All experiments involving animals were approved by The Rockefeller University Institutional Animal Care and Use Committee and were in accordance with the National Institutes of Health guidelines. Constitutive Ahnak knockout (KO) and Floxed Ahnak mice were generated and maintained at The Rockefeller University as described previously (Jin et al., 2020). Floxed Ahnak mice were crossed with EMX-Cre (stock 005628, The Jackson Laboratory) to generate forebrain glutamatergic neuron-specific Ahnak KO line. The crossings of floxed Ahnak with EMX-Cre were assisted by in vitro fertilization (IVF) and embryo transfer techniques (Transgenic and Reproductive Technology Center, The Rockefeller University), which provide genotype- and age-matched animals. All mice are male of C57BL/6 background, except for CD1 aggressors (strain 022, Charles River, Kingston, NY), used for CSDS. Mice were housed 3–5 per cage with a 12:12 h light/dark cycle and ad libitum access to food and water. All behavioral tests were performed during the light cycle. All behavioral experiments with the CSDS paradigm commenced with male mice aged 8–12 weeks old. In addition, ~ 4-month-old constitutive Ahnak KO and WT controls were used for the test of ketamine and fluoxetine. Transgenic mice were assigned randomly to experimental stress conditions based on their genotype. For AAV-mediated gene delivery experiments, transgenic mice were randomly assigned to experimental virus groups and then experimental stress conditions. Mice from each group were evenly assigned to equipment and run in parallel during behavioral tests.

All stereotaxic surgeries were performed on an Angle Two Small Animal Stereotaxic Instrument (Leica Biosystems, Buffalo Grove, IL) with a microinjection syringe pump (UMP3 UltraMicroPump, World Precision Instruments, Sarasota, FL), as described previously (Bhatti et al., 2022). Male mice (7–8 weeks of age) were anesthetized with a mix of ketamine (100 mg/mL) and xylazine (10 mg/mL). AAV5-hSyn-GFP and AAV5-hSyn-Cre-GFP were obtained from UNC Vector Core. Viruses (300 nL/side) were injected bilaterally into the PFC (AP + 1.6 ML + 0.4 DV-2.3 mm relative to bregma) with a 2 mL Hamilton Neuros syringe at a speed of 0.1 μL/min, targeting primarily prelimbic cortex. The needle was left for an additional 10 min and then slowly withdrawn. Mice were monitored for 48 h to ensure full recovery from the surgery. Experiments commenced 3 weeks after stereotaxic surgery to allow optimal expression of AAVs.

Chronic social defeat stress (CSDS) was carried out as described previously (Golden et al., 2011; Bhatti et al., 2022). Retired male breeder CD-1 mice were screened for 3 consecutive days, and aggressors were selected according to the following criteria: (i) the latency to the initial attack was under 60 s and (ii) the screener mouse was attacked for 2 consecutive days. For 10 consecutive days, the experimental mice were placed in the home cage of a prescreened CD-1 aggressor for 5 min of physical attack and then separated by a perforated divider for the remaining 24 h until the next defeat of 24 h. Each experimental mouse was exposed to a different aggressor each day. In parallel, stress-naïve control mice were placed in pairs within an identical home cage setup separated by a perforated divider for the duration of the defeat sessions. They were never in physical or sensory contact with CD-1 mice. After 10 days of social defeat, all aggressors and experimental mice were separated and singly housed. The social interaction (SI) test was conducted after 24 h.

Subthreshold social defeat stress (SSDS) was carried out as described previously (Cheng et al., 2019). Experimental mice were placed in the home cage of a prescreened CD-1 aggressor for 5 min of physical attack. After 15 min, the experimental mice were introduced to another 5 min of physical attack by a novel CD-1 aggressor. This procedure was repeated once more for three defeat sessions. The SI test was performed 24 h later.

