If you don't remember your password, you can reset it by entering your email address and clicking the Reset Password button. You will then receive an email that contains a secure link for resetting your password
If the address matches a valid account an email will be sent to __email__ with instructions for resetting your password
Corresponding author: Ben Julian A Palanca, M.D., Ph.D., M.Sc., Departments of Anesthesiology and Psychiatry Washington University School of Medicine in St. Louis, 660 S. Euclid Avenue, St. Louis, MO 63110-1093.
Department of Anesthesiology, Washington University School of Medicine in St. Louis, St. Louis, MO, USADepartment of Psychiatry and Taylor Family Institute for Innovative Psychiatric Research, Washington University School of Medicine in St. Louis, St. Louis, MO, USADepartment of Biomedical Engineering, Washington University in St. Louis, St. Louis, MO, USADivision of Biology and Biomedical Sciences, Washington University School of Medicine in St. Louis, St. Louis, MO, USANeuroimaging Labs Research Center, Washington University School of Medicine in St. Louis, St. Louis, MO, USACenter on Biological Rhythms and Sleep, Washington University School of Medicine in St. Louis, USA
Department of Psychiatry and Taylor Family Institute for Innovative Psychiatric Research, Washington University School of Medicine in St. Louis, St. Louis, MO, USANeuroimaging Labs Research Center, Washington University School of Medicine in St. Louis, St. Louis, MO, USA
Department of Psychiatry and Taylor Family Institute for Innovative Psychiatric Research, Washington University School of Medicine in St. Louis, St. Louis, MO, USANeuroimaging Labs Research Center, Washington University School of Medicine in St. Louis, St. Louis, MO, USA
Nitrous oxide holds promise in the treatment of major depressive disorder. Its psychotropic effects and N-methyl-D-aspartate receptor antagonism have led to comparisons with ketamine. Despite long-standing use, persistent effects of nitrous oxide on the brain have not been characterized.
Sixteen healthy volunteers were recruited in a double-blinded crossover study. In randomized order, individuals underwent a 1-hour inhalation of either 50% Nitrous oxide/Oxygen or Air/Oxygen mixtures. At least two 7.5-minute echo-planar resting-state functional magnetic resonance imaging scans were obtained prior to, and at 2-hours and 24-hours after each inhalation (total 130 minutes/participant). Using time series of preprocessed, motion artifact-scrubbed, and nuisance covariate-regressed imaging data, inter-regional signal correlations were measured and converted to t-scores. Hierarchical clustering and linear mixed effects models were employed.
Nitrous oxide inhalation produced changes in global brain connectivity that persisted in the occipital cortex at 2- and 24-hours post inhalation (p<0.05, FDR-corrected). Analysis of resting-state networks demonstrated robust strengthening of connectivity between regions of the visual network and those of the dorsal attention network, across 2- and 24-hours after inhalation (p<0.05, FDR-corrected). Weaker changes in connectivity were found between visual cortex and regions of the frontoparietal and default mode networks. Parallel analyses following Air/Oxygen inhalation yielded no significant changes in functional connectivity.
Nitrous oxide inhalation in healthy volunteers revealed persistent increases in global connectivity between regions of primary visual cortex and dorsal attention network. These findings suggest that nitrous oxide inhalation induces neurophysiological cortical changes persisting at least 24 hours. ClinicalTrials.gov registration: NCT02994433.
Nitrous oxide is an odorless, colorless, gas that has been routinely employed in clinical practice for over 150 years due to analgesic, amnestic, and hypnotic properties. Its psychomimetic effects and non-competitive antagonism of N-methyl-D-aspartate receptors (NMDARs) have led to comparisons with ketamine (
). Despite widespread and long-standing use, mechanistic understanding of nitrous oxide effects on the brain have not been fully elucidated.
