The GluN2D part of NMDA receptors may be a target for fast-acting antidepressants

NAB-14 (an N-aryl benzamide, NAB) is a GluN2C/GluN2D-selective negative allosteric modulator. As the GluN2C subunit is poorly expressed in adult mice, NAB-14 is selective for GluN2D in our experimental approach. Site-directed mutagenesis experiments indicated that NAB-14 binds to a site between subunits in the transmembrane domain, specifically interacting with the GluN2D M1 helix. To gain insight into this interaction, we computationally docked NAB-14 against a published cryo-EM structure of GluN1A/GluN2D NMDARs (PDB ID: 7YFF). The putative binding pose indicates that NAB-14 forms pi-stacking interactions with W586 and F590 via its indole and benzene moieties, respectively. It also forms an H-bond with W637 as well as hydrophobic interactions with M589, L594, I633, and M634 (Fig. ). A recently reported cryo-EM structure of the TDM domain of the GluN1b/GluN2D NMDAR in complex with the allosteric GluN2C/GluN2D antagonist YY-23 confirmed the location of the proposed NAB-14 binding site. Furthermore, using the same approach, we found that the binding pocket of the GluN2B-NAM Ifenprodil is clearly distinct from that of NAB-14. In hippocampal brain slices, the addition of NAB-14 to the bath solution dose-dependently reduced the amplitude of NMDAR currents in INs to a higher extend than in PCs (Fig. ). Increasing concentrations of NAB-14 inhibited NMDAR currents significantly in INs but not in PCs (Supplementary Fig. ). Other studies have shown that NAB-14 has lower inhibitory potency on triheteromeric GluN1A/2B/2D NMDARs than on GluN1A/2D NMDARs. Par9

2+ , KET binds to the phencyclidine-binding site within the channel pore of the NMDAR. This results in a use-dependent open-channel block that decreases the mean channel open time. Furthermore, an allosteric closed block might decrease the open frequency, causing trapping of KET in the channel pore. KET is rather unselective for GluN2 subunits; however, under physiological conditions with 1 mM Mgadded to the bath solution, KET shows 5- to 10-fold selectivity for GluN2C- and GluN2D-containing receptors over GluN2A and GluN2B. In our experiments, KET preferentially inhibited NMDAR currents in INs compared to PCs at most concentrations (Fig. ). Increasing concentrations of KET dose-dependently and significantly decreased the amplitude of NMDA EPSCs both in PCs and in INs (Supplementary Fig. ). Par10

Negative allosteric modulators of GluN2B are currently under investigation as potential antidepressants. To distinguish between the mechanisms of action of GluN2D and GluN2B inhibition, we assessed the modulation of NMDAR-mediated currents by different concentrations of the GluN2B-selective NAMs Ifenprodil and RO25-6981. At a high concentration (100 µM), Ifenprodil significantly reduced NMDAR currents in PCs and INs compared to baseline, with no significant difference in effect between the two cell types (Supplementary Fig. ). RO25-6981 did not significantly affect NMDAR currents compared to baseline in either PCs or INs, and no significant differences were observed between cell types at any tested concentration (Supplementary Fig. ). This differential cell-type sensitivity clearly distinguishes the pharmacological profiles of GluN2D and GluN2B NAMs. Par11

The FFL was selectively activated by subthreshold Schaffer collateral stimulation, i.e., in the absence of PC action potentials (APs). The wash-in of 20 µM but not of 10 µM NAB-14 dose-dependently increased EPSP amplitudes in CA1-PCs (Fig. ). Under these recording conditions, the increase in EPSPs is primarily driven by the inhibition of PV-INs by NAB-14. PV-INs express lower levels of GluN2D compared to SOM-INs. This may account for the lower efficacy of 10 µM NAB-14 in this experiment relative to the direct measurement of NMDAR currents in stratum oriens (Fig. ), where SOM-INs predominate. In contrast, low- and very high-dose KET did not significantly modify the EPSP amplitudes (Fig. ). During the very-low-frequency stimulation of Schaffer collaterals performed in these experiments, which evoked single APs in FFL-INs and allowed a reset of the NMDAR to a closed state between each stimulation event, the open channel NMDAR blocker KEThad no effect. In contrast, the negative allosteric modulator NAB-14 reduces NMDAR channel conductance independent of its opening state. The different mechanisms of NMDAR inhibition by the open channel blocker KET and the negative allosteric modulator NAB-14 might therefore explain the differential modulation of EPSP amplitudes by selective activation of the FFL in the absence of PC APs. Par13

