The endogenous opioid system in the medial prefrontal cortex mediates ketamine’s antidepressant-like actions

Recent studies have implicated the endogenous opioid system in the antidepressant actions of ketamine, but the underlying mechanisms remain unclear. We used a combination of pharmacological, behavioral, and molecular approaches in rats to test the contribution of the prefrontal endogenous opioid system to the antidepressant-like effects of a single dose of ketamine. Both the behavioral actions of ketamine and their molecular correlates in the medial prefrontal cortex (mPFC) were blocked by acute systemic administration of naltrexone, a competitive opioid receptor antagonist. Naltrexone delivered directly into the mPFC similarly disrupted the behavioral effects of ketamine. Ketamine treatment rapidly increased levels of β-endorphin and the expression of the μ-opioid receptor gene (Oprm1) in the mPFC, and the expression of the gene that encodes proopiomelanocortin, the precursor of β-endorphin, in the hypothalamus, in vivo. Finally, neutralization of β-endorphin in the mPFC using a specific antibody prior to ketamine treatment abolished both behavioral and molecular effects. Together, these findings indicate that presence of β-endorphin and activation of opioid receptors in the mPFC are required for the antidepressant-like actions of ketamine.


INTRODUCTION
Ketamine, a noncompetitive antagonist of N-methyl-D-aspartate glutamate receptors (NMDARs), has been found to have rapid antidepressant and anti-suicide effects in many patients, and similar rapid antidepressant-like effects in preclinical models [1,2].In addition to its well-established ability to block NMDARs, ketamine also interacts with a range of other targets, including opioid receptors [3].The endogenous opioid system consists of the µ-, δ-, and κ-opioid receptors (ORs), encoded by the Oprm1, Oprd1, and Oprk1 genes; their primary endogenous ligands are the opioid peptides β-endorphin, enkephalins, and dynorphins, respectively.These receptors and peptides are implicated in a wide range of physiological and pathological processes, including the regulation of pain and of reward and stress responses [4][5][6].Accumulating evidence implicates the endogenous opioid system in the pathophysiology and treatment of depression [7].In particular, β-endorphin is implicated in both MDD and its treatment [8], and the µ-OR differs between healthy controls and patients with MDD in multiple brain regions [9,10].Previous preclinical studies extensively documented the role of medial prefrontal cortex (mPFC) in regulating ketamine's antidepressant-like effects, implicating synaptic protein synthesis in the mPFC in ketamine's actions [11,12].However, no study to date has examined the involvement of endogenous opioid signaling in the mPFC in ketamine's behavioral and molecular effects.
In a seminal clinical study, Williams et al. reported that pretreatment with naltrexone, an opioid receptor antagonist, attenuates the antidepressant and anti-suicidal effects of ketamine in depressed patients [13,14] (although another clinical study did not show such an effect in patients treated with naltrexone for alcohol use [15]).In preclinical work, systemic naltrexone pretreatment was found to block the antidepressant-like effects of ketamine in three studies [16][17][18], though not in a fourth [19].Here, we test the hypothesis that β-endorphin and activation of opioid receptors in the mPFC is required for the antidepressant-like actions of ketamine, and for the underlying molecular mechanisms in the mPFC.

