flow and metabolism (Figure 4B).

9We next investigated whether the rCBF changes during the unconscious state returned to baseline during the recovery state. We identified 10 volunteers who responded to verbal instructions (opened eyes and gave a thumbs-up signal, though they were still mildly sedated) during at least 6 min of the recovery ASL scan. Surprisingly, when we compared the recovery state to the unconscious state in these volunteers, we found that the rCBF did not increase (cluster corrected, Figure 5A). In fact, when explored at a more liberal threshold (uncorrected, p < 0.05), further decreases in rCBF were observed (Figure 5B). When we compared the recovery state to the awake state, we found that the decrease in rCBF showed a spatial distribution similar to that observed during the unconscious state (Figure 5C). Next, we aligned all subjects to unconscious and recovery time points in order to study the dynamics of rCBF changes within the statistically significant clusters. We confirmed that rCBF decreased during loss of consciousness and that this decrease was maintained during recovery from this state in all regions analyzed (Figure 5—figure supplement 1). These results suggest that cortical rCBF increase was not sufficient for the early stages of recovery from unconsciousness induced by dexmedetomidine.

10We found that thalamic functional connectivity to the DMN was restored during the recovery state (Figure 6A–B, Table 7). We also found that the opposing left and right FPN changes in the cerebellar cluster (Figure 3B–C) were also reversed (Figure 6C–F, Table 7). When we analyzed the functional connectivity of our thalamic seed of interest, we found that thalamic functional connectivity to the posterior cingulate cortex and thalamo-thalamic functional connectivity were restored during the recovery state (Figure 6G–H, Table 7). Taken together, these results provide further evidence that dexmedetomidine-induced unconsciousness is associated with decreased thalamic functional connectivity to the DMN and supports the notion that the thalamus plays a critical role in mediating unconsciousness and recovery from this state.

11Resting-state functional brain imaging is increasingly being utilized to probe the mechanisms of consciousness. By using a very site-specific drug, performing for the first time simultaneous measurements of CMRglc, rCBF, resting state BOLD low frequency oscillations, and analyzing both dexmedetomidine-induced loss of consciousness and recovery, we have been able to identify a minimal disruption of neural networks that is shared between altered consciousness states. Our results show that during dexmedetomidine-induced unconsciousness, cortico-cortical functional connectivity remains intact but thalamo-cortical functional connectivity is disrupted. This disrupted thalamo-cortical functional connectivity appropriately reverses during recovery from dexmedetomidine-induced unconsciousness.

12The notion that disruption of thalamo-cortical functional connectivity could constitute a neural-network mechanism responsible for unconsciousness that is distinct from disruption of cortico-cortical functional connectivity is supported by a number of findings. During, NREM I and II sleep in humans, DMN cortico-cortical functional connectivity remains preserved while thalamo-cortical functional connectivity is disrupted. However, during NREM III, there is a functional disconnection between frontal and parietal nodes of the DMN, and disrupted thalamo-cortical functional connectivity. Likewise, in-depth electrode studies in humans show that changes in thalamic activity during sleep precedes changes in cortical activity, suggesting that disrupted thalamo-cortical functional connectivity may underlie changes in consciousness. Also, during propofol anesthesia-induced unconsciousness, decreases in thalamo-cortical functional connectivity, alongside disruptions in cortico-cortical functional connectivity in the DMN and FPNs, have been described in healthy volunteers Functional reintegration of the thalamus to key regions of the cortex appears to be necessary for the recovery of consciousness from this state.