Social interaction (SI) test was conducted as described previously (Golden et al., 2011). The test was composed of two phases, each consisting of 150 s, where the experimental mice were allowed to explore an open field (42 cm × 42 cm × 42 cm) with a wire mesh enclosure (10 cm wide × 6.5 cm deep × 42 cm high). In the first phase, the wire mesh enclosure was empty. In the second phase, a novel CD-1 aggressor mouse was placed inside the wire mesh. The amount of time the experimental mice spent in the interaction zone surrounding the wire mesh enclosure was collected and analyzed by the video-tracking apparatus and software EthoVision XT 7 (Noldus Information Technology, Leesburg, VA). The SI ratio was calculated by dividing the amount of time the experimental mice spent in the interaction zone in phase two by the time in phase one [SI Ratio = (phase 2 time)/(phase 1 time)]. Susceptible mice were defined by an SI ratio under 1, whereas resilient mice were defined by an SI ratio greater than 1 (Golden et al., 2011).

Sucrose preference test (SPT) was adapted from previous studies (Jin et al., 2020). During the 1-day habituation period, mice were given a choice of two water bottles. The following day, bottles were replaced with new bottles containing either water or 2% sucrose solution. The consumption of water and sucrose solution was measured after 24 h. The sucrose preference was represented as percent preference for sucrose [(sucrose consumed/(water+sucrose consumed)) × 100].

Novelty suppressed feeding (NSF) test was conducted as described previously (Lee et al., 2015). The Ahnak KO or WT mice were given saline (vehicle), ketamine (10 mg/kg, i.p.) or fluoxetine (15 mg/kg, i.p., daily for 3 weeks) and then subjected to food deprivation for 24 h in the home cage (after the last injection in case of fluoxetine). The mice were habituated in the test room for 30 min. After placing the mice in the corner of the test box (42 × 42 × 30.5 cm), the latency to eat a food pellet located in the center of the test box was measured. After the test, the mice were returned to their home cage and allowed to eat food pellets for 10 min, and food consumption was measured for home cage feeding.

Forced swim test (FST) and tail suspension test (TST) were conducted as described previously (Lee et al., 2015). We used an automated TST/FST device (Clever Sys, Reston, VA, USA) for measuring the duration of behavioral immobility. For the TST, the mice were suspended by the tail and video-recorded for 6 min. Immobility during the last 4 min was analyzed. For FST, the mice were placed in a glass cylinder (15 cm diameter, 35 cm height), which was filled with water to a height of 15 cm (22–24°C), and video-recorded for 6 min. Immobility time during the last 4 min was analyzed.

WT and Ahnak KO mice were given chronic fluoxetine (15 mg/kg, i.p., daily for 3 weeks) or a single injection of ketamine (10 mg/kg, i.p., Sigma–Aldrich). After 24 h of the last injection, the first behavioral test (NSF) was conducted. In the CSDS experiment, susceptible mice were identified based on the SI test. Ketamine administration (10 mg/kg, i.p.) or (2R, 6R)-hydroxynorketamine (HNK, 10 mg/kg, i.p.) were used. Fluoxetine, ketamine, or HNK were dissolved in saline (0.9% sodium chloride), and saline was used as a vehicle.

(2R, 6R)-HNK was prepared using racemic NK (norketamine) as described in the study conducted by Zanos et al., 2016. Commercially available racemic NK (norketamine) was resolved using (L)-(+)-tartaric acid, and the resulting enantiomerically pure (R)-NK. tartrate salt was basified by Aq. NaOH and concomitantly Boc-protected using Boc₂O under the basic condition. Resulting N-Boc-protected (R)-NK was converted into TMS-enol ether using LDA and TMSCl treatment, and the product was epoxidized using mCPBA and treated with TBAF giving N-Boc-protected (2R, 6R)-HNK. The latter product was N-Boc deprotected using TFA, and resulting (2R, 6R)-HNK.TFA salt was basified using Aq. NaHCO₃. Subsequently, the mixture was extracted using EtOAc and precipitated using 4 M HCl in dioxane to afford the (2R, 6R)-HNK.HCl salt. Identity of all products was determined using spectral (¹HNMR and MS) analysis and compared with the reported data (Zanos et al., 2016). Purity and enantiomeric purity were determined using HPLC and chiral HPLC (chiralpak AD column, eluants: 60% EtOH in hexanes) (Zanos et al., 2016).