Animals
Adult male Sprague-Dawley rats (Charles River Laboratories; 250-500g at the time of experiments) were obtained and allowed at least one week acclimation to housing facilities.Rats were singly housed at least 5 days before behavioral experiments or tissue collection and maintained on a 12h light/dark cycle, with ad libitum access to food and water.Animal use and procedures were in accordance with NIH guidelines and approved by the Yale University Animal Care and Use Committee.
Naltrexone at this dose has been frequently used in preclinical studies [20][21][22], although it may lose some of its selectivity for the µ-OR and also bind to δ-, and κ-ORs at this dose [23][24][25][26].Behavioral testing was carried out starting 24 hours after ketamine treatment.
Experiment 2: To examine the effects of local opioid receptor blockade in the mPFC on ketamine's behavioral actions, mPFC-cannulated rats were bilaterally infused with naltrexone (20 µg/0.5 µl/side) 30 minutes prior to ketamine (10 mg/kg, i.p.) or saline treatment.This naltrexone dose was chosen based on previous studies demonstrating the effects of intracranially microinjected naltrexone [27,28].Behavioral testing was carried out starting 24 hours after ketamine treatment.Experiment 3: ELISA and qPCR were performed with mPFC and hypothalamus collected 1 or 24 hours following ketamine (10 mg/kg, i.p.) or saline treatment to examine ketamine's effects on the presence of β-endorphin and expression of opioid receptors in the mPFC and on the expression of β-endorphin precursor, proopiomelanocortin (POMC), in the hypothalamus.Experiment 4: To examine the effects of local neutralization of β-endorphin on ketamine's behavioral, mPFC-cannulated rats were bilaterally infused with anti-β-endorphin antibody (0.5 µg/0.5 µl/side; Phoenix Pharmaceuticals #G-022-33, Burlingame, California) or control IgG 30 minutes prior to ketamine (10 mg/kg, i.p.) or saline treatment.Behavioral testing was carried out starting 24 hours after ketamine treatment.
Experiment 5: To examine the effects of systemic opioid receptor blockade and local neutralization of βendorphin on ketamine's molecular actions, rats received treatments as described in Experiments 1 and 4, and mPFC was collected 1 hour or 24 hours after ketamine treatment for Western blot analysis.

mPFC Cannulation and Infusion Procedures
Rats were anaesthetized with a mixture of ketamine (75 mg/kg) and xylazine (5 mg/kg), and bilateral guide cannulae (P1 Technologies Inc., Roanoke, Virginia: 22-gauge) were implanted 0.5 mm above the infusion site at the following coordinates: from bregma: +3.0 mm anterior/posterior; +/-1.0 mm medial/lateral; and − 4.0 mm dorsal/ventral.Following 9-14 days of recovery, rats were bilaterally infused with naltrexone or an anti-β-endorphin antibody at a rate of 0.2 µl/min, using a microinfusion pump (Harvard Apparatus, Holliston, Massachusetts).The needle was left in place for 2.5 minutes after the injection to allow complete dispersion of the solution.Naltrexone was dissolved in saline; control rats were infused with 0.9% saline.The anti-β-endorphin antibody was reconstituted following manufacturer's instructions; rabbit control puri ed IgG (Phoenix Pharmaceuticals #NRG-500) was used as a control.

Forced Swim Test (FST)
FST was conducted as previously described [29].Rats were exposed to a 15-minute pre-swim in 25 ± 1°C water in a Plexiglas cylinder (65 cm height, 30 cm diameter).24 hours following the pre-swim, rats were treated as described above and then placed in the swim tank for 10 minutes.Data were analyzed by scoring immobility time during minutes 2-6.

Female Urine Sni ng Test (FUST)
FUST was conducted as previously described [29].Rats were habituated to a sterile cotton-tipped applicator placed into their home cage for 1 hour, and then exposed to a water-dipped cotton-tipped applicator for 5 minutes.After a 45-minute interval, rats were exposed to a cotton-tipped applicator infused with 75 µl fresh urine from females of the same strain for 5 minutes, during which the time spent sni ng the cotton-tipped applicator was measured.Time spent biting the cotton-tip was excluded from analysis.

Novelty Suppressed Feeding Test (NSFT)
NSFT was conducted as previously described [29].Rats were food deprived for at least 20 hours and then placed in an open eld with one food pellet in the center.The latency to feed was measured, with a cut-off time of 15 minutes.After NSFT, home cage feeding during a 15-minute period was measured to verify motivation to feed.

Locomotor Activity
Rats were placed in testing cages (46 cm × 23 cm × 20 cm) for 30 minutes, during which the number of beam breaks was measured using Med-PC software (Med Associates, Fairfax, Vermont).
Protein and RNA sample preparation, Western Blot, and qPCR analysis Crude synaptosomal fraction or total homogenate of rat mPFC were prepared and analyzed by Western blot.RNA from mPFC or hypothalamus was extracted by RNeasy Mini Kit (Qiagen #74104, Hilden, Germany), reverse transcribed and subjected to qPCR, as detailed in Supplemental Material.

Primary hypothalamic culture and in vitro ketamine treatment
Primary hypothalamic culture prepared from E18 embryos was treated with 0.5 µM ketamine on day 12 in vitro, as detailed in Supplemental Material.