13Norepinephrinergic neurons in the locus ceruleus project to thalamic mediodorsal, midline, intralaminar nuclei, which in turn have neurons that project to the cingulate cortex. Our results show that these connections may comprise a functional circuit that is disrupted during dexmedetomidine-induced unconsciousness. Moreover, this functional circuit appears specifically to involve the posterior cingulate cortex, distinct from anterior or mid-cingulate cortex. In our thalamic seed-based functional connectivity analysis, we found that during dexmedetomidine-induced unconsciousness, thalamic regions consistent with intralaminar, midline, mediodorsal, and ventral anterior nuclei were functionally disconnected from posterior DMN structures (precuneus and the posterior cingulate cortex). Perhaps more importantly, recovery from dexmedetomidine-induced unconsciousness was associated with restored thalamo-thalamic and thalamic functional connectivity to the posterior cingulate cortex. Structural connectivity between the thalamus and the posterior cingulate cortex has also recently been correlated with the severity of DOC and used to subcategorize patients in minimally conscious states. The above observations lead us to conclude that a noradrenergic circuit involving the thalamus and posterior cingulate cortex may be important for recovery from both dexmedetomidine-induced unconsciousness and DOC states.

14An FDG-PET study has recently described a linear relationship between metabolism in the precuneus and central thalamus in severely brain-injured patients with DOC. We note that our observed reduction in blood flow in the precuneus and thalamus during dexmedetomidine-induced unconsciousness parallels this result. However, during recovery from dexmedetomidine-induced unconsciousness, we did not observe an increase in rCBF to any brain regions. Since our current and previously reported results suggest that dexmedetomidine does not decouple rCBF and CMRglc, we surmise that metabolism does not return to baseline during the earliest phases of recovery from dexmedetomidine-induced unconsciousness. Thus, the reduced rCBF we report after recovery from dexmedetomidine-induced unconsciousness could reflect a residual drug effect state in which subjects are responsive to external stimuli but remain in a sedated state. This residual effect may be mediated by the broad and diffuse nor-adrenergic projections from the locus ceruleus to the rest of the brain.

15This result is interesting because it suggests that recovery from unconsciousness may occur in a graded fashion. For example, performance on more complex cognitive tasks likely differs substantially between the awake and recovery states. This suggests that re-establishment of functional connectivity between vital brain regions precedes increases in rCBF and CMRglc. Return of rCBF and CMRglc to baseline levels may be indicative of the final stages of recovery from dexmedetomidine-induced unconsciousness. Future investigations incorporating cognitive and psychomotor vigilance tasks during recovery from experimental models of unconsciousness would lend further insight on whether this is a drug-specific effect. Nevertheless, this finding is interesting because it suggests that recovery from unconsciousness states may proceed prior to significant increases in rCBF.

16Drawing from previous work on sleep and anesthesia, our results suggest a hierarchy of brain network disruptions that can result in different states of altered consciousness. In NREM II (non-slow wave) sleep and dexmedetomidine-induced unconsciousness, patients can be easily aroused to respond to verbal commands by sufficiently strong external stimuli. In these states, cortico-cortical functional connectivity is preserved, but thalamo-cortical functional connectivity is disrupted. In propofol anesthesia-induced unconsciousness and some DOC states, where patients cannot be aroused by external stimuli, both cortico-cortical and thalamo-cortical functional connectivity are disrupted.

17In the case of sleep, dexmedetomidine-induced unconsciousness, and some minimally conscious states, maintained cortico-cortical functional connectivity likely allows for the cortex to be ‘primed’, ready to recover from the altered consciousness states with restoration of thalamic functional connectivity, which could be triggered by ascending midbrain and brainstem inputs or direct therapeutic intervention at the thalamic level. In other altered consciousness states, such as during general anesthesia or comatose states, markedly disrupted cortico-cortical functional connectivity could further impair the ability to recover consciousness. We note that while it might be theoretically possible to attain unconsciousness by disrupting only cortico-cortical functional connectivity while preserving thalamo-cortical functional connectivity, it appears, from our study and others, that this does not happen during sleep and anesthesia-induced unconsciousness.

18Compared to the findings supporting a role for disrupted cortico-cortical functional connectivity during unconsciousness, our findings suggest that thalamo-cortical connectivity constitutes a more fundamental disruption required to produce unconsciousness. This insight could lead to a more objective characterization, diagnosis, and prognosis of DOC. Also, the state of cortico-cortical and thalamo-cortical functional connectivity could be used to better select patients who may benefit from therapeutic interventions. For instance, in the event of maintained cortico-cortical functional connectivity but impaired thalamo-cortical functional connectivity with intact thalamo-cortical structural connections, therapies aimed at restoring thalamo-cortical functional connectivity might be more likely to help patients recover. These therapies could be pharmacological, electrical, or could involve other forms of stimulation. Since dexmedetomidine appears to disrupt functional network activity in a manner similar to NREM II sleep, our results also suggest that alpha-2-receptor agonists could be further developed as effective sleep therapeutic drugs.

