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, 16–18], 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 influences 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 [23–26], and β-endorphin binds to both µ-ORs and δ-ORs, with a lower affinity 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 specific antagonists for µ-ORs, κ-ORs, and δ-ORs and more comprehensive behavioral testing and molecular characterization will be needed to unambiguously determine the specific 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 [41–43]. 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 [44–47], could synergize with the direct effects of ketamine on GABAergic interneurons and consequent disinhibition of pyramidal neurons and surge in synaptic glutamate [41–43], 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 affinity for both µ-ORs (Ki = 42.1 µM) and κ-ORs (Ki = 28.1 µM) [3]. However, even if ketamine does meaningfully engage ORs during single-dose treatment, since ketamine has a higher affinity 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 find 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 [41–43], 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 fluid 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 β-endorphin 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 β-endorphin 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 profile 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, 34–36, 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 conflicting evidence on this question. In mice, morphine reduces immobility time in the FST and tail suspension test (TST) [80, 81]. In rats, however, morphine does not influence 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 β-endorphin possesses rapid antidepressant potential.
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 findings 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.