Persistent effects on brain activity (e.g., improved mood) occurring with brief exposure to nitrous oxide are thought to mirror those of ketamine, i.e., they are rapid in onset and sustained for a week or more. Two studies have now demonstrated that antidepressant effects of a single nitrous oxide inhalation can persist for at least 1-2 weeks (
). These persistent effects parallel those observed with ketamine: a sustained antidepressant benefit for days after a single ketamine exposure has been a reproducible finding, with peak effects at 24-hours after administration (
). Persistent antidepressant effects of ketamine are, in part, attributed to synaptic plasticity within networks of brain regions. For ketamine, functional neuroimaging has revealed sustained changes in brain connectivity in depressed individuals (
). In contrast, there have been no nitrous oxide neuroimaging studies to determine whether such changes persist after inhalation. Such investigations may help identify the loci of action of nitrous oxide’s persistent antidepressant effects that extend up to weeks after a single nitrous oxide inhalation (
Here, we conducted a double-blinded cross-over investigation of subacute (2 hours) and persistent (24 hours) effects of nitrous oxide on cortical functional connectivity, using resting-state functional magnetic resonance imaging (rs-fMRI) of healthy, non-depressed volunteers.
METHODS AND MATERIALS
Participants and Study Design
The parent study was registered (ClinicalTrials.gov NCT02994433) and approved by the Washington University School of Medicine Human Subjects Human Research Protection Office. Data from the depressed cohort will not be presented here.
Following eligibility screening, twenty-one healthy participants were recruited throughout the greater St. Louis, Missouri community and through the Volunteer for Health registry at Washington University School of Medicine in St. Louis. All participants fulfilled the following inclusion criteria: age 18-65, right-handed, good command of English, no history of depression as determined by structured clinical interview (The Mini-International Neuropsychiatric Interview [MINI] (
)). Additionally, participants did not meet any of the following exclusion criteria: a primary DSM-IV Axis I diagnosis, as documented in medical records and as determined by structured clinical interview, known primary neurological disorders or medical disorders impacting brain imaging including dementia, stroke, encephalopathy, Parkinson’s Disease, brain tumors, multiple sclerosis, seizure disorder, severe cardiac or pulmonary disease; central nervous system active medications as determined by study investigator; known disease affecting drug metabolism and excretion (e.g. renal or liver disease) as determined by study investigator; ineligibility for MRI scans (e.g. history of claustrophobia or MRI-incompatible metal implanted); current use of psychotropic medications, antidepressants, or prescription or non-prescription drugs/herbals intended to treat depression or anxiety; recent (within past 12 months) history of substance dependence or abuse, determined by reported history or urine drug screen, or ability to become pregnant and not using effective contraception. In addition, participants could not have contraindications against nitrous oxide or study procedures: pneumothorax, bowel obstruction, middle ear occlusion, elevated intracranial pressure, chronic cobalamin and/or folate deficiency treated with folic acid or vitamin B12, pregnancy or breastfeeding, inability to provide informed consent, or any other factor that in the investigators’ judgment may affect patient safety or compliance (e.g., distance greater than 100 miles from clinic). Written informed consent was obtained from all study participants prior to undergoing any trial activities. Participants received remuneration for time and effort.
The study design was a prospective, placebo-controlled, double-blind crossover study with five study visits (Figure 1). Inhalation order was randomized for the two arms (intervention versus placebo), using a random number generator (block size of 2). The sessions were 4-6 weeks apart to minimize carryover effects between conditions previously observed in nitrous oxide inhalation studies (
). Six individuals withdrew after the first inhalation or post-inhalation imaging session (CONSORT flow diagram in Figure S1. Recruitment from October 2015 to February 2020 was halted during the COVID-19 pandemic; imaging and inhalation facilities were closed to investigation for an extended period. Despite completing all inhalation and imaging sessions, one individual was excluded due to use of a different scanner system and imaging protocol. The final sample size (N=14) had 7 females, with a median age of 44.5 years. When self-identified by race and ethnicity, 2 (14%) were Asian, 5 (36%) were Black, 7 (50%) were White, while 1 of 14 (7%) was Hispanic. No participants had active or recent tobacco use. Participant racial and ethnic demographics are included in Table S1. Table S2 lists concurrent medications, medical comorbidities, and adverse events encountered during inhalation sessions.
