Significance Ketamine, an NMDA receptor antagonist, has generated intense excitement as a therapy for treatment-resistant depression. However, ketamine and its metabolites can act on a wide range of targets, including opioid receptors, which has raised concerns. Using behavioral and cellular assays in rodents, we find that blocking opioid function prevents the antidepressant-like effects of ketamine. However, in contrast to ketamine, administration of a µ-opioid agonist is hedonic and ineffective on anhedonia/avolition. Furthermore, ketamine’s cellular actions are both mimicked and occluded by an NMDAR antagonist but not by a µ-opioid agonist. These results suggest that ketamine does not act as a μ-opioid agonist, but functional μ-opioid receptors are permissive for the antidepressant effects of ketamine.

Abstract Slow response to the standard treatment for depression increases suffering and risk of suicide. Ketamine, an N-methyl-d-aspartate (NMDA) receptor antagonist, can rapidly alleviate depressive symptoms and reduce suicidality, possibly by decreasing hyperactivity in the lateral habenula (LHb) brain nucleus. Here we find that in a rat model of human depression, opioid antagonists abolish the ability of ketamine to reduce the depression-like behavioral and LHb hyperactive cellular phenotypes. However, activation of opiate receptors alone is not sufficient to produce ketamine-like effects, nor does ketamine mimic the hedonic effects of an opiate, indicating that the opioid system does not mediate the actions of ketamine but rather is permissive. Thus, ketamine does not act as an opiate but its effects require both NMDA and opiate receptor signaling, suggesting that interactions between these two neurotransmitter systems are necessary to achieve an antidepressant effect.

Low-dose ketamine is being increasingly used in the treatment of acute suicidality and refractory depression (1, 2). The rapid therapeutic onset is particularly well suited for use in urgent settings, as standard treatments often require weeks to achieve clinical effects (3). Unlike other drugs currently used for depression treatment, ketamine displays high affinity for, and inhibits, the N-methyl-d-aspartate (NMDA) receptor (NMDAR; K i ∼ 0.5 μM) (4, 5). While NMDAR antagonists including APV and MK-801 produce short-lived antidepressant responses in preclinical studies, clinical studies have generally not demonstrated an antidepressant response of NMDAR-blocking agents, possibly due to different pharmacokinetics of the drugs (6, 7). Ketamine also inhibits other targets, albeit with significantly lower affinity [e.g., μ-opioid receptor, μOR; K i > 10 μM (8)]. Ketamine analgesia, achieved with higher than antidepressive doses, is diminished by coadministration of µOR antagonists, suggesting that ketamine may have activity at both opiate and NMDA receptors at biologically relevant concentrations (9⇓⇓⇓–13). It is thus unclear if the antidepressant effects of ketamine are mediated solely through NMDARs. Indeed, a recent small clinical study found that the antidepressant and antisuicidal effects of ketamine were blocked by the opioid antagonist naltrexone (refs. 14 and 15, but see refs. 16⇓⇓–19). Furthermore, opiates have long been clinically used for mood augmentation with ongoing clinical trials of novel formulations (20⇓⇓–23). The possibility that ketamine acts as an opiate has raised valid concerns among clinicians that could significantly impact its use (24).

The lateral habenula (LHb; coined the “disappointment center”) is part of a midbrain circuit that inhibits dopamine neurons in the ventral tegmental area after punishment or absence of an expected reward (25, 26). These features are thought to be key in normal reinforcement learning (27). In animal models, excessive LHb activity contributes to a number of behaviors that mimic core aspects of human depression (28), and inhibiting the LHb reduces such behaviors in rodents (29⇓⇓–32) and can ameliorate human depression (33). Notably, systemic ketamine delivery in humans (34) or intrahabenular delivery in rodents reduces LHb activity and depression-like behaviors (35).

A well-characterized rodent model to study the circuitry and pharmacology of depression is provided by the congenitally learned helpless (cLH) rat. cLH animals have been inbred from Sprague–Dawley rats based on susceptibility to learned helplessness after inescapable shock training (36). Unlike the wild type (WT), cLH rats exhibit a helpless phenotype without prior exposure to stress, as demonstrated in the shuttle box or forced swim test (37). Consistent with its modeling maladaptive valence processing, cLH animals display abnormal reward responses like those observed in depressed patients (38⇓–40). Furthermore, this line displays several depression-like symptoms (e.g., anhedonia, avolition, weight changes) (41) that improve with drugs (42⇓–44) used to treat human depression, including ketamine (35).

