Our results indicate that the glutamatergic system is involved in the mechanism of action of rapid antidepressant response. In addition, this system may be a feasible target for developing treatments that have rapid and robust efficacy in individuals who have treatment-resistant depression and suicidal thoughts. We found that the glutamatergic modulator ketamine resulted in rapid, robust and relatively sustained antidepressant, antisuicidal, and antianhedonic effects. Response with ketamine occurred within 2 hours and lasted approximately 1 week. Study: (Biomarkers of rapid response in major depressive disorder): protocols 04-M-0222 (NCT00088699), 17-M-0060 (NCT03065335), and 19-M-0107 (NCT 03973268). OBJECTIVE: To identify the neural correlates and cellular and molecular targets of rapid antidepressant response to the NMDA antagonist ketamine in subjects with major depressive disorder. Aims are 1) to examine the antisuicidal effects of ketamine, and 2) to examine correlates of antidepressant response to ketamine in both major depressive disorder and bipolar disorder and include these data/outcome measures: clinical (e.g., family history), imaging (magnetic resonance imaging/spectroscopy), electrophysiological (magnetoencephalography MEG, electroencephalography EEG), neuropsychological, and biochemical (e.g., genetics, microRNA, BDNF, metabolomics), 3) To demonstrate more robust neuropharmacodynamic effects measured by neuropharmacodynamic imaging (fMRI+EEG and MEG) of ketamine 0.5 mg/kg as compared to placebo administered over 40 minutes, and 4) To understand the involvement of AMPA receptors in ketamine's antidepressant response. Secondary aims: To determine if increases in synaptic plasticity, using electrophysiological measures in response to TMS and in association with sleep (i.e., slow wave sleep EEG activity) are associated with better antidepressant response. Results in the past year: 1. NMDA receptor activation-dependent antidepressant-relevant behavioral and synaptic actions of ketamine. Ketamine is an NMDA receptor (NMDAR) antagonist, although the significance of this pharmacology to its rapid (within hours of administration) antidepressant actions, which depend on mechanisms convergent with strengthening of excitatory synapses, is unclear. Activation of synaptic NMDARs is necessary for the induction of canonical long-term potentiation (LTP) leading to a sustained expression of increased synaptic strength. We tested the hypothesis that induction of rapid antidepressant effects requires NMDAR activation, by using behavioral pharmacology, western blot quantification of hippocampal synaptoneurosomal protein levels, and ex vivo hippocampal slice electrophysiology in male mice. We found that ketamine exerts an inverted U-shaped dose-response in antidepressant-sensitive behavioral tests, signifying that an excessive NMDAR inhibition can avert ketamine's antidepressant effects. Ketamine's actions to induce antidepressant-like behavioral effects, up-regulation of hippocampal AMPAR subunits GluA1 and GluA2, as well as metaplasticity measured ex vivo using electrically-stimulated LTP, were eliminated by pretreatment with other non-antidepressant NMDAR antagonists, including MK-801 and CPP. We found that NMDAR activation is necessary for the beneficial effects of ketamine and several other putative antidepressant compounds. 2. Experimenters' sex modulates mouse behaviors and neural responses to ketamine via corticotropin releasing factor. We demonstrate that the sex of human experimenters affects mouse behaviors and responses following administration of the rapid-acting antidepressant ketamine and its bioactive metabolite (2R,6R)-HNK. Mice showed aversion to the scent of male experimenters, preference for the scent of female experimenters and increased stress susceptibility when handled by male experimenters. This human-male-scent-induced aversion and stress susceptibility was mediated by the activation of corticotropin-releasing factor (CRF) neurons in the entorhinal cortex that project to hippocampal area CA1. Exposure to the scent of male experimenters before ketamine administration activated CA1-projecting entorhinal cortex CRF neurons, and activation of this CRF pathway modulated in vivo and in vitro antidepressant-like effects of ketamine. 3. 6-O-(2-18FFluoroethyl)-6-O-Desmethyl-Diprenorphine (18FFE-DPN) preferentially binds to Mu opioid receptors In Vivo. 6-O-(2-18FFluoroethyl)-6-O-desmethyl-diprenorphine (18FFE-DPN) is regarded as a non-selective opioid receptor radiotracer. Here, we report the first characterization of 18FFE-DPN synthesized from the novel precursor, 6-O-(2-tosyloxyethoxy)-6-O-desmethyl-3-O-trityl-diprenorphine (TE-TDDPN), using a one-pot, two-step nucleophilic radiosynthesis to image opioid receptors in rats and mice using positron emission tomography. We also show that 18FFE-DPN and 3HDPN exhibit negligible brain uptake in mu opioid receptor (MOR) knockout mice. Taken together with prior findings, our results suggest that 18FFE-DPN and 3HDPN preferentially bind to MOR in rodents in vivo. 4. Exome-wide association study of treatment-resistant depression suggests novel treatment targets. Here we investigated the hypothesis that uncommon, putatively functional genetic variants are associated with TRD. At the gene level, 5 genes, ZNF248, PRKRA, PYHIN1, SLC7A8, and STK19 each carried exome-wide significant excess burdens of variants in TRD cases (q < 0.05). Analysis of 41 pre-selected gene sets suggested an excess of uncommon, functional variants among genes involved in lithium response. Among the genes identified in previous TRD studies, ZDHHC3 was also significant in this sample after multiple test correction. ZNF248 and STK19 are involved in transcriptional regulation, PHYIN1 and PRKRA are involved in immune response, SLC7A8 is associated with thyroid hormone transporter activity, and ZDHHC3 regulates synaptic clustering of GABA and glutamate receptors. 5. Mu opioid receptor activation mediates (S)-ketamine reinforcement in rats. (S)-ketamine is an NMDA receptor antagonist, but it also binds to and activates mu opioid receptors (MORs) and kappa opioid receptors in vitro. However, the extent to which these receptors contribute to (S)-ketamine's in vivo pharmacology is unknown. We investigated the extent to which (S)-ketamine interacts with opioid receptors in rats by combining in vitro and in vivo pharmacological approaches, in vivo molecular and functional imaging, and behavioral procedures relevant to human abuse liability. We found that the preferential opioid receptor antagonist naltrexone decreased (S)-ketamine self-administration and (S)-ketamine-induced activation of the nucleus accumbens, a key brain reward region. A single reinforcing dose of (S)-ketamine occupied brain MORs in vivo, and repeated doses decreased MOR density and activity and decreased heroin reinforcement without producing changes in NMDA receptor or kappa opioid receptor density. These results suggest that (S)-ketamine's abuse liability in humans is mediated in part by brain MORs.