For the intervention condition, participants inhaled 50% Nitrous oxide (balance Air/Oxygen) for 1 hour. We utilized a dose of nitrous oxide that conferred an antidepressant response in a previous trial, 50% inhalation for 1 hour (
). In the placebo condition, subjects inhaled Air/Oxygen mixture for 1 hour. Except for the composition of the gas mixtures, inhalations were otherwise identical. A face mask and strap were used to generate a semi-closed system connected to an anesthesia machine or an FDA-approved Porter/Praxair MXR breathing circuit. Total gas flow was kept at 2-8 L/min with nitrous oxide concentrations increased gradually over the first 10 minutes of the inhalation. Participants were monitored according to American Society of Anesthesiologists standards, including an available attending anesthesiologist, 3-lead electrocardiography, pulse oximetry, end-tidal carbon dioxide, and non-invasive blood pressure. Following inhalation, subjects underwent a one-hour observation period for monitoring vital signs and potential adverse events.
Extent of blinding was dependent on the roles in the study. The anesthesia team conducted the inhalation per treatment arm assignment. Concealment was undertaken by shielding the study flow meters from the study participant and recusal of assessor during the inhalation. Thus, blinding to the inhalation conditions was maintained for participants, the psychiatry team performing the ratings, and the fMRI data analyst. To evaluate blinding, participants were assessed with a 5-point Likert scale where they were asked to rate the extent to which they knew they inhaled nitrous oxide (strongly believe nitrous oxide, somewhat believe nitrous oxide, somewhat believe placebo, strongly believe placebo, and don’t know).
Resting-state Functional Magnetic Resonance Imaging (rs-fMRI) Data Acquisition
Images were acquired using a Siemens 3T Prisma scanner with a 64-channel head coil. At each imaging time point (Figure 1), at least two 7.5-minute resting-state functional magnetic resonance imaging scans were acquired pre-inhalation (baseline), 2-hours post-inhalation, and approximately 24-hours post-inhalation (range 22-28 hours afterwards), for a total of 6 scanning sessions. We acquired imaging at 2- and 24-hours following inhalation as antidepressant response were observed at these times in a previous trial (
). Blood-oxygen-level-dependent (BOLD) echo-planar imaging was acquired (2.4mm3 voxels, TE=30 ms, TR=0.8 s, 552 volumes). During all resting-state scans, participants were instructed to keep eyes open while focusing on a fixation cross. They were told that they could relax but to remain awake. Compliance to this instruction was not monitored. Framewise Integrated Real-time MRI Monitoring (FIRMM software) was employed to assist in evaluating head motion during data acquisition. Four foam pads were utilized to minimize head movement during imaging. Whole-head T1-weighted structural images (1 mm3 voxels) were also acquired during the pre-inhalation session.
Image Analysis Preprocessing
Preprocessing and subsequent analyses were performed using the Conn toolbox (
). rs-fMRI inter-regional correlations were calculated from BOLD image time series that had been slice time corrected, realigned, co-registered with the high resolution T1-weighted volume, spatially normalized, and spatially smoothed with an 8 mm kernel. The beginning and end of the time-series was temporally smoothed with a digital Hanning filter to reduce potential edge effects (
). This preprocessing incorporated a quality assurance protocol that excluded outlier time points >3 standard deviations from the mean global signal intensity or exhibiting >0.3 mm scan-to-scan head motion. Imaging quality statistics are included in Table S3. Participants had a median imaging time of 131 minutes (9827 imaging volumes) across the 6 imaging sessions. Physiological sources of noise, estimated from gray and white matter segments using anatomical CompCor (
), were treated as confounds. Time series characterizing estimated subject motion, 6 parameters representing their first-order temporal derivatives, scan-to-scan motion outliers, and the BOLD-contrast signal within the subject-specific WM and CSF masks were used as nuisance variables. The resulting residual time series were band-pass filtered from 0.008-0.20 Hz. Inter-regional correlations were then computed and converted to t-scores.
Voxel-to-voxel (LCOR and GCOR) and inter-regional (32 regions comprising the canonical networks, Table S4) correlations were computed to identify areas showing changes in functional connectivity after nitrous oxide inhalation.
Local connectivity strength was estimated using local correlation (LCOR) metric (
). LCOR was computed as the average of correlation coefficients between each individual voxel and its neighboring voxels. LCOR maps demonstrate the estimated average correlation strength and sign between a voxel and the neighboring areas in the brain.