Here we test if the effects of low-dose ketamine are mediated by the opioid system, using behavioral and cellular assays. Is the µOR necessary and sufficient for the effects of ketamine? We find that µOR activity is necessary for the effects of ketamine, but µOR activation is not by itself sufficient to produce ketamine-like effects. These results suggest that µORs permit, but do not directly transmit, the actions of ketamine.

Materials and Methods Subjects. Male cLH and WT Sprague–Dawley rats, aged 3 to 4 wk for virus injection and aged 8 to 12 wk for behavioral studies, were kept on a 12/12-h, reverse-light/dark cycle (lights off 9 AM to 9 PM, lights on 9 PM to 9 AM). Red filters were used for lighting during daytime care. All procedures involving animals were approved by the Institutional Animal Care and Use Committees of the University of California San Diego. Drugs. Ketamine (Zoetis; 15 mg/kg, intraperitoneally [i.p.]), morphine (Hospira; 10 mg/kg, subcutaneously [s.c.]), and naltrexone hydrochloride (Tocris; 1 mg/kg, s.c.) were dissolved in 0.9% saline for injection. Mice in the control group were injected with 0.9% saline. All doses were calculated according to the base weight of the drug. Drugs for slice experiments were as follows: 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX; Tocris; 10 μM), d-(−)-2-amino-5-phosphonopentanoic acid (d-APV; Tocris; 25 μM), tetrodotoxin (TTX; Tocris; 1 μM), CTAP ([d-Phe-Cys-Tyr-d-Trp-Arg-Thr-Pen-Thr-NH2]; Tocris; 100 nM), [d-Ala2, NMe-Phe4, Gly-ol5]-enkephalin (DAMGO; Tocris; 1 μM), ketamine (Tocris; 10 μM), naltrexone (Tocris; 1 μM), and morphine (Hospira; 0.9 μM). Forced Swim Test. Rats were subjected to a 1-d modified forced swim test (mFST) as cLH animals do not require a “pretest” swim day to display immobility behavior (32). Subjects were brought up from the vivarium during the dark cycle and allowed to acclimate in a quiet, dark room for 1 h before drug injection and testing. Rats were placed in a clear cylinder filled with 12″ of 35 °C temperature water and observed by video camera for 15 min, upon which the test was stopped. Drug injections were performed as described in the main text. Analysis of mFST movies was performed using the open-source Bonsai software package as described previously (45). Movies were cropped to include the subject and automatically thresholded into a binary image where the subject was represented as pixels with a value of 1 and background pixels as 0. Identical threshold parameters were used for each movie. Subject movement was quantified as standard motion pixels [SMPs (46)]. Subsequent frames of the thresholded movie were subtracted from each other and movement was determined by the number of pixels that changed. The resulting mobility over time (15 min) graph was normalized for each animal by setting the minimum to 0 and the maximum to 1. Percent immobility for the session was determined by calculating the number of SMP values below a predetermined threshold that remained consistent across all subjects. Restricting analysis to the last 4 min of testing, rather than the full 15 min, did not significantly alter conclusions. Experiments with vehicle and ketamine were interspersed across testing sessions and results were pooled for graphing and statistical comparisons. Conditioned Place Preference. Conditioned place preference (CPP) took place over the course of 6 d. On the first day, rats were placed into a box divided into two chambers by a metal divider. The two chambers were decorated uniquely with both tactile and visual cues. The rats could freely roam for 15 min, and their position in the chambers was monitored by video camera, after which a side preference for each rat was calculated depending on which of the two chambers they preferred. Most rats did not appear to have a significant preference on the first day. On the subsequent 4 d, rats were either injected with morphine or ketamine and detained in their nonfavored side. On the sixth, and final, day, rats were injected with saline and again allowed to roam freely across both sides of the chamber. Place preference is reported as the absolute time the rat spends on the conditioned side the first day and the final day. Progressive Ratio. Rats were food-restricted to 90% of their body weight following protocols established by the Institutional Animal Care and Use Committees of the University of California, San Diego. Once at testing weight, rats were trained in a Pavlovian task to associate the illumination of a magazine light with availability of a reward at the delivery port (20 to 30 μL 7% sucrose). Most subjects had >100 port entries during the first training session, and were continued on to fixed ratio (FR1) training. During the 30-min FR1 training sessions, subjects