This review (2021) explores the possibilities of using Nitrous Oxide (NO2) for treating treatment-resistant depression (TRD). The use of NO2 as a psychiatric intervention is discussed along with its possible mechanism of action. Its antidepressant effects are believed to be mediated through the NMDA receptor.
Stemming from the results of the historic STAR-D trial, it is evident that a significant subset of individuals (20-25%) with major depressive disorder (MDD) do not respond to conventional antidepressant medications. As a result, an emphasis has been placed on the development of novel therapeutics for MDD over the last two decades. Recently, substantial research efforts have been focused on the use of ketamine as an antidepressant whose mechanism of action is via the N-methyl-d-aspartate (NMDA) receptor. Another potential therapeutic compound of interest is nitrous oxide, which has been utilized for more than a century in multiple fields of medicine for its analgesic and anesthetic properties. Recent clinical studies suggest that nitrous oxide may be effective for treatment-resistant depression. In this review, we will discuss the administration of nitrous oxide as a psychiatric intervention, current use in psychiatry, putative mechanisms of action, and future directions highlighting knowledge gaps and other potential utilities in the field of psychiatry.
Over several decades antidepressant development focused on monoamine systems, yet an estimated 20-25% of people with major depressive disorder (MDD) do not respond to standard pharmacotherapies and are commonly described as having treatment-resistant depression (TRD). Interest in novel mechanisms has therefore intensified, with NMDA receptor antagonists such as ketamine and its enantiomer esketamine showing rapid antidepressant effects and prompting investigation of other NMDA-targeting agents. Nitrous oxide (N2O), a long‑used inhalational anaesthetic and analgesic, has emerged as a candidate because preclinical and early clinical evidence suggest it may have antidepressant properties. Quach and colleagues set out to review the emerging literature on nitrous oxide as a treatment for depression. The review aims to describe how N2O is administered in psychiatric settings, summarise clinical evidence for its antidepressant efficacy and tolerability, outline proposed mechanisms of action, and identify gaps and priorities for future research and clinical development.
Papers cited by this study that are also in Blossom
Garcia-Romeu, A., Kersgaard, B., Addy, P. H. · Experimental and Clinical Psychopharmacology (2016)
Nagele, P., Duma, A., Kopec, M. et al. · Biological Psychiatry (2015)
Guimarães, M. C., Guimarães, T. M., Hallak, J. E. et al. · brazilian Journal of Psychiatry (2021)
Zanos, P., Moaddel, P. J., Morris, P. J. et al. · Nature (2016)
The review synthesises clinical and preclinical findings rather than reporting new primary data. Key clinical trials summarised include a pilot study, a Phase II dose-ranging trial, and a parallel-group randomised trial. Nagele and colleagues conducted an early double-blind, placebo-controlled crossover pilot in 20 subjects with severe TRD (defined as failure of at least two adequate current trials and at least three lifetime trials, with an average of eight prior failed antidepressant trials). One‑hour inhalations of 50% N2O in oxygen were compared with placebo (50% oxygen/50% room air). Treatment produced a statistically significant reduction on the Hamilton Depression Rating Scale, 21 items (HDRS-21). The overall response rate was 20% and three participants achieved remission. Reductions in depressive symptoms were apparent at 2 hours and 24 hours post‑treatment. Nausea and vomiting were the most common adverse events; no psychosis or vitamin B12 changes were reported. In a Phase II, prospective, randomised, double‑blind crossover trial, the antidepressant effects of 50% and 25% N2O (one‑hour inhalations) were compared with placebo in 24 TRD subjects (median of 4.5 prior adequate antidepressant trials). The primary outcome was HDRS-21 measured up to two weeks post‑inhalation. Both 25% and 50% concentrations produced statistically significant reductions in HDRS-21 versus placebo, with week‑2 differences versus placebo of −5.19 points (P=0.02) for 25% N2O and −7.00 points (P=0.001) for 50% N2O. No significant HDRS-21 difference was observed between the two active doses, but adverse effects were significantly lower with 25% N2O (P<0.0001). Antidepressant effects were sustained up to two weeks in the group average, and some participants maintained benefit longer: 8 of 20 remained in remission at three months in the follow‑up described. Guimaraes and colleagues tested adjunctive N2O in a randomised, placebo‑controlled, double‑blind, parallel trial in 23 people with MDD (not selected for treatment resistance). Participants received 50% N2O (60 minutes) or placebo (100% O2) twice weekly for four weeks; 21 subjects were included in the final analysis and the primary measure was the HDRS‑17. The N2O group showed a statistically significant reduction in depressive symptoms versus placebo. After four weeks, remission and response rates in the N2O arm were 75% and 91.7% respectively, compared with 