Global connectivity strength was estimated using the global correlation (GCOR) metric. For a single voxel in space, GCOR is the average of correlation coefficients between that voxel and all voxels across the brain (
). GCOR provides a measure of node centrality, operationalized as the strength and sign of connectivity between a given voxel and the rest of the brain.
Region-based functional connectivity was subsequently used to characterize the spatial pattern of connectivity changes associated with resting-state networks. These estimates involved computing inter-regional correlations among the regions (Table S4) that were then converted to t-scores. A hierarchical clustering approach was then used to identify clusters with overall changes across conditions. To mitigate bias introduced by serial testing, false discovery rate (FDR) corrections for multiple tests were employed (
We estimated the effects of nitrous oxide on neural activity at a group level, leveraging maps of GCOR and LCOR obtained from first-level modeling. A repeated measures mixed-effects model was implemented, with fixed effects of replication, session, condition, and subject as a random effect. The effect of replication accounted for any differences in effects among the three scans that comprised each session; the model included coding of session (-1, 0, +1). The effect of session examined linear effects among the imaging days that comprised the experiment. The linear effect of condition allowed the contrast of pre-nitrous oxide to post-nitrous oxide data in the different sessions.
The principal contrasts compared the pre-nitrous oxide condition to the post-nitrous oxide condition at both two hours and 24 hours post-inhalation. Parallel analyses utilized this approach for the pre-Air/Oxygen inhalation and post-Air/Oxygen inhalation imaging data acquired at similar time points.
We assessed the effectiveness of masking our treatment conditions across the 28 inhalation sessions; Subjects were asked if they had received nitrous oxide or placebo. Participants correctly guessed the condition in 21/28 sessions (75%), but assumed incorrectly in 5/28 (18%), or were unable to ascertain after two of the inhalations (7%).
Global connectivity following nitrous oxide inhalation in visual cortical regions
Using a voxel-based GCOR analyses, voxels clustered within the occipital cortex demonstrated stronger correlation across the brain following nitrous oxide inhalation (Figure 2A-B). No voxels displayed a reduction in correlation (data not shown). These changes in the calcarine sulcus were present at 2-hours and persisted at 24-hours post-inhalation (p < 0.05 for linear relationship across time, Figure 2C). A parallel pre-post analysis of the Air/Oxygen mixture inhalations revealed no effects at the same multiple-test corrected critical threshold (data not shown). Thus, across the brain, visual cortex voxels show the greatest change in correlated brain activity with persistence at 24-hours following nitrous oxide inhalation but not placebo.
Local connectivity following nitrous oxide inhalation
Using a voxel-based LCOR analyses, no suprathreshold voxels were detected following nitrous oxide inhalation (voxel threshold p<0.001 (uncorrected); cluster threshold p<0.05 family-wise error rate-corrected). A parallel analysis of the Air/Oxygen mixture revealed no effects at the same multiple-test corrected critical threshold (data not shown).
Functional connectivity among canonical networks following nitrous oxide inhalation
Further analyses were undertaken to characterize changes in connectivity across canonical resting-state networks represented by 32 cortical regions (Table S4). As a quality control, pre-inhalation imaging verified established connectivity patterns across resting-state networks (Figure S2). Employing these same 32 cortical regions, pre-post-nitrous oxide inhalation connectivity analysis demonstrated that regions of the visual network and dorsal attention network showed stronger correlated activity at both 2- and 24-hours (Figure 3), which withstood FDR correction for multiple comparisons during network-based statistical analysis. Linear increases in functional connectivity were noted among visual cortical regions, as well as between the visual network and the dorsal attention network regions in the intraparietal sulcus and frontal eye fields. Less pronounced connectivity changes were noted between the visual cortical regions and lateral prefrontal cortical regions of the frontoparietal network, the posterior cingulate cortex and left lateral parietal region of the default mode network. Small connectivity changes were also observed between visual cortex and regions of the sensorimotor network, regions in the language network, and posterior regions of the cerebellum. Lastly, weaker correlations were noted between the medial visual cortical region and the posterior parietal cortex of the frontoparietal network. A correlation matrix of inter-regional changes across all 32 cortical regions is provided in Figure S3. A parallel analysis of the Air/Oxygen mixture (placebo condition) data revealed no effects at the same multiple-test corrected critical threshold (data not shown). Thus, following nitrous oxide, but not placebo inhalations, persistent changes in network connectivity were identified linking primary and higher-order visual processing regions visual attention areas and secondarily to motor and language networks.