,, FBL-INs are highly dynamically recruited by excitatory PC activity and generate slow inhibitory postsynaptic currents (IPSCs) in PCs mainly by activation of SOM-INs, effectively controlling PC NMDAR activation and synaptic plasticity. To activate FBL-INs, we repeatedly injected a depolarizing current into the soma of CA1-PCs, evoking a burst of APs. Subsequently, after each AP burst, an EPSP was induced by Schaffer collateral stimulation, which served as a readout for the effects of the modulation of the excitation/inhibition (E/I) balance on glutamatergic transmission in PCs (microcircuit-activating [MICA] protocol; Fig. ). After achieving a stable baseline for 10 min, NAB-14 was added to the bath solution, which increased the EPSP amplitudes and decay time constants compared to those of the control condition without NAB-14 (10 µM, Fig. and Supplementary Fig. ). Similar effects were observed with KET (10 µM; Fig. and Supplementary Fig. ). In contrast to the FFL-activating protocol (Fig. ), the MICA protocol induces a train of APs in PCs and INs so that the open channel blocker KET can effectively inhibit NMDAR currents in INs. Par14

m Coefficient of variation (CV) analysis of the EPSP slopes before and after the wash-in of NAB-14 was consistent with a predominant mechanism at the dendrites of postsynaptic neurons (Supplementary Fig. ). PC excitability, as assessed by the PC input resistance (R) and the number of APs during the burst, did not significantly change in the presence of either NAB-14 or KET (Supplementary Fig. ). We therefore concluded that GluN2D inhibition of interneurons shapes the amplitude and time course of synaptic transmission in PCs. Par15

Next, we assessed how GluN2D inhibition in interneurons modulates excitatory activity in hippocampal PCs. Using a protocol similar to that used in the MICA experiments, Widman and McMahon demonstrated that KET increases the probability of synaptically driven AP. In our experiments, NAB-14 and KET caused a pronounced conversion of EPSPs to APs at a fixed extracellular stimulation intensity (Fig. and Supplementary Fig. ). Par16

Taken together, these results suggest that GluN2D-mediated recruitment of GABAergic interneurons powerfully controls feedback and feedforward inhibitory circuits to moderate hippocampal network activity.

These results from the mPFC suggest that the microcircuit modulation by selective GluN2D inhibition, as demonstrated in the hippocampus as a model region, translates into increased event-related bulk excitatory network activity.

A , , , Since PV-INs modulate PC input at proximal dendrites, reduced PV-IN activity is concordant with an increase in EPSP amplitude and a conversion of subthreshold EPSPs to APs. SOM-INs preferentially activate α5-subunit-containing GABAreceptors at CA1 PC dendrites and play a key role in dendritic computation and the shaping of LTP. Moreover, the suppression of SOM-IN activity by KET has resulted in increased synaptically evoked calcium transients in the apical dendritic spines of pyramidal neurons in the mPFC. Therefore, it is unclear whether GluN2D inhibition, especially by the partially selective antagonist KET, increases LTP induction in the hippocampus or potentially decreases LTP via direct inhibition of NMDA receptors in CA1 PCs. Par20

To assess the putative role of increased glutamate release in the modulation of LTP by NAB-14 or KET, we determined the paired-pulse ratio (PPR) before and after the induction of weak-aLTP. The PPR was unchanged in both the absence and presence of 10 µM NAB-14 or 5 or 10 µM KET (Supplementary Fig. ), indicating a postsynaptic effect. This suggests that an altered E/I balance and its computation in the dendrites of postsynaptic cells are sufficient for the modulation of synaptic plasticity and that a decisive role for a 'glutamate burst', as proposed for KET, is unlikely. Par22

LTP induction was completely abolished in ex vivo brain slices from mice injected with the control solution used to dissolve NAB-14 and sacrificed two days after the RS protocol. RS-induced LTP blockade could be reversed in a dose-dependent manner after intraperitoneal injection of 5 or 10 mg/kg NAB-14 (Fig. and Supplementary Fig. ). KET injection (10 mg/kg) equally restored LTP induction (Fig. ). Par25

Grin2d Grin2d Grin2d As a nonpharmacological method for interfering with GluN2D, we used a small interfering RNA (siRNA) to achieve posttranslational gene silencing of, the gene encoding GluN2D. We administered mouse-siRNA (50 nM/animal) by intrathecal injection using in vivo jetPEI as a nonviral delivery vector. Injections were performed the day after the termination of the RS protocol; thereafter, the animals were left in their home cages for three more days (Supplementary Fig. ). Real-time PCR was used to verify thatRNA expression was downregulated in the hippocampus and frontal cortex (Supplementary Fig. ). The RS-induced decrease in LTP was fully reversed after siRNA treatment (Fig. ). Overall, the results of the RS-LTP experiments showed that KET and GluN2D inhibition reversed LTP deficits in an animal model of depression. Par26