11.1% remission and 44.4% response in the placebo arm. The intervention was generally tolerable; five of 96 N2O sessions were discontinued early. Common adverse events across studies included somnolence, paresthesia, nausea, headache and transient dizziness. Safety and pharmacological profile summaries note that N2O is commonly administered in oxygen mixtures up to inspiratory concentrations of 70% and has a low blood–gas solubility producing rapid onset and recovery. In a large non‑psychiatric survey of 35,828 hospital patients receiving N2O, side effects occurred in 4.4% and were typically mild and brief. Chronic or heavy exposure is associated with inactivation of vitamin B12 and risk of subacute combined degeneration of the spinal cord. Preclinical mechanistic findings reported include electrophysiology and behavioural studies supporting non‑competitive NMDA receptor antagonism by N2O, with evidence that N2O inhibits NMDA agonist‑induced currents and that NMDA receptors are required for some behavioural effects in invertebrate and murine models. N2O weakly inhibits AMPA/kainate receptors and modulates other targets including opioid receptors, low‑voltage‑activated calcium channels, GABAA/C receptors, glycine receptors and 5‑HT3 receptors. Human neurophysiological data are limited but include electroencephalography findings of decreased functional connectivity in superficial parietal networks and frontal slowing in delta frequencies during N2O exposure.
The authors interpret the assembled evidence as indicating that nitrous oxide is a promising candidate for treating MDD and TRD, with rapid onset of antidepressant effects demonstrated across small controlled trials and an apparently favourable tolerability and safety profile in short‑term use. Phase II data provided preliminary information on dose, showing comparable efficacy between 25% and 50% N2O but fewer adverse effects at the lower concentration, and suggest that therapeutic effects can persist for days to weeks in some participants. Quach and colleagues position these findings alongside the ketamine literature: both agents are non‑competitive NMDA antagonists used historically as anaesthetics and more recently explored as rapid‑acting antidepressants, but they differ at the molecular level (ketamine acts as an open‑channel “trapping” blocker whereas N2O is a partial, weakly voltage‑dependent inhibitor). Clinically, N2O may offer practical advantages such as rapid recovery and lower regulatory constraints, and no research reports to date of N2O‑induced psychosis have been cited. Key limitations acknowledged include the small size and heterogeneity of the clinical trials to date, limited duration of follow‑up, and an incomplete understanding of mechanism. The authors note that human mechanistic data are sparse and preclinical findings do not yet clarify which receptor systems or downstream pathways mediate antidepressant effects. Safety concerns that require continued attention include the known risks of chronic exposure (vitamin B12 inactivation) and the need to monitor for adverse events in larger samples. For future research and clinical practice the authors advocate replication in larger, multi‑centre trials to define optimal dosing, frequency and maintenance schedules and to compare N2O directly with established rapid‑acting treatments such as ketamine. They also recommend mechanistic studies in humans and animals, exploration of potential indications beyond unipolar depression (for example post‑traumatic stress disorder, bipolar disorder), and assessment of effects on suicidality. If larger trials confirm efficacy and safety, the authors envisage possible integration of N2O delivery into specialised clinical settings or supervised outpatient practice.
aberrant functioning of the "biogenic amines" that include serotonin, dopamine, and norepinephrine. Over the subsequent four decades, pharmacotherapies remained focused primarily on these systems. These medications appear to be effective for most individuals with Major Depressive Disorder (MDD); however, a significant subset (estimated 20-25%) of patients do not respond to these standard pharmacotherapies and can be referred to as having "treatmentresistant depression" (TRD). Though the field has yet to clearly define TRD, evidence does support that patients who fail to respond to two different antidepressant medication trials, with adequate dose and duration (6 to 8 weeks), are less likely to respond to subsequent treatmentsand some consensus has emerged that failure to respond to two successive treatments defines TRD. Over the last two decades, a concerted effort has been placed on the investigation of novel agents/interventions to treat TRD. These include NMDA antagonists, neurosteroids, neurostimulation treatments, and psychedelics, among others. The NMDA receptor antagonists have shown tremendous promise in successfully treating TRD. Early work in ketamine has served as the foundation for exploring other NMDA antagonists as novel antidepressants. In fact, esketamine, which is an enantiomer of ketamine, recently gained FDA approval in 2019 for treatment-resistant depression. Another potential therapeutic that is actively under investigation is nitrous oxide.