Mice were euthanized with CO₂. After decapitation and removal of the brains, transversal slices (300 μm thickness) were cut using a Vibratome 1,000 Plus (Leica Microsystems, USA) in cold NMDG-containing cutting solution (in mM): 105 NMDG (N-Methyl-D-glucamine), 105 HCl, 2.5 KCl, 1.2 NaH2PO4, 26 NaHCO3, 25 Glucose, 10 MgSO4, 0.5 CaCl2, 5 L-ascorbic acid, 3 sodium pyruvate, and 2 thiourea (pH was approximately 7.4, with osmolarity of 295–305 mOsm). After cutting, slices were left to recover for 15 min in the same cutting solution at 35°C and for 1 h at room temperature (RT) in recording solution (see below). Whole-cell patch-clamp recordings were performed with a Multiclamp 700B/Digidata1550A system (Molecular Devices, Sunnyvale CA, USA). The extracellular solution used for all recordings contained (in mM): 125 NaCl, 25 NaHCO3, 2.5 KCl, 1.25 NaH2PO4, 2 CaCl2, 1 MgCl2, and 25 glucose (bubbled with 95% O₂ and 5% CO₂). The slice was placed in a recording chamber (RC-27 L, Warner Instruments, USA) and constantly perfused with oxygenated CSF at room temperature.

For measuring the action potential firing, we used whole-cell current-clamp recordings. The intracellular solution contained (in mM): 126 K-gluconate, 4 NaCl, 1 MgSO4, 0.02 CaCl2, 0.1 BAPTA, 15 glucose, 5 HEPES, 3 ATP, and 0.1 GTP (pH 7.3). Small currents were injected to the cells to bring the membrane potential at −70 mV. Consecutive current steps of 25 pA were injected to induce depolarization. The frequency of action potentials was measured using the first two action potentials evoked by the respective injected current. Action potential properties were measured from the first action potential to avoid any confounding effects of adaptation.

For measuring mini excitatory postsynaptic currents (mEPSC), TTX (0.5 mM) and bicuculline (10 mM) were added to the recording solution to block action potentials and GABA receptors, respectively. Cell membrane potential was measured at −70 mV.

Data were acquired at a sampling frequency of 100 kHz, filtered at 1 kHz, and analyzed offline using pClamp10 software (Molecular Devices, Sunnyvale, CA, USA). All electrophysiological data are expressed as means ± SEM. Statistical analysis was conducted by Student’s t-test using GraphPad Prism 5. In all experiments, p < 0.05 was considered significant.

Mouse PFC tissues were lysed with a lysis buffer (Pierce IP Lysis Buffer, Thermo Fisher Scientific) supplemented with a protease and phosphatase inhibitor cocktail (78,442, Thermo Fisher Scientific). The tissue lysates were homogenized with a Tissue Grinder (10 strokes, 02–911-529, Thermo Fisher Scientific) and centrifuged at 800 × g for 5 min. Protein levels in the supernatant were measured by the BCA method. The samples were mixed with the standard protein sample buffer and boiled on a hot plate for 2 min and subjected to SDS-PAGE with 4–20% Novex Tris-Glycine gels (Thermo Fisher Scientific), followed by protein transfer onto a nitrocellulose membrane. The membranes were immunoblotted with incubation of anti-Ahnak (rabbit polyclonal, RU2064, 1:5,000) or anti-Gapdh (mouse monoclonal, MAB374, Millipore, 1:5,000) primary antibody followed by incubation of horseradish peroxidase-linked goat anti-rabbit or anti-mouse secondary antibody (1:5,000, Thermo Fisher Scientific). Antibody binding was detected using the enhanced chemiluminescence immunoblotting detection system (Perkin Elmer LLC WESTERN LIGHTNING PLUS-ECL, Thermo Fisher Scientific) and the Kodak autoradiography film. The bands were quantified by densitometry using NIH Image 1.63 software.