19At present, there is a debate pertaining to whether anesthetized and unresponsive patients (without motor impairments) can remain internally conscious to subjective experiences (internally generated or provoked by external stimuli). This debate has been fueled by clinical studies of anesthetized patients that have reported rare intraoperative episodes of awareness (resulting from light anesthetic depth) under anesthesia and dreaming under anesthesia. Noreika et al. studied this phenomenon with dexmedetomidine, sevoflurane, propofol, and xenon and confirmed that subjective experiences of consciousness occur during clinically defined unconsciousness states. However, the experimental paradigm that was used to test for subjective experiences of consciousness involved a gradual increase in drug concentrations. This empiric gradual increase did not directly target anesthetic drug dosing to a neurophysiologically defined brain state nor does it inform us on the amount of time that was spent in lighter levels of anesthesia. Thus, the reported subjective experiences could have occurred when the drug concentration levels were very low. This line of reasoning is supported by evidence confirming that subjective experiences of dreaming under general anesthesia likely occur when patients are emerging from general anesthesia.

20Subjective experiences of consciousness also occur during REM sleep. To a limited degree, they are also thought to occur during NREM sleep, especially at sleep onset in NREM I and during NREM II sleep periods encountered later at night. However, the current methods for scoring sleep states remain imprecise and highly subjective, with limited state and temporal resolution, and high intra- and inter-scorer variability, raising the possibility that clinically scored NREM I and II sleep may include periods of arousal. In clinical sleep scoring, trained technicians visually score the sleep time-series data in 30-s epochs according to semantically defined sleep stages. These scoring standards grossly simplify sleep electroencephalographic dynamics. For example, consider the characterization of sleep onset. The American Academy of Sleep Medicine defines sleep onset as the first appearance of any 30-s epoch that contains at least 15 s of sleep. This implies that sleep onset is a binary process. However, recent EEG studies in humans clearly demonstrate that sleep onset, at both a neurophysiological and behavioral level, is a continuous process. As such, methods designed to track moment-to-moment continuous variations in the EEG are significantly better at predicting when subjects lose wakefulness, measured in terms of a behavioral task, than traditional sleep scoring methods. In particular, subjects can continue to perform awake behavior despite being scored as asleep (NREM I) using traditional sleep scoring methods. Thus, it is possible that the subjective experiences of consciousness that have been ascribed to NREM sleep could have occurred during subjectively scored sleep stages that included un-scored periods of arousal or wakefulness.

21Despite this rapidly evolving understanding of subjective experiences during altered arousal under sleep and anesthesia, we have tried in this study to focus on a particular state of dexmedetomidine-induced unconsciousness. We achieved this state through rapid administration of dexmedetomidine and confirmed that this dose and form of drug administration produces a NREM II-like state. We then applied a combination of fMRI and FDG-PET imaging that would characterize the overall time-integrated brain activity during this state. Clinical evidence shows that motor-evoked potentials, and thus motor function from cortex down, remain intact in this state, making it unlikely that the loss of responsiveness we observed is only a result of disrupted motor function. Therefore, we feel it is appropriate to refer to the state of altered arousal described in this manuscript as dexmedetomdine-induced unconsciousness.

22A long-standing debate in anesthesiology relates to the fundamental systems-level mechanisms for anesthesia-induced unconsciousness, with some investigators proposing a thalamic switch and others proposing disrupted cortico-cortical connections as the central mechanism. Our results, in combination with previous work, resolve this conflict by showing how both mechanisms could co-exist but at different parts of the continuum of altered arousal or unconsciousness. Moreover, it suggests a mechanistic interpretation for varying levels of anesthesia-induced unconsciousness, where disrupted thalamo-cortical functional connectivity, but intact cortico-cortical functional connectivity reflects lighter states of unconsciousness and disruptions of both cortico-cortical and thalamo-cortical functional connectivity reflect deeper states of unconsciousness. These insights could guide development of system-specific anesthetic drugs that possess minimal side effects and improved monitoring of patients in both the operating room and intensive care settings.