β-endorphin ELISA analysis
Rat mPFC was collected 1 hour or 24 hours following ketamine injection and homogenized in PBS.βendorphin levels were measured using QuickDetect™ beta-Endorphin (Rat) ELISA Kit (BioVision, Milpitas, California #E4460) according to the manufacturer's instructions.Protein concentrations in each sample were measured using a Pierce BCA Protein Assay Kit (Thermo Scienti c, Waltham, Massachusetts) and results are presented as pg of β-endorphin in 1 mg of protein in the sample.

Statistics
Statistical analyses were performed using GraphPad Prism (San Diego, California).Values were excluded only if they were detected as outliers by Grubb's test.Comparisons between two groups were made using Student's t test.Correlation was calculated using Pearson's r.Comparisons for four groups were made using two-way analysis of variance (ANOVA) followed by Sidak's multiple comparisons, as indicated in the Figure Legends and Supplemental Material (Table S1).All tests are two-sided.All data are presented as mean ± s.e.m..

Systemic naltrexone pretreatment blocks the antidepressant-like actions of ketamine
To investigate whether the endogenous opioid system is required for the antidepressant-like actions of ketamine, we treated rats with naltrexone (20 mg/kg, i.p.) 30 minutes before ketamine (10 mg/kg, i.p.; Fig. 1A).Rats were tested in a series of behavioral paradigms, including forced swim test (FST), female urine sni ng test (FUST), novelty suppressed feeding test (NSFT), and locomotor activity (LMA), starting 24 hours after ketamine treatment.In the FST, a model of behavioral despair, ketamine signi cantly reduced immobility time in saline-pretreated rats; this effect was completely blocked by naltrexone pretreatment (Fig. 1B).In FUST, a model in which less time spent sni ng female urine indicates anhedonia in males, ketamine signi cantly increased female urine sni ng time in saline-but not naltrexone-pretreated rats (Fig. 1C).In the NSFT, a paradigm in which longer latency to feed is considered an anxiety-like behavior, ketamine signi cantly shortened latency to feed in saline-but not naltrexonepretreated rats (Fig. 1D), without changing home cage food consumption (Figure S1).Locomotor activity was not affected by either naltrexone pretreatment or ketamine treatment (Fig. 1E).Together, these results suggest that activation of opioid receptors is required for the behavioral effects of ketamine.

Intra-mPFC naltrexone infusion blocks the antidepressantlike actions of ketamine
As the mPFC plays an important role in regulating ketamine's antidepressant-like effects [11,12], we sought to determine whether localized blockade of ORs using targeted naltrexone infusion into the mPFC would be su cient to attenuate the behavioral effects of ketamine.We infused naltrexone (20 µg/0.5 µl/side) or saline into the mPFC of cannulated rats 30 minutes before ketamine treatment (10 mg/kg, i.p.) and subjected the rats to behavioral tests starting 24 hours later (Fig. 2A).In saline-pretreated rats, ketamine induced signi cant behavioral effects in FST (Fig. 2B), FUST (Fig. 2C), and NSFT (Fig. 2D); all effects were absent in naltrexone-pretreated rats.There were no effects of ketamine, with or without naltrexone, on home cage feeding (Figure S2) or locomotor activity (Fig. 2E).

Ketamine induces β-endorphin release in mPFC
While naltrexone is a nonselective OR antagonist, it has the highest a nity for µ-ORs [30,31].β-endorphin is the primary endogenous agonist for µ-ORs, and its presence in the cortex has been documented [32].
Ketamine signi cantly increased β-endorphin levels in the mPFC, measured using ELISA in dissected tissue collected 1 hour after treatment (Fig. 3A).β-endorphin is derived from a precursor protein, proopiomelanocortin (POMC), encoded by the Pomc gene, which is primarily expressed in the arcuate nucleus (ARC) of hypothalamus, as well as in the pituitary gland [33].To investigate whether ketamine concurrently in uences Pomc expression in the hypothalamus, we measured Pomc mRNA using qPCR in tissue collected 1 hour following ketamine treatment.Ketamine signi cantly increased hypothalamic Pomc mRNA relative to saline-injected controls (Fig. 3B); change in Pomc was positively correlated with the increased β-endorphin level in the mPFC (Fig. 3C).In further support of this nding, increased βendorphin, measured by ELISA, was released into the supernatant after ketamine treatment in hypothalamic neuronal cultures, at trend level (Figure S3A, B).Ketamine also signi cantly increased mPFC Oprm1 mRNA (Fig. 3D), but not Oprk1 mRNA (Figure S3C), at 1 hour.
At 24 hours, the elevation in β-endorphin levels in the mPFC was completely absent in the ketamine group (Fig. 3E), and there was no change in Pomc mRNA expression in the hypothalamus (Fig. 3F); however, hypothalamic Pomc expression remained positively correlated with β-endorphin level in the mPFC (Fig. 3G).Ketamine treatment did not signi cantly change the mRNA expression of Oprm1 (Fig. 3H) and Oprk1 (Figure S3D) in the mPFC at 24 hours.Thus, ketamine induced a rapid, non-sustained increase in βendorphin and the expression of the gene that encodes its primary receptor, µ-OR, in the mPFC, and of the expression of the gene that encodes the β-endorphin precursor, POMC, in the hypothalamus.