All data are expressed as means ± SEM except pie charts and correlation graphs. Sample sizes for biochemistry, electrophysiology, and CSDS were determined based on our empirical data accumulated in the laboratory and previous studies using CSDS (Berton et al., 2006; Krishnan et al., 2007; Menard et al., 2017). Sample sizes and statistical methods are provided in each figure legend. The statistical analysis was conducted using two-tailed unpaired Student’s t-test, one-way ANOVA, two-way ANOVA, or repeated-measures two-way ANOVA. ANOVA of paired Gaussian distributions was followed with Bonferroni’s or Newman–Keuls post-hoc test. The D’Agostino–Pearson omnibus normality test was used to determine normality. GraphPad Prism was used for statistical analysis and graphical preparations. p < 0.05 was considered the cutoff for statistical significance. Statistical significance is shown as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, otherwise indicated in figures or figure legends.

In this study, we used the mouse CSDS paradigm, in which mice are subjected to repeated exposure of ethologically relevant stressors such as physical defeat and sensory contact with aggressive conspecifics to elicit social avoidance and anhedonic-like behavior (Krishnan et al., 2007; Golden et al., 2011). These behavioral phenotypes are determined by decreased time in social interaction (SI) with a novel aggressor and the amount of sucrose reward consumed in the home cage, respectively. Notably, an SI ratio comprising of the ratio of time spent in the interaction zone in the presence or the absence of a novel aggressor allows experimenters to divide cohorts on mice based on stress-induced behavioral outcomes. Specifically, using this SI ratio after CSDS, a proportion of mice is deemed as stress-susceptible (SI ratio < 1) or stress-resilient (SI ratio ≥ 1) (Krishnan et al., 2007; Golden et al., 2011).

Ahnak is expressed in the layer II/III of the PFC (Jin et al., 2020). First, we sought to examine whether behavioral outcomes after CSDS were associated with changes in its expression. After CSDS and SI, the PFC tissue was collected from stress-resilient, stress-susceptible, and non-defeated control mice to assess protein expression using immunoblotting (Figure 1A). Ahnak protein levels were significantly higher in the PFC of resilient mice compared with the levels in susceptible mice or non-defeated control mice (Figures 1B,C). Ahnak expression in the PFC of mice exposed to CSDS was also positively correlated with time spent in the interaction zone containing an aggressive conspecific (Figure 1D) and SI ratio (Figure 1E), suggesting that Ahnak may play a role in CSDS-induced behavioral adaptations.

To assess whether Ahnak in the PFC is required for behavioral responses to chronic stress, we first examined whether selective deletion of Ahnak in the PFC would alter the behavioral consequence of CSDS. To achieve conditional deletion of Ahnak in a region-specific manner, we injected adeno-associated virus (AAV) expressing Cre recombinase fused to GFP, or empty vector GFP as a control, into the PFC of floxed Ahnak mice (cKO^PFC). After 3 weeks, mice were subjected to CSDS (Supplementary Figure S1A). We found that conditional deletion of Ahnak in the PFC resulted in higher proportion of stress-susceptible compared with control (Supplementary Figures S1B–D) as indicated by increased social avoidance after CSDS.