Here, we demonstrate, in a group of non-depressed normal controls, sustained brain network changes following nitrous oxide inhalation. In a data-driven approach, we observed increased global connectivity within the bilateral calcarine sulci of the occipital lobe. In an independent analysis, a similar cluster of visual regions demonstrated a linear increase in connectivity from 2 to 24 hours post-inhalation, with the strongest increases in connectivity to dorsal attention network regions. Weaker changes in connectivity were noted between visual cortex and regions of the frontoparietal and default mode networks. In contrast, parallel analyses for imaging data obtained from the same individuals after inhalation of Air/Oxygen mixture did not recapitulate these findings. To our knowledge, this is the first evidence demonstrating sustained alterations in network connectivity following nitrous oxide inhalation, i.e., the effects persisted for at least 24 hours after inhalation of nitrous oxide.
Our findings are consistent with the limited previous reports of persistence of either brain imaging or behavioral changes following nitrous oxide inhalation. A positron emission tomography cross-over study of 10 individuals evaluated changes in global and regional cerebral metabolic rate (rCMR) during and after inhalation of 50% nitrous oxide or 30% oxygen (
). While no changes in global CMR were observed during nitrous oxide inhalation, rCMR was augmented by 14% in the basal ganglia and 22% in the thalamus. Persistent augmentation in cortical CMR was observed in a subset of participants who were imaged 1-hour after crossing-over from a previous inhalation of 50% nitrous oxide. More specifically, sustained effects in frontal, temporal, parietal, and occipital cortical regions contrasted to an absence of change in the cerebellum, thalamus, or basal ganglia. Analogous observations of nitrous oxide effects in the electroencephalogram (EEG) have been reported with increases in frontal theta (
) EEG power above baseline, 15-minutes following cessation of nitrous oxide inhalation. As these studies only measured for 15 minutes after nitrous oxide inhalation, it is unknown whether these EEG changes persist for 2- or 24-hours to mirror our imaging findings. From a behavioral standpoint, the persistence of augmented cortical CMR is consistent with a small (N = 12) randomized double-blinded healthy volunteer study comparing sleepiness following 30-minute inhalations of 40% nitrous oxide, 20% nitrous oxide, or 100% oxygen (
). After inhalation of 40% nitrous oxide, participants were less sleepy at 1-hour by the multiple sleep latency test compared to those who inhaled 0% or 20% nitrous oxide. In contrast, there were no changes in performance observed on five psychomotor tests following all three inhalation conditions. A rapid recovery in cognitive task performance following inhalation was also observed in another study evaluating recall, reaction time, and attention (
). Thus, our data advance spatial and temporal characterization of neural changes that persist beyond the pharmacokinetic elimination of nitrous oxide.