Intra-mPFC anti-β-endorphin antibody infusion blocks the antidepressant-like actions of ketamine
We next tested whether β-endorphin in the mPFC is necessary for the behavioral effects of ketamine.We infused rats with a β-endorphin neutralizing antibody (0.5 µg/0.5 µl/side) or control IgG 30 minutes before ketamine treatment (10 mg/kg, i.p.) and subjected them to behavioral tests starting 24 hours later (Fig. 4A).In control IgG-infused rats, ketamine produced robust behavioral effects, all of which were absent in the rats that received β-endorphin antibody infusion: FST (Fig. 4B), FUST (Fig. 4C) and NSFT (Fig. 4D).There was no effect on locomotor activity or home cage feeding (Fig. 4E and Figure S4).Intra-mPFC β-endorphin antibody infusion alone did not in uence these behaviors.

Systemic naltrexone pretreatment abolishes ketamineinduced molecular changes in the mPFC
To determine the molecular mechanisms underlying impaired ketamine response, we examined the effect of systemic naltrexone pretreatment on ketamine-induced phosphorylation of AMPA receptor subunit GluR1, which has been implicated in ketamine's antidepressant-like actions [2,34,35], using Western blotting.mPFC tissue was collected 1 hour after ketamine injection, preceded by naltrexone or saline (Fig. 5A).Ketamine signi cantly increased phosphorylation of GluR1 in the total homogenate (Fig. 5B); this was blocked by naltrexone pretreatment.Similarly, ketamine induced numerically increased phosphorylation of µ-OR, which was partially blocked by naltrexone pretreatment at 1 hour, though these effects did not reach statistical signi cance (Fig. 5C).
We next determined the effect of naltrexone pretreatment on the ketamine-induced elevation in new synthesis of GluR1 in the mPFC [12,36].24 hours after ketamine treatment, GluR1 protein levels were signi cantly increased in mPFC synaptosomes in saline-pretreated rats, while no signi cant changes were observed in naltrexone-pretreated group (Fig. 5D).These results indicate that opioid receptor activation is required for synaptic changes induced by ketamine in the mPFC.

Intra-mPFC anti-β-endorphin antibody infusion blocks the molecular effects of ketamine
To determine whether β-endorphin in the mPFC is required for these molecular effects, we infused rats with a β-endorphin neutralizing antibody 30 minutes before ketamine treatment and collected mPFC 1 hour and 24 hours after ketamine (Fig. 5E).Increased phosphorylation of GluR1 (Fig. 5F) and µ-OR (Fig. 5G) in the total homogenate at 1 hour were both absent in rats pretreated with β-endorphin antibody.Increased GluR1 levels in synaptosomes at 24 hours (Fig. 5H) was similarly abolished by β-endorphin antibody pretreatment.