Nitrous oxide (N 2 O), commonly referred to as laughing gas, is a volatile, colorless gas. It is predominantly used as an anesthetic and analgesic in both hospital and office-based settings, e.g., for dental procedures. N 2 O is delivered in an oxygen mixture due to its elevated minimal alveolar concentration and it is commonly administered using a specialized delivery system designed to prevent hypoxia. Mixtures up to inspiratory concentrations of 70% N 2 O are considered safe. Another typical safety feature of delivery systems includes scavenging systems to remove excess and residual N 2 O in the environment as well as preventing chronic exposure to treating personnel. N 2 O has many potential advantageous properties including: a low side effect profile, rapid onset/recovery due to low blood gas solubility, limited drug interactions, and simple metabolism not influenced by other medications as uptake and elimination is solely pulmonary making it a viable option for individuals with hepatic or renal disease. In a large survey of 35,828 patients receiving N 2 O in the hospital, side effects were observed in 4.4% of cases. Typically, adverse effects such as mild sedation, mild euphoria, nausea, vomiting, headaches, and dizziness account for a majority of the cases. Most, if not all, of the side effects are noted to be brief in duration and resolve with discontinuation of use. Long term exposure or chronic abuse of N 2 O can result in vitamin B12 deficiency and subacute combined degeneration of the spinal cord due to its inactivation of the vitamin.
J o u r n a l P r e -p r o o f In the setting of the recent success of ketamine as a novel antidepressant, work has continued to look at the potential of other NMDA receptor antagonists as targets for MDD treatment. Given that nitrous oxide is thought to work predominantly as an NMDA receptor antagonist (discussed below), Nagele et al., conducted a pilot study to specifically target N 2 O's antidepressant potential. In that study, a double-blind, placebo-controlled crossover design was utilized, in which one-hour inhalation treatments of N 2 O (50-50% oxygen mixture) vs placebo (50% oxygen 50% room air) were provided to 20 TRD subjects. Treatment resistance in this study was defined as failing at least 2 medication trials in the current depressive episode and at least 3 lifetime trials of adequate dose (based on medication package insert) and duration (8 weeks); although it should be noted, that these subjects were severely treatment-resistant with an average of 8 prior failed antidepressant trials. Confounding diagnoses such as substance use disorders, severe personality disorder, psychotic illnesses, and bipolar disorder were excluded. Treatment with N 2 O resulted in significant improvement in depression scale scores compared to placebo as defined by the primary outcome measure, the Hamilton Depression Rating Scale-21 items (HDRS-21). An overall response rate of 20% was observed, and 3 individuals achieved complete remission. In addition, a decrease in depression scores was statistically significant at 2 hours and 24 hours post-treatment, suggesting rapid onset effects. The most prevalent side effects observed included nausea and vomiting. None of the subjects experienced psychosis or alterations to vitamin B12. These initial intriguing findings from the pilot study prompted several questions, including what is the optimal antidepressant dose for N 2 O and are the effects of N 2 O sustained? To begin to answer these questions, further work by Nagele et al. investigated the use of different nitrous oxide dosages. In this phase 2, prospective, randomized, double-blind, crossover trial, antidepressant effects of two different concentrations of N 2 O were compared to placebo in a group of 24 TRD subjects. Participants had failed a median of 4.5 antidepressant treatment trials with adequate dose and duration. Treatment arms included one-hour inhalations of 50% N 2 O, 25% N 2 O vs placebo with study time measurements extending to 2 weeks post inhalations. The primary outcome measure was the HDRS-21. Overall results for this study showed statistically significant reduction in depressive symptoms for both 25% N 2 O and 50% N 2 O compared to placebo. No significant differences in HDRS-21 scores were seen between the two N 2 O concentrations; however, both dosages showed sustained effects as reflected in decreased HDRS-21 scores over the duration of the study (-5.19 points [P=0.02] and -7.00 points [P=0.001] at week 2 for 25% N 2 O and 50% N 2 O respectively compared to placebo). Of note, adverse effects declined significantly with the lower N 2 O dose (P < 0.0001). In summary, key findings from this study included: 1) there was similar antidepressant efficacy across the two N 2 O dosages; 2) both doses of N 2 O were well-tolerated, but the lower dose (25%) was associated with significantly fewer side effects; and 3) antidepressant effects were sustained up to two weeks. Additionally, there was suggestion that some subjects continued to receive benefit beyond two weeks: 8 out of 20 remained in remission