could press a lever, once a light indicated reward availability, to receive a sucrose reward from the port. Once subjects achieved >60 lever presses in a session, they were given 1 d of FR5 training to stabilize lever-pressing behavior. Subjects were then allowed to rest in their home vivarium for several days before starting the progressive ratio (PR) schedule. The PR schedule started with an assessment of the baseline number of lever presses a subject would perform to receive a sucrose reward. Sucrose reinforcements could be earned with the following number of presses: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 28, 32, 36, 40, 44, 48, 52, 56, 64, 72, 80, and 88. The final ratio achieved represented the breakpoint value. The next day, rats were treated as described in the text and the breakpoint was reassessed 2 h post treatment as well 1 and 7 d post treatment. Surgery. Rodents were anesthetized with isoflurane for stereotaxic injection of an adeno-associated virus (AAV) expressing jGCaMP7f (AddGene; pGP-AAV-syn-jGCaMP7f-WPRE; AAV8) bilaterally into the LHb (anterior–posterior, −3.2 mm; medial–lateral, ±0.5 mm; dorsal–ventral, −4.85 to −5.0 mm). A total of 0.5 to 1.0 μL virus was injected over an 8- to 10-min period. At the end of the injection, the pipet remained at the site for 5 min to allow for diffusion of the virus into the surrounding tissue. Rats were injected with 5 mg carprofen (a nonsteroidal antiinflammatory drug) per kg body weight after surgery. Calcium Imaging. Three weeks after viral injection, rats were anesthetized with isoflurane before decapitation and brain removal. Brains were chilled in ice-cold dissection buffer (215 mM sucrose, 20 mM glucose, 26 mM NaHCO 3 , 4 mM MgCl 2 , 4 mM MgSO 4 , 1.6 mM NaH 2 PO 4 , 1 mM CaCl 2 , and 2.5 mM KCl), gassed with 95%/5% O 2 /CO 2 (carbogen), and cut into 300-μm-thick coronal slices through the LHb. Slices were transferred to 35 °C in a 50%/50% mixture of dissection buffer and artificial cerebrospinal fluid (ACSF) (124 mM NaCl, 26 mM NaHCO 3 , 10 mM glucose, 2.5 mM KCl, 1 mM NaH 2 PO 4 , 4 mM CaCl 2 , and 2 mM MgCl 2 ) and gassed with carbogen for 30 min. After an additional 30 min of recovery at room temperature, slices were transferred to room-temperature ACSF. Acute LHb slices were imaged on a Prairie Labs 2p system, using Prairie View software and a Chameleon laser tuned to 980 nm. Regions with jGCaMP7f expression in the LHb were identified and 80 frames at 1.2 Hz (66 s total) were acquired. Two or three separate movies were acquired for each slice imaging session, a baseline, followed by one or two drug conditions. Each movie was separated by 15 min to permit drug penetration. For some experiments, two baseline movies were recorded 15 min apart to measure the baseline level of change in the absence of drugs. Vehicle experiments were interspersed among imaging sessions and the average baseline value across all experimental sessions was reused for graphing and statistical comparisons. Drug effect quantified as ratio of fluorescence intensity after drug application divided by intensity during baseline period. A ratio of 1 indicates no change; a ratio below 1 indicates the drug reduced activity. Analysis. Calcium imaging analysis was performed using FIJI (ImageJ) and MATLAB. The movies for each slice were concatenated, registered (linear stack alignment with SIFT) in FIJI, and divided by the average projection across time image to create a ΔF/F movie. Segmentation of neuronal somas was performed automatically in MATLAB using the Calcium Signal Extract toolbox (Stephan Meyer, GitHub), and intensity over time was measured for each region of interest (ROI). To exclude ROIs with constant decrease or increase, ROI ΔF/F intensity traces were detrended (MATLAB), and traces displaying less than 20% change over baseline were excluded. The rest of the analysis was performed on the nondetrended ROI ΔF/F traces encompassing baseline and drug treatment images. These traces were normalized by setting the lowest value of the 5-point moving average to 0 and the maximum intensity to 1. For each ΔF/F trace, the mean values during baseline and during drug treatment were calculated, and the ratio of mean activity in drug condition over baseline was calculated for each neuron. In the figure legends for imaging experiments, N/N′ indicates the number of neurons (N) and number of animals (N′). For behavioral experiments, N is the number of animals. Materials and Data Availability. All experimental procedures and data from this study are provided in the main text and SI Appendix. All rat strains and plasmids used in the study are available upon request, or can be obtained from Addgene (https://www.addgene.org/) by qualified researchers for their own use.