Additionally, we recorded spontaneous EPSP activity in hippocampal slices from naïve animals, following the RS protocol, and 24 hours after i.p. treatment of RS mice with 10 mg/kg KET or NAB-14. We observed a significant reduction in spontaneous excitatory activity after RS, which was restored by both NAB-14 and KET (Fig. ). These findings support the notion that GluN2D inhibition modulates the E/I balance in a way that facilitates excitatory network activity. Par27

, Both LTP and spine growth are highly BDNF dependent. Protein kinase B/AKT and the extracellular signal-regulated kinase ERK are important signaling molecules downstream of the BDNF-TrkB receptor. KET rapidly upregulates the phosphorylated and activated forms of both p-AKT and p-ERK. Pharmacological inhibition of DUSP6, which is dephosphorylating ERK, could prolong KET's effect on synaptic plasticity, synaptogenesis, and behavior. The activation of ERK is tightly linked to GluN2B. To further differentiate the mechanism of action of GluN2D and GluN2B inhibition, we incubated hippocampal slices with NAB-14, KET and the GluN2B-NAM RO25-6981 for 45 min and assessed the level of phosphorylated AKT (p-AKT) and ERK1/2 (p-p44, p-p42) by Western blot. We found that NAB-14 significantly increased p-AKT, but not p-p44 and p-p42, whereas RO25-6981 increased p-p44 and p-p42, but not p-AKT. KET increased the phosphorylated forms of all molecules (Supplementary Fig. ). These results link GluN2D to AKT and confirms the coupling between GluN2B and ERK. It further supports that the mechanism of action of KET involves several targets. Par29

, Grin2d In the RS animal model of depression, the immobility time, conceptualized as a measure of behavioral despair, gradually increased during the daily swim sessions, reaching a plateau at days 4-5, and remained stable in the delayed swim session on day 8 (Supplementary Fig. ). In previous experiments, we observed a persistent increase in immobility time for up to four weeks. Thereafter, the immobility time returns to baseline levels without further intervention. 24 or 48 h after a single intraperitoneal injection of either 5 or 10 mg/kg NAB-14 or 10 mg/kg KET, as well as 72 h after the intrathecal administration ofsiRNA, immobility time was significantly reduced compared to day 5 in the respective control condition. I.p. administration of saline had no effect (Fig. ). Par31

Grin2d As an alternative behavioral readout after RS, we performed the nose-poke sucrose preference test(NP-SPT) using a progressive ratio reinforcement schedule to assess wanting-type anhedonia and reward behavior. Compared with naïve animals, RS mice showed a significantly reduced sucrose preference. Reward behavior was restored by a single injection of NAB-14 or KET and by the application ofsiRNA but not by the application of a vector or scrambled siRNA (Fig. ). Par32

We conclude that escape and reward behavior is compromised in an animal model of depression. Inhibition of GluN2D after RS normalized behavior to a level comparable to that of naïve animals; this indicates that GluN2D-mediated recruitment of inhibitory circuits powerfully controls depression-related behavior. GluN2D inhibition interferes with this process and has antidepressant-like activity in an animal model of depression.

Finally, we tested for acute behavioral alterations following GluN2D inhibition alone. We assessed motor coordination with the rotarod test. After a single KET injection, the latency to fall was significantly shorter than that after NAB-14 or vector injection (Fig. ). This might be due to increased oscillatory activity in the basal ganglia induced by KET. As GluN2D is not expressed in the striatum, these effects might be mediated by the action of KET on GluN2B receptors, which are abundantly expressed in striatal projection neurons. Locomotion was unchanged after both NAB-14 and KET administration but decreased after administration of the benzodiazepine lorazepam, which was used as an active control (Fig. ). The time spent in the center in the open field test, which has been conceptualized as a measure of anxiety, was significantly decreased after KET but not after NAB-14 injection in comparison to that after vector injection (Fig. ). Previous studies have reported acute anxiogenic effects of ketamine, whereas its anxiolytic properties at later time points—consistent with its antidepressant effects—are well documented. In terms of motor coordination and anxiety-like behavior, the selective GluN2D antagonist NAB-14 produced fewer acute side effects than KET. Par35

All experimental procedures were approved by the relevant authorities in Germany (Regierungspräsidium Freiburg, TV-G-20-106, TV-G-20-87, TV-G-19-10, TV-G20-141, TV-G21-69, TV-G22-116, TV-G-23-038, TV-G-24-033, TV-G-24-094) or France (CREMEAS, APAFIS n°2020042818477700), and conducted in accordance with EU Directive 2010/63/EU. Prior power calculations for behavioral assessments were performed to minimize the number of animals used.