following the 3-month study period. The use of N 2 O as an adjunctive therapy in subjects with MDD was recently investigated by Guimaraes et al. In this randomized, placebo-controlled, double-blind, parallel clinical trial, 23 subjects with MDD received either 50% N 2 O or placebo (100% O 2 ) for 60 minutes twice a week over 4 weeks. At the completion of the study, 21 subjects were included in the final analyses and the primary outcome measure was the Hamilton Depression Rating Scale -17-item (HDRS-J o u r n a l P r e -p r o o f 17). There was a statistically significant reduction in depressive symptoms in the N 2 O group as compared to placebo. After 4 weeks, the remission and response rates for subjects in the N 2 O group were 75% and 91.7%, respectively. In the placebo group, 11.1% and 44.4% were observed to be in remission and response, respectively. The treatment was relatively well-tolerated as 5 out of a total 96 sessions were discontinued prior to the end in the N 2 O group. The most common adverse effects reported were somnolence, paresthesia, nausea, and headache. Of note, this study, in contrast toand 2021, did not attempt to enroll depressed patients with treatment-resistance. Nonetheless, this double-blinded, placebo-controlled study also supports the antidepressant benefits of N 2 O.
While there is a long history of safe use as an anesthetic and analgesic, the utility of N 2 O as an antidepressant is relatively novel and its antidepressant mechanism of action remains largely unknown. Stemming from preclinical tissue culture and rodent models, putative mechanisms have been proposed to account for its activity at the molecular level. Using a female rat model, Jevtovic-Todorovic et al. demonstrated that excitotoxicity elicited by NMDA agonists, N-methyl-D,L aspartic acid, and MK0801, could be abrogated with exogenous nitrous oxide. By taking direct electrophysiologic measurements from cultured hippocampal neurons, they demonstrated that the addition of N 2 O inhibited electrical current triggered by NMDA agonism. Dose-response studies performed indicated that saturating concentrations of NMDA receptors inhibited by N 2 O were not able to overcome this inhibition. Furthermore, subsequent studies from independent groups showed that the NMDA receptor is required for the behavioral effects of N 2 O; these effects were observed both in an invertebrate Caenorhabditis elegans and a murine model. Taken together, these studies support that some of the effects observed with N 2 O occur in conjunction with NMDA antagonism. Further, subsequent studies supported that N 2 O acts as non-competitive NMDA antagonist. Similar to the NMDA receptor, alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA)/kainite-type receptors are another type of glutamate receptor that are weakly inhibited by N 2 O. In preclinical tissue culture and animal models, AMPA receptor activation in tissue culture led to increased brain derived neurotrophic factor (BDNF) and neurogenesiswhile AMPA receptor antagonism attenuated the antidepressant effects of ketamine in mice. In human studies investigating the role of AMPA receptors in depression, treatment with ketamine led to increased glutaminergic signaling in regions with elevated levels of AMPA receptors. Further studies are needed as thus far, the few studies investigating the role of AMPA receptor activity in depression have involved the use of ketamine. It is unknown whether N 2 O has similar effects in terms of mediating possible antidepressant effects through the AMPA receptor. In addition to the NMDA receptor, N 2 O has also been shown to target other receptor-mediated pathways. For example, a substantial amount of research has been devoted to understanding the role that opioid receptors play in mediating the analgesic effects of N 2 O. Given that kappa opioid receptors have been shown to be important for antidepressant effects mediated by buprenorphine and the antidepressant effects of ketamine can be attenuated by naltrexone (an J o u r n a l P r e -p r o o f opioid antagonist), it is reasonable to hypothesize that N 2 O's antidepressant effects may also be mediated via opioid receptor agonism. Aside from receptor-mediated signaling pathways, nitrous oxide has been shown to modulate various ion channels including low voltage activated calcium channels, gamma-aminobutyric acid receptors A and C, glycine receptors, and 5-hydroxytryptamine 3 receptors. Like other anesthetics, it also changes membrane fluidity. It is unclear, though, if any of these ion channels modulated by N2O is relevant to its antidepressant activity. Human studies investigating the mechanism of action of N 2 O are limited. To date, studies utilizing electroencephalography have associated N 2 O treatment with decreases in functional connectivity in the superficial parietal network and a slowing in frontal slow (delta) wave activity. Whether these signaling pathways or changes in brain activity are related to the antidepressant effects of nitrous oxide remains unknown, but they do provide compelling targets to explore in future studies.