Discussion Here we use rodents to test the hypothesis that ketamine acts on opioid receptors to achieve its antidepressive effects. In our behavioral studies, we confirm that cLH animals display immobility and amotivation, behaviors thought to model human hopelessness. We find that ketamine administration rapidly improves these behaviors, and that these beneficial effects are blocked by naltrexone in multiple assays. Together, our results indicate that functional opioid receptors are required for ketamine to produce antidepressive-like behavioral effects in rodents. However, it does not appear that ketamine is directly acting on opiate receptors to produce these effects, as activation of µOR by morphine, sufficient to induce a hedonic response, did not mimic the rapid antidepressive-like effects of ketamine. Nor did it appear that ketamine, at an antidepressant dose, had a hedonic effect typically associated with µOR activation. Therefore, at least in rodents, low-dose ketamine does not have the same behavioral effects as µOR activation. In our cellular studies, we focused on activity of neurons in the LHb, as hyperactivity of this region is thought to contribute to depression (34, 53), and normalization of this hyperactivity may be beneficial in treating depression (54). Furthermore, the LHb not only receives inputs from many opioid-sensitive brain regions but also contains a high density of µORs that can be directly modulated by opioids (55⇓–57). Using a brain slice imaging method, we confirmed that LHb neurons from cLH animals display hyperactivity, when compared with neurons from WT animals, and were reduced to the activity level of WT animals by ketamine (35). As with the behavioral studies, this cellular effect of ketamine was blocked by naltrexone as well as by CTAP, indicating an intact µOR system is required for these effects. However, activation of µORs with a specific agonist failed to reduce LHb neuronal activity. Thus, in the results from our studies of LHb cellular activity, ketamine does not appear to act as a µOR agonist, though we cannot exclude the possibility that ketamine may be activating µORs in a manner that does not produce CPP or a hedonic effect. Our observation that the effects of the specific NMDA antagonist APV are also blocked by CTAP suggests that some activity of µOR is necessary for NMDAR antagonism, or the effects of NMDAR antagonism, as APV has no known action on µOR. It may be that this tonic action of µOR alters the biophysical properties of NMDAR to gate antagonism of both ketamine and APV. Therefore, the most direct interpretation of our results is that ketamine, at an antidepressive dose, is not mediating its behavioral or cellular effects by directly activating µOR. Rather, some level of µOR activity appears permissive as multiple lines of evidence with opioid antagonists do demonstrate the necessity of intact µOR signaling for ketamine to produce its rapid antidepressant response. How then can µOR gate the response of ketamine? Indeed, there is a rich body of literature describing multiple interactions between these two signaling systems (58). Pain research studies, using ketamine at higher than antidepressive doses, suggest both direct and indirect interactions between ketamine and opioid receptors (1, 13, 59). At the electrophysiological level, NMDAR activation can be modulated by actions of opioid receptors (60⇓⇓–63). And, in some brain regions (including the habenula), NMDARs and opioid receptors display colocalization at the light and ultrastructural microscopic levels (64⇓⇓⇓–68). Such results support the view that NMDARs and opioid receptors display significant interactions, either by direct binding of the receptors or by downstream signaling pathways. Such a scenario, as well as our results, are consistent with there being some level of µOR activity permitting blockade of NMDARs by ketamine or permitting the effects of NMDAR blockade by ketamine. In this way, blocking µOR function with naltrexone would prevent the effects of ketamine on NMDAR function, and could account for the cellular and behavioral effects of this study. Since naltrexone can antagonize more than just the µ-opioid subtype (albeit at lower affinities), antagonists with greater specificity will be necessary to elucidate the requirement of each subtype in behavioral processes. In conclusion, our study finds that the actions of ketamine are not mimicked by activating µORs, indicating ketamine is not acting as an opioid to produce antidepressive effects in a rodent model. However, the opioid system is required for the actions of ketamine, indicating an interaction between the NMDA receptor and opioid receptor systems.

Acknowledgments This work was supported by the National Institute of Mental Health [R.M., R01MH091119; M.E.K., R25MH101072 (principal investigator Neal Swerdlow)]. We thank Dr. Neal Swerdlow for his help with the manuscript and statistical analyses, and Sahil Sheth for his help with the behavioral tasks.

Footnotes Author contributions: M.E.K. and R.M. designed research; M.E.K., J.C., and S.S. performed research; M.E.K. and R.M. analyzed data; and M.E.K. and R.M. wrote the paper.

Reviewers: H.H., Zhejiang University; and B.L., Cold Spring Harbor Laboratory.

The authors declare no competing interest.

This article contains supporting information online at https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1916570117/-/DCSupplemental.