DISCUSSION
We demonstrate that blockade of opioid receptors by a single dose of systemic naltrexone 30 minutes prior to ketamine treatment abolishes the effects of ketamine on behavioral despair, anhedonia-like, and anxiety-like behaviors in rats.This is consistent with several previous clinical and preclinical reports [13,14,[16][17][18], though not with others [15,19].These discrepancies in the literature could be explained by the differences in the routes and doses of naltrexone administered, the timepoints at which naltrexone is administered, comorbidity of alcohol use disorder with depression in one clinical study [15], and prior exposure to stress, which in uences expression of opioid receptor [37], in one preclinical study [19].Additional preclinical studies employing stress exposure, comprehensive behavioral phenotyping, and various doses of naltrexone are needed to clarify these discrepancies.
Intra-mPFC infusion of naltrexone prior to ketamine treatment also blocks the behavioral effects of ketamine, and systemic naltrexone pretreatment blocks ketamine-induced molecular changes in the mPFC.These results indicate that opioid signaling in the mPFC plays a central role in regulating ketamine's actions.This importantly extends recent research on the interplay between ketamine and the opioid system by pinpointing a critical site of action, although our current data do not exclude contributions from other locations.
Naltrexone can bind to µ-ORs, κ-ORs, and δ-ORs at the dose we used [23][24][25][26], and β-endorphin binds to both µ-ORs and δ-ORs, with a lower a nity for κ-ORs.Recent studies have reported that pharmacological blockade of κ-ORs abolished the behavioral effects of repeated ketamine administration in the FST in mice [17], and that δ-OR agonists produce antidepressant-like effects [38].We cannot rule out the contribution of κ-ORs and δ-ORs in mediating ketamine's actions.Future studies using more speci c antagonists for µ-ORs, κ-ORs, and δ-ORs and more comprehensive behavioral testing and molecular characterization will be needed to unambiguously determine the speci c type(s) of ORs that mediate ketamine's actions.However, phosphorylation of µ-ORs and upregulation of POMC are suggestive of a µ-OR mediated mechanism; this remains our leading hypothesis to explain these effects.
Oprm1 is expressed in GABAergic and glutamatergic neurons and on astrocytes in the cortex [39,40].Recent studies suggest that ketamine blocks NMDARs on GABAergic interneurons, resulting in disinhibition and a burst of glutamate that produces synaptic and behavioral effects [41][42][43].Our results support an intriguing model that merits further study: ketamine induces release of β-endorphin into the mPFC, which activates µ-ORs on GABAergic interneurons and/or astrocytes.Inhibition of interneurons and release of glutamate from astrocytes, both of which are known effect of µ-OR activation [44][45][46][47], could synergize with the direct effects of ketamine on GABAergic interneurons and consequent disinhibition of pyramidal neurons and surge in synaptic glutamate [41][42][43], thus contributing to the initiation of rapid and sustained antidepressant-like effects.However, alternative interpretations exist.Ketamine may directly activate ORs: it has an appreciable binding a nity for both µ-ORs (K i = 42.1 µM) and κ-ORs (K i = 28.1 µM) [3].However, even if ketamine does meaningfully engage ORs during singledose treatment, since ketamine has a higher a nity for κ-ORs than for µ-ORs, and κ-OR agonists have dysphoric and aversive properties [48], one would expect direct agonist effects to be biased towards the pro-depressant effects produced by κ-OR activation.This is not what we observe.
We nd evidence for stimulation of β-endorphin release by ketamine in hypothalamic neuronal culture (Figure S3A, B), as has previously been reported in pituitary cell culture [49].Previous studies suggest that ketamine induces endogenous opioid release in vivo [50,51].We document elevated β-endorphin levels in the mPFC, and a correlated increase in Pomc mRNA levels in the hypothalamus, 1 hour following ketamine treatment in vivo; both return to baseline at 24 hours but remain correlated with one another.These observations suggest that ketamine activates ARC POMC neurons, directly or indirectly, leading to rapid and transient increase in β-endorphin in the mPFC, which collectively with other processes [41][42][43], initiates the antidepressant-like effects.ARC POMC neurons send projections to cortical areas and limbic system [52], so it is possible that β-endorphin is directly released by the ARC POMC neuron terminals in the mPFC.Alternatively, β-endorphin released in the hypothalamus may be transported via cerebrospinal uid in the ventricular system to the mPFC by volume transmission [53].