23This study was conducted at the Athinoula A Martinos Center for Biomedical Imaging at the Massachusetts General Hospital. The Human Research Committee and the Radioactive Drug Research Committee at the Massachusetts General Hospital approved the study protocol. After an initial email/phone screen, potential study subjects were invited to participate in a screening visit. At the screening visit, informed consent including the consent to publish was requested after the nature and possible consequences of the study was explained. All subjects provided informed consent and were American Society of Anesthesiology Physical Status I with Mallampati Class I airway anatomy. After providing informed consent, a standard pre-anesthesia assessment was administered and a blood toxicology screen was performed to ensure that subjects were not taking drugs that might confound the interpretation of study results. A complete metabolic panel and a urine pregnancy test were also obtained to confirm that all values were within normal ranges and to confirm non-pregnant status, respectively. A total of 20 subjects participated in screening visits and 18 subjects were deemed eligible to participate in the study protocol. Two subjects were deemed ineligible after medical evaluation and one subject was lost to follow-up after providing informed consent. During all study visits, subjects were required to take nothing by mouth for at least 8 hr. A urine toxicology screen was performed to ensure that subjects had not taken drugs that might confound interpretation of the results. A pregnancy test was also administered (serum for PET visits, urine for fMRI only visits) for each female subject to confirm non-pregnant status.

24We studied awake, unconscious, and recovery states in humans, using an integrated positron PET/MR approach, in healthy volunteers, 18–36 years of age. Brain imaging was performed with the Biograph mMR scanner (Siemens Healthcare, Erlangen, Germany), which allows simultaneous acquisition of whole-body PET and 3 Tesla MRI data. The fully integrated PET detectors use avalanche photoiodide technology and lutetium oxyorthosilicate crystals (8 × 8 arrays of 4 × 4 × 20 mm3 crystals). The PET scanner's transaxial and axial fields of view are 594 mm and 25.8 cm, respectively. Approximately, 5 mCi of FDG was purchased from an outside approved vendor and was administered as a bolus immediately before the initiation of the dexmedetomidine (described below). A PET-compatible 16-channel head and neck array was used to acquire the MR data.

25At the beginning of the imaging visit, we acquired structural MRI (MPRAGE volume, TR/TE = 2100/3.24 ms, flip angle = 7°, voxel size = 1 mm isotropic) for the purpose of anatomical localization, spatial normalization, and surface visualization of the imaging data. A 6:08 min pASL scan and a 6:15 min BOLD fMRI scans were performed (‘awake’ pASL and BOLD scans; Figure 1C), in order to assess rCBF and functional connectivity at baseline. The pASL scans were collected using the ‘PICORE-Q2TIPS’ MRI labeling method (TR/TE/TI1/TI2 = 3000/13/700/1700 ms, voxel size = 3.5*3.5*5 mm, number of slices = 16). ‘Tag’ images were acquired by labeling a thick inversion slab (110 mm), proximal to the imaging slices (gap = 21.1 mm). ‘Control’ images were acquired interleaved with the tag images, by applying an off-resonance inversion pulse without any spatial encoding gradient. At the beginning of each pASL scan, an M0 scan (i.e., the longitudinal magnetization of fully relaxed tissue) was acquired for rCBF quantification purposes. The pASL imaging volume common to all subjects scanned covered most of the cerebrum, and extended ventrally to the midbrain and dorsally to the vertex (Figure 1D). BOLD fMRI data were collected using a whole brain T2*-weighted gradient echo BOLD echo planar imaging pulse sequence was used (TR/TE = 3000/35 ms, flip angle = 90°, voxel size = 2.3 × 2.3 × 3.8 mm, number of slices = 35).