All pharmacokinetic studies were conducted in rats at Aptuit (Verona, Italy). All experimental procedures complied with the highest standards of animal welfare and were performed in accordance with the Italian Legislative Decree No. 26/2014 and European Directive No. 2010/63/EU. The studies were approved by the internal Aptuit Committee on Animal Research and Ethics and authorized by the Italian Ministry of Health (Project Authorization Code No. 35222). General procedures for animal care and housing followed the current recommendations of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).

Wild-type C57Bl6N mice were obtained from Janvier (Le Genest-Saint-Isle, France) and Thy1-GCaMP6 mice from Jackson Laboratory (C57BL/6J-Tg(Thy1-GCaMP6f)GP5.5Dkim/J; Stock No: 024276/GP5.5). SOM-Cre (SST tm2.1(cre)Zjh/J) mice were provided by the Department of Biomedicine Basel and SOM-IRS-Cre/J from the Department of Physiology of the University of Freiburg. Animals were housed at the accredited facilities of the Universities of Freiburg and Strasbourg. Adult mice (10-14 weeks) were group-housed (up to five per cage) under standardized conditions (12 h light/dark cycle, controlled temperature (21 °C ± 2°) and humidity (55% ± 10%), food and water ad libitum, nesting material and enrichment).

All animals were handled by daily tunnel handling for 5 min over five days prior to behavioral testing to reduce stress. Welfare was monitored at least once daily by trained staff and additionally after invasive procedures, with health assessments including body weight, coat condition, locomotor activity, and posture. For stereotactic and other invasive procedures, anesthesia was induced and maintained with isoflurane, and peri-operative analgesia was provided (carprofen, buprenorphine). At the end of experiments, euthanasia was performed according to downstream analysis: for electrophysiology, mice were preoxygenated in 100% O₂ for 5 min before cervical dislocation and decapitation; for histology, mice were deeply anesthetized and transcardially perfused with fixative; for behavioral cohorts not undergoing perfusion or slice preparation, euthanasia was performed by cervical dislocation and decapitation without preoxygenation.

ad libitum For studies conducted in rats, male Sprague Dawley rats (Crl: CD(SD), 9 weeks old, 250–300 g) were bred and housed at Aptuit facilities in Verona, Italy. Animals were maintained in solid-bottomed plastic cages with sawdust bedding and either external watering systems or water bottles. Two to three rats of the same treatment group were housed per cage under controlled environmental conditions (temperature 20–22 °C; humidity 45–65%; 12 h light/dark cycle with species-appropriate illumination). Rats received a standard maintenance diet (Altromin 1324 IRR, rat diet) and filtered tap water. Par54

Slices from mice expressing tdTomato in SOM-INs (SOM-Cre (SST tm2.1(cre)Zjh/J)) or from wild-type mice were prepared as described above. Interneurons were visually identified by fluorescence imaging at an excitation wavelength of 553 nm and an emission wavelength of 580 nm by using a polychromator (Polychrome IV, Till Photonics, Germany), a fluorescence camera (Orca Flash 4.0, Hamamatsu, Japan), and the SmartLUX extension for Patchmaster NEXT software (HEKA, Germany). In slices from wild-type animals, SOM-INs were identified by their morphological characteristics, their location in the stratum oriens, and an initial membrane potential in the range of −60 mV.

Five EPSPs and five postsynaptic APs were paired at 100 Hz with a 5 ms delay (AP after EPSP). Five of these bursts of synchronized EPSP → AP pairs were applied at theta frequency (5 Hz), followed by an interval of 10 s and 4 more theta blocks, resulting in 125 EPSP/AP pairings.

EPSPs were elicited by Schaffer collateral stimulation at a frequency of 0.1 Hz. The mean baseline EPSP amplitude was calculated at 0-5 min. Substances were continuously applied to the bath solution after baseline recording for an additional 30 min. Mean EPSP amplitudes were calculated between 25 and 30 min after wash-in.

At a holding potential of −70 mV (PCs) or -60 mV (INs), the cells were depolarized to +40 mV for 1 s. Five hundred milliseconds after the beginning of depolarization, extracellular stimulation was applied by Schaffer collateral stimulation (PCs) or by extracellular stimulation (≤ 10 V) in the stratum oriens at a distance of approximately 200 µm from the clamped interneuron (IN). Ten peak current amplitudes with an interval of 30 s were averaged before and after the bath application of substances.

A 300 ms depolarizing current between +100 and +600 pA was injected via the patch pipette into a PC to elicit a train of 5–10 APs. After an interval of 600 ms, one EPSP (5–10 mV amplitude) was elicited by Schaffer collateral stimulation. This pattern was continuously repeated at a frequency of 0.2 Hz. Substances were added by bath application after a stable 10 min baseline recording for an additional 20 min.