Given the similarities in purported mechanisms of action for nitrous oxide and ketamine (for example NMDAR antagonism), these nitrous oxide data warrant comparison to sustained brain effects observed following ketamine exposure. Several findings in this study are consistent with previous data demonstrating enhanced synaptic plasticity in visual cortical areas following administration of ketamine. Sumner et al. (2020) analyzed visual evoked EEG responses in TRD patients 3-4 hours after intravenous subanesthestic ketamine or placebo (remifentanil) (
), evaluating changes in evoked potentials as a marker of synaptic change. Components of the visual evoked response were enhanced by periodic visual stimulation, with persisting enhancement only after ketamine. This study suggested significant modulation of EEG potentials at bilateral inferior temporal cortex and superior parietal cortex evoked by bilateral middle occipital gyrus sources over a time course consistent with our present neuroimaging results. These changes in processing deviant/novel stimuli may also extend to auditory stimuli (
). Parallels between nitrous oxide and ketamine are limited, however, as NMDAR antagonism is only one molecular mechanism of action for these two agents that have many other targets (e.g., GABA, nicotinic acetylcholine, and muscarinic acetylcholine receptors) (
We cautiously speculate on how brain connectivity alterations following nitrous oxide inhalation in non-depressed individuals may identify potential loci of action that would underlie antidepressant response. Aberrant functional connectivity within and between resting-state networks has been associated with depression (
), with hyperconnectivity within and between the frontoparietal and default mode networks and hypoconnectivity between frontoparietal and dorsal attention networks. Strengthening of connectivity to nodes with aberrantly strong interconnectivity could conceivably disrupt the latter. The lack of any modulation of connectivity to the salience network would seem to suggest that action at this network is less likely to be observed in imaging investigations of depressed individuals treated with nitrous oxide. These lines of conjecture warrant evaluation in future studies to also address whether these persistent change in brain connectivity emerge after 25% inhalation of nitrous oxide in the same manner as antidepressant effects (
). This precludes subgroup analyses on sex or other factors. However, this study did include up to 30 minutes of BOLD resting-state scanning time for each scan session, considerably longer than most trials. In addition, the lack of an active placebo must be acknowledged (
). As we did not image individuals during inhalation, we are not able to ascertain whether functional connectivity changes emerged during inhalation or post-inhalation (rebound). Finally, this study was focused on resting-state, i.e., there is a lack of behavioral tasks to correlate with these imaging findings. These weaknesses are offset by the crossover design of the study, with individuals serving as their own controls.
These hypothesis-generating data predict that there may be emergence of functional changes in visual attention and processing in healthy participants. It is not clear yet whether these connectivity changes are related to antidepressant effects or perhaps represent another behavioral change, e.g., increased alertness/reduced sleepiness as previously described (
). Studies are underway to determine whether the observed changes in brain connectivity also manifest in those with clinical depression/TRD and whether there are associated changes in depressive symptom severity. Though preliminary, these findings suggest a rapid and persistent effect on intracortical connectivity could potentially contribute to antidepressant effects, i.e., the depressed patient by undergoing alteration in brain activity in the visual and attention regions may “see” and interpret the world differently. In parallel, these data may point to alternative ways to consider depression circuitry that may be relevant to psychiatric therapeutics.
This work was supported by National Institute of Mental Health R21MH108901 (CRC, PN), P50MH122379 (CFZ), Taylor Family Institute for Innovative Psychiatry Research (CRC and CFZ, 1U01MH128483 (BJAP), Washington University Center for Perioperative Mental Health grant number P50 MH122351 (BJAP), and University of Chicago (PN).
CRC receives research funding through Washington University from LivaNova, PLC and is the Lead Investigator of the RECOVER Trial. He has also received consultation fees from Sage Therapeutics. Sage and LivaNova were not involved in this work. CFZ serves on the Scientific Advisory Board of Sage Therapeutics and has equity in the company. PN is currently receiving funding from NIMH, American Foundation for Prevention of Suicide, and Brain Behavior Foundation; has received research funding and honoraria from Roche Diagnostics and Abbott Diagnostics; and has previously filed for intellectual property protection related to the use of nitrous oxide in major depression (“Compositions and methods for treating depressive disorders”; US20170071975A1). All other authors report no biomedical financial interests or potential conflicts of interest.
We appreciate the efforts of Frank Brown, Jacob Bolzenius, and Linda Barnes in assisting on the execution of the study. We thank the following colleagues for administering our inhalation sessions: Branden Yee, Michael Montana, Andreas Kokoefer, Jaime Brown-Shpigel, Christian Guay, Tracy Lanes, Sara Pitchford, and Marlette Williams. Preliminary presentation of these data was submitted for the 2022 American Society of Anesthesiologists Meeting and the 2023 Society of Biological Psychiatry Meeting.
PN, CRC, and CFZ obtained funding. BJAP had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. PN, CRC, and CFZ were responsible for study concept and design. All authors were responsible for acquisition, analysis, and interpretation of data. BJAP, CRC, TZ, CFZ, and PN drafted the original manuscript. TZ performed statistical analysis. All authors critically revised the manuscript for important intellectual content.