β-endorphin has been implicated in the pathophysiology and treatment of depression [7,8].Plasma βendorphin levels are correlated with certain clinical symptoms of depression [54] and are increased by several antidepressant treatments [55,56].Release of endogenous opioid(s) targeting µ-ORs in the PFC has been observed after high-intensity exercise [57], which has been linked to improved mood [58].Fluoxetine, a selective serotonin reupdate inhibitor, has been demonstrated to induce β-endorphin release in the ARC and nucleus accumbens [59].Although our observations that ketamine increases β-endorphin levels in the mPFC in vivo and that intra-mPFC pretreatment with anti-β-endorphin neutralizing antibody blocks the antidepressant-like effects of ketamine suggest a causal relationship between β-endorphin in the mPFC and ketamine's actions, we did not directly monitor changes in the extracellular level of βendorphin in the mPFC in response to ketamine (the increase shown in Fig. 3A was in total mPFC tissue), nor did we examine potential ketamine-induced β-endorphin release in other brain regions.Future study using in vivo microdialysis could better characterize the temporal pro le of ketamine-induced β-endorphin release in the mPFC.Future examination of multiple brain regions might also identify other regions where β-endorphin may have a role in mediating ketamine's actions.
Previous studies have implicated brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF) in the antidepressant-like actions of ketamine [60,61].Whether these trophic factors and their respective signaling act in parallel with or subsequently to β-endorphin remains to be determined.β-endorphin has been reported to increase BDNF expression in the PFC and hippocampus [62], and µ-OR agonists activate VEGF receptors [63], suggesting the possibility that BDNF and VEGF signaling could be downstream of β-endorphin.However, it is also possible that ketamine induces release of β-endorphin, BDNF, and VEGF independently and they then act interdependently, together with other processes, to mediate ketamine's antidepressant-like effects.
It has been shown that ketamine and other agents with rapid antidepressant-like properties rapidly induces GluR1 phosphorylation in multiple brain regions, including mPFC [2,[34][35][36]64], and that GluR1 phosphorylation is required for the rapid and sustained antidepressant-like effects of ketamine and subsequent increase in synaptic GluR1 levels [35].Our results indicate that activation of opioid receptors and presence of β-endorphin in the mPFC are required for ketamine-induced increase in GluR1 phosphorylation and elevated synaptosomal GluR1 levels in the mPFC.µ-OR agonists have been shown to increase protein kinase A and calcium/calmodulin-dependent protein kinase II activity in vivo [65, 66], which can in turn phosphorylates GluR1 [67, 68], mediating its role in regulating synaptic delivery, and incorporation of GluR1-containing AMPA receptors into synapses [69].β-endorphin leads to phosphorylation of µ-ORs at Ser375 [70].
Preclinical studies have begun to reveal sex differences in response to ketamine.Females are sensitive to lower dose of ketamine and exhibit stronger behavioral response in some contexts [77].Sex differences have also been reported in β-endorphin levels in multiple brain regions, both at baseline and under various experimental conditions [78,79].Because of these reported effects of sex, we focused here on male rats, to reduce the number of variables at play.It will be important to examine potential sexual dimorphisms in the reported effects in future studies.
Our data suggest that β-endorphin in the mPFC can contribute to antidepressant-like effects.Previous studies have provided con icting evidence on this question.In summary, our study demonstrates that β-endorphin and opioid receptor activation in the mPFC are required for the behavioral and molecular actions of ketamine in a well-established rat model.These ndings are consistent with accumulating evidence implicating endogenous opioid signaling in the rapid antidepressant effects of ketamine.Importantly, our results suggest a potential mechanism by which ketamine produces antidepressant-like actions: by increasing β-endorphin release, which in turn activates µ-ORs in the mPFC.This work lays the foundation for future studies to further delineate these mechanisms to inform the development of next-generation rapidly acting antidepressant agents.

Figure 5 Systemic
Figure 5 In mice, morphine reduces immobility time in the FST and tail suspension test (TST)[80, 81].In rats, however, morphine does not in uence the immobility time in the FST[16, 82].Early clinical studies documented antidepressant effects induced by intravenous β-endorphin infusion [83-85].Within central nervous system, intracerebroventricular infusion of β-endorphin increases Bdnf mRNA expression in the PFC and hippocampus[62]; this is similar to the effects seen following chronic conventional antidepressant treatments [86] and acute ketamine administration[87].Interestingly, one recent study found that endogenous and exogenous opioids act on GABAergic and glutamatergic neurons, respectively, to mediate behavioral effects[88].Therefore, the lack of consistent effects from exogenous µ-OR agonists cannot rule out the possibility that endogenous βendorphin possesses rapid antidepressant potential.