Silica cannulas (400 µm core diameter, 0.37 NA, MFC_400/470-0.37_3.0 mm_MF2.5_FLT, Doric Lenses, Canada) were directly implanted in the left hemisphere of the prefrontal cortex (+1.7 AP, -0.3 ML and -2.9 DV relative to bregma) of Thy1-GCaMP6 transgenic mice. After insertion, the cannula was fixed and protected using dental cement (Light Curing Nano Flowable Composite, Dline, Lithuania), solidified using a UV-light emitting device.

, Fiber photometry allows for the measurement of neuronal calcium transients in real time. The fiber photometry apparatus consisted of a Doric fiber photometry console (FPC, Doric Lenses, Canada), a 3-port fluorescent minicube with a built-in LED and detector (ilFMC3, Doric Lenses, Canada), and a USB DAQ board (USB-6210, National Instruments, USA). The minicube was connected to the cannula through a 5-meter low autofluorescence patch cord (400 µm core diameter, 0.37 NA, MFP_400/440/1100-0.37_5m_FCM-MF2.5_LAF, Doric Lenses, Canada). Two LEDs emitted light at 405 nm (isosbestic excitation wavelength) and 470 nm (GCaMP signal). The emission light was collected by the same optical fiber, passed through a GFP filter and reached the built-in photodetector. Data were then collected by the DAQ and a custom-written LabVIEW script (National Instruments, USA) at a sampling rate of 2 kHz. Data were then demodulated using a custom MATLAB (Mathworks, USA) script, and the GCaMP and isosbestic signals were analyzed using the Guppy fiberphotometry python suite. For baseline recordings, the GCaMP channel was divided by the isosbestic channel and expressed as GCaMP/isosbestic. For the event-related recordings, the ∆F/F of the GCaMP/isosbestic signal was calculated (subtraction of the median of the recordings from the raw trace). To measure changes in GCaMP signals during behavior, mouse behavior was video tracked using DeepLabCut v2.3.5. Timestamps were recorded each time the mouse entered a 35-pixel circular boundary around the object to explore. Peristimulus/event time histograms (PSTHs) were then computed using a window of 5 seconds before and 10 seconds after the event timestamp with baseline correction. Par68

For the ERA experiment, other mice were temporarily removed from the home cage prior to the experiment. The test mouse cannula was plugged into the patch cord using a mating sleeve. LED excitation was then activated for a period of 15 minutes for habituation and to minimize initial photobleaching. Then, the camera and fiber photometry recordings were started. The mouse was allowed to move freely in its home cage for baseline recording. After 20 minutes of baseline, the drug (ketamine, NAB-14, or saline) was injected intraperitoneally, and the mouse was allowed to move freely in the home cage for another 20 minutes (after the injection period). Finally, an object was placed into the home cage, and the recording was continued for another 20 minutes.

Neurons were filled with biocytin during electrophysiological recordings. Biocytin (10 mg/2 mL; Sigma, #B4261) was dissolved in internal solution by vortexing, followed by sonication for 10 min, and a final vortex. Neurons were injected with biocytin for 10–15 min while recording. To preserve cellular morphology, electrodes were carefully and rapidly withdrawn after injection. Following recordings, slices were fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for 24 h at 4 °C under gentle agitation. Par70

Fixed slices were incubated in a blocking solution containing Alexa Fluor 488-conjugated streptavidin (1:500) for 48 h at 4 °C or over two days, with gentle agitation. Slices were then washed thoroughly in PBS at room temperature and mounted in ProLong Antifade Gold (Invitrogen).

The blocking solution was prepared by dissolving 5 g sucrose and 2 g bovine serum albumin (BSA; Sigma) in 90 mL PBS, followed by the addition of 9 mL 10% Triton X-100. The solution was warmed to dissolve all components, aliquoted in 2 mL portions, and stored at −20 °C.

Biocytin-filled neurons were visualized using a confocal microscope Zeiss LSM800 equipped with an argon laser. Image stacks were acquired with a physical size of 89.4 × 89.4 μm and a logical size of 2100×2100 pixels. Each stack consisted of 20–50 image planes captured through a Plan-Apochromt 63× oil immersion lens (NA 1.4, working distance 180 μm, refractive index 1.515). To optimize resolution, a zoom factor of 1.5 was applied, resulting in a voxel size of 75.1 × 75.1 × 136.4 nm with a z-step of 0.15 μm. For each mouse, 1–3 randomly selected dendrites were scanned approximately 30-50 µm from the soma. The acquired stacks were processed using a 3D blind deconvolution algorithm (Imaris 9.7.2, Bitplane AG, Zurich, Switzerland).