We also employed subthreshold social defeat stress (SSDS), a submaximal stressor, which does not evoke social avoidance in wild type (WT) mice but has been used to assess stress-susceptible factors (Krishnan et al., 2007; Golden et al., 2011; Cheng et al., 2019). Ahnak cKO^PFC and AAV-GFP control mice were subjected to SSDS, and subsequently social interaction and sucrose preference were measured (Figure 2A). SSDS exposure induces social avoidance as measured by decreased SI ratio and time and increases the proportion of susceptible mice in Ahnak cKO^PFC mice, while littermate controls maintained social interaction with novel aggressors (Figures 2B–D). Additionally, defeated Ahnak cKO^PFC mice display anhedonic-like behavior as measured by decreased sucrose consumption compared with defeated control mice (Figure 2E). However, non-defeated Ahnak cKO^PFC mice and control mice display comparable behaviors in SI test (Figures 2B,D) and SPT (Figure 2E). These results suggest that Ahnak is required for maintaining adaptive resilience to low-intensity stressors since its deletion results in increased behavioral measures of susceptibility such as social avoidance and anhedonic-like behavior. Across nearly all the experiments, there was no significant difference in locomotor activity between control and Ahnak cKO^PFC groups during social interaction test or in open field test (Supplementary Figures S1E–G). This is consistent with no difference in locomotor activity between WT and Ahnak null mice and Ahnak cKO^EMX mice (Jin et al., 2020).

In our previous study, we observed baseline anhedonic-like and passive coping behaviors in mice with Ahnak deletion in Emx1-expressing forebrain glutamatergic neurons (cKO^EMX) compared with floxed (fl/fl) littermate controls (Jin et al., 2020). To assess whether Ahnak cKO^EMX mice also display behavioral stress-susceptibility, we exposed cKO^EMX and control mice to SSDS, and subsequently, behavioral tests, SI and SPT, were performed (Figure 3A). Defeated Ahnak cKO^EMX mice showed decreased social preference, as measured by SI ratio and time interacting with a novel aggressor conspecific and an increased proportion of mice with behavioral-susceptibility compared with defeated control fl/fl mice (Figures 3B–D). Non-defeated Ahnak cKO^EMX mice and control mice display comparable behaviors in SI test (Figures 3B,C). However, both non-defeated and defeated Ahnak cKO^EMX mice show a decrease in sucrose consumption compared with controls in SPT (Figure 3E). The reduced sucrose consumption in non-defeated Ahnak cKO^EMX mice is due to their baseline depression-like behavior (Jin et al., 2020). The behavioral responses to social stress in Ahnak cKO^EMX mice are consistent with those found in Ahnak cKO^PFC mice (Figure 2). Altogether, our biochemical (Figure 1) and behavioral data derived from cell- and region-specific Ahnak knockout mice (Figures 2, 3) suggest an idea that Ahnak expressed in glutamatergic neurons in the PFC may play a critical role in behavioral resilience to social stress.

Previous study has implicated p11, a key binding partner of Ahnak, in the actions of different classes of antidepressants including fluoxetine, a traditional SSRI antidepressant (Oh et al., 2013; Svenningsson et al., 2013), and ketamine, a fast-acting antidepressant (Sun et al., 2016). To determine whether Ahnak also mediates the behavioral effects of antidepressants, we examined the behavioral effects of ketamine and fluoxetine on WT and constitutive Ahnak KO mice. Both chronic fluoxetine and acute ketamine administrations result in a series of behavioral outcomes including decreased latency to feed in the novelty-suppressed feeding (NSF) test, a measure of suppressed hunger-induced exploration in a novel anxiogenic arena (Dulawa and Hen, 2005; Oh et al., 2013; Lee et al., 2015), and decreased immobility in the forced swim test (FST), a measure of engagement of passive coping strategies under threat (Commons et al., 2017). These behavioral outcomes are often used to measure antidepressant efficacy. We observed that the behavioral effects of ketamine were prevented in Ahnak KO mice in both the NSF test and FST (Figures 4A–C). However, chronic administration of fluoxetine similarly reduced both latency to feed and immobility time in WT and Ahnak KO mice (Figures 4D–F). While p11 together with its binding partner SMARCA3 mediate neurogenic and behavioral effects of SSRIs (Oh et al., 2013), our data suggest that Ahnak is dispensable in the actions of fluoxetine, but it is essential for the behavioral effects of the rapid antidepressant, ketamine. Together, these findings suggest that Ahnak-mediated mechanism for ketamine is distinct from the SMARCA3-mediated mechanism for SSRIs.