26After the awake scans, dexmedetomidine was administered as a 1 mcg/kg loading bolus over 10 min, followed by a 0.7 mcg/kg/hr infusion to maintain unconsciousness. Another pASL scan was initiated at the onset of the dexmedetomidine infusion, with identical imaging parameters as the awake pASL scan, except for a longer duration (20:08 min). This longer acquisition was performed in order to ensure that perfusion data were collected for a long enough period to capture the transition from the awake to the unconscious state. During the infusion period, the study anesthesiologists monitored cuff blood pressure, capnography, electrocardiogram, and pulse-oximetry. Volunteers were instructed to keep their eyes open during the course of the study; loss of consciousness was defined as the onset of sustained eye closure and lack of response to a verbal request to open the eyes. After the onset of unconsciousness, we then performed a 6:15 min ‘unconscious’ BOLD fMRI scan (to evaluate unconsciousness-related changes in functional connectivity). After acquisition of all images, the dexmedetomidine infusion was discontinued, and a 20:08 min pASL scan was performed to assess changes in rCBF upon recovery. Spontaneous eye opening and a positive response to give a thumbs-up signal were used to determine recovery of consciousness. All 10 subjects who experienced spontaneous eye opening during the final 6:08 min of the recovery of consciousness pASL successfully executed on the thumbs-up signal request. An additional 6:15 min ‘recovery’ BOLD fMRI scan was performed, in order to assess variations in functional connectivity upon recovery.

27PET data collected from 10 subjects (two visits each) and stored in list mode format were binned into sinograms. In addition to the static frame corresponding to 40–60 min post FDG administration, dynamic frames of progressively longer duration were generated for quantitative analysis. PET images were reconstructed using an ordered subsets expectation maximization algorithm, with 3 iterations and 21 subsets. Corrections were applied to account for variable detector efficiencies and dead time, photon attenuation and scatter and radioactive decay using the software provided by the manufacturer. We employed a method that is similar to the generation of attenuation maps for computed tomography data to generate our head attenuation maps from the MPRAGE data. This head attenuation map was combined with the hardware (i.e., RF coil, patient table, etc) attenuation map provided by the manufacturer. Spatial smoothing was performed post-reconstruction using a 4 mm full-width-at-half-maximum (FWHM) Gaussian kernel.

28Standardized uptake values (SUV; i.e., mean radioactivity/injected dose/weight) were computed voxelwise from the emission data collected 40–60 min post-injection. In order to quantitatively assess metabolic changes, cerebral metabolic rate of glucose (CMRglc) were computed from the dynamic PET frames with the non-invasive method proposed by, using the whole-brain as our reference region. SUV and CMRglc maps were then coregistered with the high resolution MRI scan using BBREGISTER tool from the FreeSurfer suite (http://surfer.nmr.mgh.harvard.edu/), normalized to the Montreal Neurological Institute (MNI) space using nonlinear registration (FNIRT, from the FSL suite; FMRIB's Software Library, www.fmrib.ox.ac.uk/fsl/) and then smoothed with an 8 mm FWHM kernel. A paired, whole-brain, voxel wise, mixed effects analysis was conducted to compare SUV and CMRglc across visits (n = 10). Statistical parametric maps were thresholded using clusters determined by a voxel-wise threshold (Z > 2.3) and a (corrected) cluster significance threshold of p = 0.05.

29fMRI data were collected from 17 subjects. However, BOLD data (awake, unconscious, and recovery) were successfully collected in only 15 subjects. This is because BOLD data was collected with a different set of parameters in one subject and another subject exited the scanner immediately prior to the recovery BOLD scan. ASL data preprocessing was performed using a combination of analysis packages including FSL, and Freesurfer. The ‘tag’, ‘control’, and M0 scans were first motion-corrected using MCFLIRT. Then, tag and control scans were surround subtracted (i.e., given each tagX, [(controlX−1 + control X+1)/2 − tagX]) to achieve perfusion-weighted images. Quantification of rCBF was performed using the ASLtbx toolbox from the following data: the whole 6 min awake scan, the last 6 min of the 20 min induction of unconsciousness scan (i.e., the portion of the scan during which all subjects were unconscious; ‘unconscious’ scan) and the last 6 min of the 20-min recovery of consciousness scan (i.e., the portion of the scan during which 10 subjects had recovered; ‘recovery’ scan).