, Dendrites were traced using Imaris 9.7.2 software (Bitplane AG, Zurich, Switzerland) to quantify dendritic spine density. Oblique apical dendrites from pyramidal neurons in the stratum radiatum of the CA1 region were traced from their proximal to distal tips. Dendritic spines were marked during the tracing process and considered as protrusions for inclusion in the analysis. Spine density was determined by counting all protrusions along the traced dendritic segments. This analysis was performed on 1–2 neurons per mouse, and no factors were applied to correct for spine counts. Par74

Spine head volume was measured using Imaris 9.7.2 software (Bitplane AG, Zurich, Switzerland). For each dendritic segment, various intensity thresholds were applied to generate a model that was visualized as a solid surface using the IsoSurface module. The solid surface corresponding to the contour of each spine head was then selected. The three-dimensional image of each dendrite was rotated and carefully examined to ensure the accuracy of the surface selected for each spine head. Spines without visible heads, which were extremely rare, were excluded from the analysis.

Naive mice were preoxygenated with 100% oxygen for 8 minutes and euthanized by cervical dislocation. Brains were rapidly extracted and placed in carbogenated artificial cerebrospinal fluid (aCSF). The cerebellum was removed to create a flat surface, and the brain was mounted bulbs-up onto a vibratome carrier using water-activated adhesive. Sectioning was performed at 300 µm thickness with a cutting speed of 0.40 mm/s; the blade was raised and retracted after each cut to minimize tissue damage. Slices were immediately transferred into carbogenated aCSF for incubation.

Slices were incubated in aCSF containing either ketamine-hydrochloride (10 µM), NAB-14 (10 µM), or Ro 25-6981 maleate (3 µM). Each compound was initially dissolved in DMSO to prepare concentrated stock solutions before dilution in aCSF. Control slices were incubated in aCSF containing 0.1% DMSO to ensure equal solvent concentrations across all treatment conditions.

Dissected brain regions or whole acute slices were mechanically homogenized in either homogenization buffer (320 mM sucrose, 4 mM HEPES [pH 7.4], 2 mM EDTA) or immunoprecipitation (IP) buffer (50 mM Tris HCl [pH 7.4], 120 mM NaCl, 5 mM EDTA, 0.5% Triton X-100) and centrifuged at 800 × g for 10 min at 4 °C. All buffers contained a phosphatase and protease inhibitor cocktail (Sigma‒Aldrich, USA). The protein concentrations were determined using a BCA assay kit (Thermo Fisher Scientific, USA) according to the manufacturer's instructions. DTT (10 mM) and bromophenol blue were added to total (mixed 1:1 with lysis buffer) lysates, boiled for 10 min at 95 °C, separated by SDS‒PAGE on 7.5–12% acrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes (Merck Millipore, USA). The membranes were blocked with 5% nonfat dry milk in TBS-T (1% Tween 20 in Tris-buffered saline (TBS)) or Roti-Block (Carl Roth, Germany) and then incubated with the following primary antibodies diluted in TBS: mouse anti-GluA1-NT (Millipore, USA, MAB2263, 1:2000, #3847897), rabbit anti-PSD95 (Cell Signaling, USA, 2507, 1:2000, lot #4), Anti p44/42 MAPK (ERK1/2) antibody (Cell Signaling, USA, 9102, 1:10000, lot #30), Anti Phospho-p44/42 MAPK (Erk1/2) antibody (Cell Signaling, USA, 437, 1:10000, lot #21), Anti beta-Tubulin antibody (Sigma Aldrich, USA, T4026, 1:2.000, Lot# 0000324939), Anti AKT Antibody (Cell Signaling, USA, 9272, 1:1.000, Lot #30), Anti Phospho-AKT antibody (Cell Signaling, USA, 4060, 1:2000, Lot #27), Anti-GAPDH antibody (Cell Signaling, Cat 2118, 1:1000, Lot #16) and mouse anti-GAPDH (Abcam, United Kingdom, ab8245, 1:1000, lot #1035914-6). After three washes, the membranes were incubated with the following horseradish peroxidase-conjugated secondary antibodies diluted in TBS-T: sheep anti-mouse (GE Healthcare, USA, NA931, 1:20.000, lot #17556470 and #18111935) and donkey anti-rabbit (GE Healthcare, USA, NA9340, 1:25000) and Anti-rabbit antibody (SeraCare, USA, 5450-0010, 1:10.000, Lot #10571726) for 1 h at room temperature. In some experiments, the membrane was washed and stripped for further reincubation with another antibody. In that case, the membrane was incubated two times for 15 min in a glycin stripping solution (glycin 15 g, SDS 1 g, Tween20 10 mL, 800 mL dionized water), washed three times ten minutes in TBS-T, and then blocked and incubated following the regular procedure. The washed membranes were developed with a ChemiDoc MP imaging system (Bio-Rad) using an enhanced chemiluminescence detection kit (Thermo Fisher Scientific, USA). Band intensity was quantified by densitometry with ImageJ 1.47 v software (National Institute of Health, USA) and normalized to the appropriate loading control.