Despite Ketamine’s success for treatment-resistance depression (Krystal et al., 2020), the underlying mechanisms remain elusive. One key hypothesis underlying the restorative effects of fast-acting antidepressants is the enhancement of glutamatergic transmission in the PFC (Gerhard et al., 2020; Krystal et al., 2023). Indeed, ketamine enhances prefrontal plasticity via glutamatergic mechanisms (Moda-Sava et al., 2019), and the enhancement of excitatory neuron activity in the PFC is suggested as a critical mediator of antidepressant actions of ketamine (Abdallah et al., 2018; Wu et al., 2021). We, thus, sought to determine whether Ahnak may contribute to these underlying mechanisms due to its requirement for behavioral resilience via PFC and glutamatergic neuron mechanisms (Figures 2, 3). To assess the possibility of Ahnak as a molecular substrate of ketamine actions in the PFC, we first tested whether ketamine promotes Ahnak expression in susceptible mice to a level comparable to that of resilient mice. To do so, WT mice were subjected to CSDS, and then stress-susceptible mice were selected based on SI test scores. Saline (vehicle), ketamine (10 mg/kg, IP), or its active metabolite (2R, 6R)-hydroxynorketamine (HNK) (10 mg/kg, IP) (Zanos et al., 2016) were injected to susceptible mice (Figure 5A). After 24 h, PFC tissues were collected for immunoblotting. Ahnak protein levels were significantly increased in the PFC of ketamine- or HNK-injected susceptible mice compared with the vehicle-injected susceptible mice (Figures 5B,C).

To determine whether this increase in Ahnak expression in the PFC is required for ketamine-induced behavioral changes, we subjected Ahnak cKO^EMX and their littermate controls to CSDS. We again selected stress-susceptible mice based on SI ratio (SI-1) (Supplementary Figures S2A–C), administered drugs (vehicle, ketamine, or HNK), and conducted the SI test again (SI-2) (Figure 6A). Expectedly, ketamine and HNK increased SI ratio and time in fl/fl littermate controls, indicative of restoring ‘behavioral stress-resilience’ (Figures 6B,C). Ahnak deletion in the glutamatergic forebrain (cKO^EMX mice), however, abolished the behavioral effects of ketamine or HNK (Figures 6B,C), suggesting that Ahnak in glutamatergic forebrain neurons is required for antidepressant-like effects of ketamine or HNK. Locomotor activity between WT and Ahnak cKO^EMX mice was comparable during social interaction tests before and after drug administration (Supplementary Figures S2D,E). Altogether, these results suggest an essential role of Ahnak in glutamatergic neurons of the PFC in antidepressant-like behavioral effects of ketamine or its metabolite HNK.

One mechanism by which Ahnak may be critical for fast antidepressant action is through the modulation of glutamatergic transmission. We, thus, examined the effect of Ahnak deletion on glutamatergic transmission and excitability in the PFC. We used whole-cell patch-clamp and examined the electrophysiological properties in glutamatergic neurons in layer II/III of the PFC (Figure 7A). Layer II/III PFC neurons have been proposed to mediate depression-like or stress-induced behavioral abnormality in mice (Shrestha et al., 2015; Seo et al., 2017), and Ahnak and p11 are expressed in layer II/III (Seo et al., 2017; Jin et al., 2020). Ahnak was characterized as a regulator of L-type voltage-gated calcium channels, and ~ 50% of L-type calcium influx was reduced in the layer II/III pyramidal neurons in Ahnak cKO^EMX mice (Jin et al., 2020). Ahnak cKO^EMX did not alter the frequency of action potentials (Figures 7B,C), suggesting that neuronal excitability is unaffected by Ahnak deletion. However, we found that the frequency, but not the amplitude, of mini excitatory postsynaptic currents (mEPSCs) in glutamatergic neurons in layer II/III of the PFC of Ahnak cKO^EMX were markedly reduced compared with fl/fl controls (Figures 7D–F). Thus, Ahnak expression may contribute to behavioral resilience or behavioral effects of ketamine via the modulation of glutamatergic transmission.