There are certainly parallels that can be drawn between N 2 O and ketamine since they are both non-competitive NMDA receptor antagonists used as analgesic and anesthetic agents, and more recently, as antidepressant agents. However, mechanistically, there are differences at the molecular level. Typically, the binding of glutamate to NMDA receptors, in conjunction with membrane depolarization, promotes opening or activation of the transmembrane ion channel leading to an influx and efflux of cations including Na+, K+, and Ca2+. Ketamine specifically enters this open channel and binds to a site deep within the ion channel pore to occlude ion flow in a phenomenon that is referred to as a "trapping ion channel block". In stark contrast to this, N 2 O effects are weakly voltage-dependent and do not appear to exhibit features consistent with an open channel blocker according to electrophysiology measurements. This is depicted in Figure. Additionally, there have also been some notable differences observed in the clinical trials performed so far. For example, access to ketamine is more tightly regulated due to its abuse potential compared to N 2 O. From a safety perspective, there have been no research reports of psychosis with N 2 O. Additionally, N 2 O has not been associated with hypertension, and the effects of N 2 O wear off quickly, making it possible to return back to normal everyday functioning within a short time of use, vis-à-vis ketamine, which requires more extensive monitoring following administration (2 hours post-esketamine delivery).
Recent studies demonstrating the potential utility of N 2 O as a novel therapeutic for depression have been extremely promising (Table). Additional insight regarding dose, tolerability, and duration of response was gained in a recent Phase 2 trial. However, further studies are needed to determine whether these results can be replicated and to further clarify treatment J o u r n a l P r e -p r o o f guidelines (e.g., optimal dosage, dosing schedule, "maintenance" schedule, etc.). Ongoing studies regarding the mechanistic details of ketamine's action have been helpful for conceptualizing potential mechanisms of action for N 2 O. Nevertheless, how N 2 O works as an antidepressant is currently largely unknown; hence, future studies utilizing nitrous oxide in both preclinical and clinical models will be crucial. Perhaps a clinical study comparing N 2 O to ketamine may also be of interest to investigate the context in which each is optimally effective. One possible area of exploration may include attempting to correlate euphoric experiences observed with N 2 O inhalation and subsequent antidepressant response. The possibilities of N 2 O use in other disorders such as post-traumatic stress disorder or bipolar disorder are currently under investigation. Another area of interest may be studying the impact of N 2 O on suicidality, especially given the rapid anti-suicidal effect of ketamine in previous studies. Considering its overall safety and tolerability, relative ease of use, and favorable results from initial studies in the treatment of both TRD and MDD, it is reasonable to propose the eventual potential use of N 2 O as an adjunctive treatment modality in addition to oral antidepressant therapy. At this stage of development, a larger, multi-center trial of inhaled nitrous oxide is warranted. If this trial is successful, one could envision the eventual development of a specialty clinic for delivery of N 2 O or perhaps eventual incorporation into a private practice setting under medical supervision similar to many dental practices. Typically, binding of glutamate (Glu) (in the presence of co-agonists glycine or D-serine) to NMDA receptors promotes channel activation and an influx/efflux of cations (A). Ketamine (yellow 1) enters the open channel activated by Glu and acts as a "trapping ion channel block" (B) to prevent ions from flowing through the channel. While ketamine blocks individual NMDA channels completely, its antidepressant effects are observed at submaximal concentrations and involve actions on only a subset of NMDA receptors. Nitrous oxide, like ketamine, is thought to act through the NMDA receptor to exert its antidepressant effects. However, unlike ketamine, its (green 2) effects are thought to be weakly voltage-dependent and is only a partial inhibitor of the NMDA receptor (C) at effective concentrations; thus, akin to ketamine, only a subset of NMDA receptors is inhibited. Its exact mechanism is unknown, but existing studies indicate that it is not an open ion channel blocker like ketamine.
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