Mice were forced to swim in a glass cylinder (Ø 26 cm, 60 cm high) filled to a depth of 25 cm with 25 °C water for 10 min on 5 consecutive days. The immobility time was measured in each session. After swimming, the animals were gently dried and kept in a cage under a heating light for an additional ten minutes. Afterward, the mice were allowed to rest for two days in their home cages. Mice were treated after the resting period, depending on the experimental group. For the measurement of escape behavior, an additional swim session was performed. For intraperitoneal injections, (±)-ketamine was diluted in NaCl (0.9%) and NAB-14 was dissolved in PEG 400 (40%), dimethylacetazolamide (10%), and 50% glucose (5%). Lorazepam was purchased as an injection solution (Tavor pro injectione®, 2 mg, Pfizer, Germany). A maximum volume of 0.2 ml was injected per mouse; the volume was individually adjusted to the weight of the animal to achieve the intended dose. As a control, the respective vehicle was administered.

After the intervention, the animals were either subjected to an additional swim session (test day) to measure immobility time or subjected to the nosepoke sucrose preference test (NP-SPT) using the IntelliCage system (TSE Systems, Germany). For assessment of immobility time, the mice were videotaped, and two independent experienced raters who were blinded to the experimental groupings analyzed the videos. The mean values determined by both raters were used for further analysis. Immobility time was defined as the cumulative time that the animals remained stationary, during which only movements of the tail or forepaws necessary to keep the head above the water surface were made, the animals did not travel any distance and merely passively floated, no directed movement of the front paws was observed, and the body was mostly oriented parallel to the walls of the cylinder. A detailed description of this protocol and its analysis can be found here. Par87

The IntelliCage system (TSE Systems, Germany), which allows automatic analysis of the spontaneous and exploratory behavior and drinking preference of rodents, was used. Radiofrequency identification transponders were implanted in the mice. IntelliCage comprises a common space in the middle and four measuring corners. Up to 16 group-housed mice can be given free access to food in the center, while water is supplied in the corners (by two zipper bottles each) behind electronically managed doors. The corners can be visited by only one mouse at a time. Using IntelliCage Plus software, the system records i) the number and duration of corner visits, ii) the number of nosepokes (approaches) on the doors, and iii) the number of licks on the drinking bottles. Initially, the mice were adapted to the IntelliCage for five days and provided free access to food and water in all corners. Then, for three days, the animals were habituated to sucrose (each corner contained one bottle with 1% sucrose solution and one with water; the contents of all bottles were exchanged every day) and subjected to the nosepoke protocol. The mice had to perform a nosepoke to open the doors and access both water and sucrose. Next, an NP-SPT paradigmin which gradually increasing effort (number of nosepokes) was needed to access the sucrose-containing bottles for a short period (24 h) was used for the measurement of sucrose preference. In this protocol, each door opened in response to a nosepoke and closed after the animals drank from the sucrose-containing bottle for 5 s, while the mice were allowed to consume water from the water-filled bottles for an unlimited amount of time. The number of nosepokes needed to access the sucrose-containing bottle progressively increased (1, 2, 3, 4, 5, 6, 7) after each of the 10 licking sessions. This test was used to assess the drive for obtaining a reward; a decreased sucrose preference indicates reduced reward behavior. For every side, the number of licks was measured, and the mean sucrose preference was calculated as the percentage of the licks of the sucrose solution-containing bottles compared to the total number of licks. post 1 and post 2 represent two consecutive 24 h test periods. Par88

The test was performed in a square arena (50 × 50 cm) surrounded by a 35 cm high wall made of gray PVC. The mice were placed in the center of the field and allowed to move freely. The mice were video tracked for 10 min, and the time spent in the center (16% of the entire area of the box) was compared to the time spent outside the center (time in the center/time outside the center). Tracking and analysis were performed with EthoVision XT software (Noldus, Netherlands).