30rCBF maps were coregistered with the high resolution MR scan using Freesurfer's BBREGISTER tool, normalized to MNI space using nonlinear registration (FNIRT) and then smoothed with a 7 mm FWHM kernel. A paired, voxel wise, mixed effects analyses were conducted to compare rCBF between the awake and unconscious states data (n = 17). This contrast was computed only within voxels imaged in all subjects (see Figure 2D for coverage common to all subjects), as assessed through the computation of a conjunction mask from all the MNI-registered M0 scans. Given that rCBF estimation in the white matter presents methodological challenges (particularly given the poor signal-to-noise ratio and longer arterial transit time), voxels classified as white matter in (the arbitrary value of) ≥ 70% of subjects by the Harvard–Oxford Subcortical Structural Atlas (Center for Morphometric Analyses, http://www.cma.mgh.harvard.edu/fsl_atlas.html) were excluded from the analyses.

31Subsequently, another paired analysis was performed to compare rCBF between the unconscious and recovery states. In this analysis, only the 10 subjects who were awake for the full duration of the last 6 min of the 20-min recovery scans were included. This analysis was focused within search areas determined by the statistically significant clusters in the ‘unconscious vs awake’ contrast. Unless otherwise specified, all rCBF statistical parametric maps were thresholded using clusters determined by a voxel-wise threshold (Z > 2.3) and a (corrected) cluster significance threshold of p = 0.05.

32Resting-state BOLD fMRI data were used to perform functional connectivity analyses. Datasets were slice-timing corrected (SLICETIMER), motion-corrected (MCFLIRT), brain-extracted (BET), registered to MPRAGE (BBREGISTER) and then to the MNI152 atlas (FNIRT), smoothed with a 5 mm FMWH kernel and high-pass filtered (cutoff = 0.008 Hz). As ASL and PET analyses revealed that unconsciousness was associated with a reduction in glucose metabolism and rCBF in regions belonging to distinct resting-state networks (Default Mode and both Frontoparietal Control networks; DMN, FCN), we assessed how intrinsic functional connectivity of these specific networks was affected by unconsciousness, using the dual regression approach. All the preprocessed datasets were concatenated to create a single 4D dataset. A probabilistic Independent Component Analysis (pICA) was performed using Multivariate Exploratory Linear Optimized Decomposition into Independent Components (MELODIC) on this concatenated 4D dataset, limiting the number of independent components (ICs) to 25, as in previous publications using the dual regression approach. From the pool of the 25 ICs, the DMN, right and left FCNs were clearly identified. Group-level spatial maps for each of these RSNs were used as a set of spatial regressors in a General Linear Model (GLM), in order to identify the individual subjects' time course associated with each group-level map. These time courses were then variance normalized and used as a set of temporal regressors in a GLM, to find subject-specific maps associated with the different group-level independent components. In this GLM, explanatory variables also included six motion parameters and the time courses from ventricles and white matter as covariates of no interest. Subject-specific maps were compared across states (awake, unconscious, recovery) using paired t-tests. As our dual regression ICA approach identified altered DMN functional connectivity to the thalamus, we used this thalamic region as a seed for seed-based functional connectivity analysis. This follow-up analysis was done with the purpose of (1) determining whether specific portions of the DMN (e.g., more posterior regions within the network) may be responsible for driving the effect and (2) assessing changes in functional connectivity between the thalamus and other, non-DMN regions. The extracted fMRI time series, variance normalized, was used as a regressor in a GLM for the awake, unconscious, and recovery data. The same nuisance regressors adopted in the dual regression analyses (see above), with the addition of global signal time course were used in the seed-based analyses. Statistical parametric maps were thresholded using clusters determined by a voxel-wise threshold (Z > 2.3) and a (corrected) cluster significance threshold of p = 0.05. From representative clusters identified in the various analyses, data were extracted and displayed for illustrative purposes. Human brain atlases were used for anatomical reference of the cerebrum and the cerebellum.