For habituation, the mice were placed in the center of an empty square arena (50 × 50 cm) surrounded by a 35 cm high wall of the gray PVC and maintained there for 10 min. On the following day, the mice were placed in the empty arena for 10 min for a second habituation phase. Immediately thereafter, the mice were removed, and two slightly different objects were introduced into the arena (for location, see Fig. ). The animals were then placed back into the area and allowed to rest there for 10 min. Directly after the exploration of the two objects, the mice were injected (KET/NAB-14/saline) and placed back into their home cages. Six hours later, the mice were placed in the arena where the location of one of the two objects was changed (see Fig. for the location of the object) and remained there for another 10 min. During all the time in the arena, the mice were video tracked using EthoVision XT software (Noldus, Netherlands). Par90

A ROTA ROD LE8205 (PanLab, Harvard Apparatus, Spain) was used for the experiments. This instrument automatically detects the latency to fall and enables simultaneous assessment of up to five animals. Before testing, the animals were exposed to three training sessions at 5 rpm for 1-3 min with a 10 min interval between each session. Animals that did not stay on the rod rotating at 5 rpm for at least 60 sec in the third training session were excluded from further testing. For testing, the animals were set on a rod at 4 rpm. The rod was set to accelerate from 4 to 40 rpm in 300 sec, and the latency to fall was measured. This procedure was repeated at 5, 20, 30 and 50 min postinjection, and mean values were calculated for each treatment condition.

The test was performed in a square arena (50 × 50 cm) surrounded by a 35 cm high wall made of gray PVC. The mice were placed in the center of the field and allowed to move freely. Behavior was recorded for 10 min, and the total distance traveled was analyzed with EthoVision XT software (Noldus, Netherlands).

Diclofenac was dissolved in water/acetonitrile (50:50, v/v) to give a 1 mg/ml stock solution (C).

The samples were thawed on wet ice, and the tubes of study samples and/or CSs and/or QCs samples were vortexed. A 10 μl aliquot of sample and/or CSs and/or QCs was placed into a tube and kept on wet ice. 100 μl of acetonitrile were added to the double blank(s). 100 μl of internal standard working solution (C1) was added to all other tubes. After capping the tubes, they were thoroughly vortexed. 100 μl of water were added to all tubes and again vortexed. The tubes were centrifuged for at least 10 minutes at approximately 3000 G and the supernatants were injected onto the LC-MS/MS system for analysis.

The pharmacokinetics of NAB-14 was investigated in Sprague Dawley male rats following single oral (PO) administrations of two different test item formulations. NAB-14 (batch No.: 312155; purity: 98.26%, appearance: off white powder) was administered to 3 male rats/route/formulation (9-weeks old on the day of first dosing occasion) in at libitum food regimen. Animals were dosed at 10 mg/kg or 50 mg/kg by a single PO administration. NAB-14 was suspended in 20% w/v PEG400/5% w/v Transcutol HP/5% w/v Vitamin E TGPS in 200 mM Citrate Buffer for the first formulation (Formulation A) and in 25% w/v PEG400/5% w/v Solutol HS-15/0.25% w/v MC 400 cp in 200 mM Citrate Buffer for the second formulation (Formulation B) and administered at a dose volume of 10 mL/kg. The pH of Formulation A was 3.53 and 3.58 at 10 and 50 mg/kg, respectively. The pH of Formulation B was 3.51 and 3.52 at 10 and 50 mg/kg, respectively. Individual serial plasma profiles were drawn from each animal. Pharmacokinetic parameters were evaluated after each administration. Body weights were within the normal range for animals of this strain and age. After treatment, NAB-14 was extracted from rat plasma, manually aliquoting samples kept on wet ice, using acetonitrile containing diclofenac as an internal standard. Extracts were analysed by LC-MS/MS using ZSprayTM interface with positive ion multiple reaction monitoring. This method (see section above) was qualified over the range 1 to 1000 ng/mL using a 10 μL aliquot of rat plasma. Calibration standards were freshly prepared; quality control samples were prepared and stored at -80 °C together with the study samples until use. All analytical batches met the acceptance criteria.

The Schrödinger Software Suite 2021-4 (Schrödinger LLC, NY, USA) was used for modeling studies. The SMILES of NAB-14 was converted to a valid 3D Lewis structure using LigPrep (Schrödinger LLC, NY, USA) and was geometrically optimized with the OPLS4 force field. The cryo-EM image of the heterotetrameric GluN1a/GluN2D NMDAR (PDB ID: 7YFF) structure was prepared for docking with the Protein PrepWizard. This step involves adding missing hydrogen atoms and adjusting the ionization state of polar amino acids at neutral pH and, finally, energetically minimizing the structure using the OPLS4 force field. The geometric center of residue C590 (located on the GluN2D-TMD M1 helix) was considered the grid centroid, and flexible docking was carried out using Glide with the SP scoring function. Par104

All experiments were performed with an equal distribution of male and female mice. For all experiments, a separate exploratory analysis focusing on the potential effects of sex on the experiments was performed, but no relevant differences were observed. However, our experiments were not adequately powered to reliably detect sex differences.

Further information on research design is available in the linked to this article. Par123