Main Research Findings

1) Administration of Rapastinel was found to improve markers related to depression and contextual fear extinction paradigms in an animal model, indicating the compound may have the potential to treat post-traumatic stress disorder.

2) Due to its ability to enhance long-term potentiation and reduce long-term depression at CA1 synapses in the hippocampus, Rapastinel may have the potential to enhance learning and memory.

 

Selected Data

1) The study conducted by Burgdorf et al utilized adult male Sprague-Dawley rats, aged 2-3 months. These animals were housed in Lucite cages with aspen wood chip bedding, maintained under a controlled 12:12 hour light:dark cycle, and provided with ad libitum access to Purina lab chow and tap water throughout the experiment. The investigational compound, Rapastinel (GLYX-13), was synthesized in its free base form and administered intravenously (IV) at a single dose of 3 mg/kg, delivered in 1 ml/kg of 0.9% sterile saline vehicle. For in vitro electrophysiological experiments, rapastinel was prepared for bath application across a concentration range of 20 to 1000 nM [1].

To establish an animal model reflective of post-traumatic stress disorder (PTSD) and depression, rats were subjected to a chronic unpredictable stress (CUS) protocol, which has been previously validated to induce depressive-like symptoms. This regimen involved exposure to two different stressors per day for 21 consecutive days prior to rapastinel dosing, with CUS continuing until the animals were sacrificed, extending the total stress duration to 37 days for some experimental cohorts. A total of nine distinct CUS stressors were employed, including physical manipulations like rotation on a shaker for 1 hour, exposure to a 4°C ambient chamber for 1 hour, and environmental stressors such as varying light conditions, such as lights off for 3 hours, lights on overnight, strobe light overnight, cage alterations such as 45° tilted cages overnight, and social manipulations such as crowded or isolation housing overnight. Additionally, food and water deprivation overnight were used as stressors. Animal weights were recorded weekly, and behavioral testing was conducted without the introduction of additional stressors to avoid confounding effects.

A comprehensive battery of behavioral tests was employed to assess the animals’ affective and cognitive states. The Porsolt forced swim test, a standard assay for antidepressant-like effects, involved placing animals in a clear glass tube filled with 30 cm of tap water. After a 15-minute habituation on the first day, subsequent test sessions lasted 5 minutes, occurring at 1 hour, 1 day, 7 days, and 14 days post-dosing. Floating time, indicative of behavioral despair, was scored offline by a blind experimenter using video recordings, ensuring high inter-rater reliability. Sucrose preference testing, an anhedonia assay, involved exposing rats to a palatable 1% sucrose solution for 48 hours, followed by a 4-hour water deprivation period and a 1-hour exposure to two identical bottles, one with sucrose, one with tap water. Sucrose preference was calculated as the volume of sucrose consumed divided by the total fluid consumed, expressed as a percentage, and assessed 3 days post-dosing [1].

The novelty-induced hypophagia (NIH) test, measuring anxiety-related feeding suppression, involved food-depriving animals overnight before placing them in the center chamber of an open field under dim-red lighting for 10 minutes. Latency to take the first bite of food and locomotor activity (line crosses) were scored offline by a blind experimenter. Control groups included home-cage feeding latency and food intake measurements 24 hours after food deprivation. Positive emotional learning (PEL) was assessed by measuring hedonic/aversive ultrasonic vocalizations (USVs) during a 3-minute heterospecific rough-and-tumble play session, alternating 15-second blocks of play and no-stimulation. USVs were recorded and analyzed via sonogram, and this test was conducted at 3 hours and 2 weeks post-dosing [1].

Contextual fear conditioning and extinction (CFE) tests, relevant for modeling PTSD-like learning, were performed in shock chambers. On the training day (D0), animals received three 0.5 mA, 1-second footshocks during a 400-second session. Extinction involved daily 5-minute non-reinforced trials for six consecutive days, followed by a consolidation trial on day 14. Freezing behavior, an index of fear, was quantified using FreezeFrame software at baseline and during the final 3 minutes of each extinction trial.

Electrophysiological studies focused on the medial prefrontal cortex (MPFC). For slice preparation, animals were deeply anesthetized with isoflurane, decapitated, and their brains rapidly removed and submerged in ice-cold artificial cerebrospinal fluid (aCSF) containing: 160 sucrose mM, 25 NaCl mM, 2.5 KCl mM, 4 MgCl2 mM, 0.5 CaCl2 mM, 1.25 NaH2PO4 mM, 26 NaHCO3 mM, 20 glucose mM, continuously gassed with 95% O2/5% CO2. Four hundred micrometer-thick coronal slices, encompassing both prelimbic and infralimbic regions of the MPFC, were prepared using a vibratome. Slices were then transferred to an interface holding chamber containing oxygenated cutting solution, and subsequently to a chamber with oxygenated aCSF at room temperature for recording. For long-term potentiation (LTP) recordings, slices were continuously perfused with oxygenated aCSF containing 10 µM picrotoxin and heated to 32±0.5 °C.

Field excitatory postsynaptic potentials (fEPSPs) were recorded in layer III/IV of the prelimbic MPFC via thin-walled borosilicate glass electrodes, while deep white matter inputs were stimulated with a bipolar tungsten electrode. Constant current stimuli were adjusted to evoke half-maximal fEPSPs every 30 seconds. LTP was induced by three high-frequency theta burst stimulus trains (each train comprising ten 100 Hz bursts of five pulses, with 200 ms inter-burst intervals and 2 seconds duration per train), applied 3 minutes apart. fEPSP slopes were measured, and stability criteria were applied prior to tetanus. For ex vivo studies, animals received 3 mg/kg rapastinel or vehicle 24 hours before slice preparation; for in vitro studies, rapastinel (20-1000 nM) or vehicle was bath-applied 30 minutes before LTP induction.

The NMDAR antagonist D-AP5 (25 µM) was used to confirm NMDAR dependence of LTP. Whole-cell patch clamp recordings of layer V pyramidal neurons were performed to assess input-output relations, paired-pulse facilitation, and NMDAR currents. The intracellular solution contained: 135 CsMeSO2 mM, 8 NaCl mM, 10 HEPES mM, 0.2 EGTA mM, 2 Mg-ATP mM, 0.3 Na-GTP mM, 275 mOsm mM, pH 7.25. Neurons were voltage-clamped at -70 mV for EPSCs and at -40 mV for NMDA currents, with specific pharmacological isolation for NMDA currents using 0 Mg2+, 3mM Ca2+, picrotoxin, and CNQX [1].

Transcriptome profiling involved triplicate microarray analyses of MPFC tissue. Brains were removed 24 hours post-dosing, frozen on dry ice, and MPFC tissue was dissected and stored at -80°C. Custom 45-mer oligonucleotide arrays, complementary to 1283 cloned rat CNS mRNAs, were used. Total RNA was extracted, amplified, and labeled before cohybridization. Arrays were scanned, normalized using LOWESS, and analyzed with the Significance Analysis of Microarrays (SAM) algorithm at a 10% false discovery rate (FDR). Ontological analyses were performed to identify biological associations of differentially expressed genes to Gene Ontology (GO) categories (biological process, molecular function, cellular compartment), with significance determined by Fisher’s exact test. Additionally, DAVID (Database for Annotation, Visualization and Integrated Discovery) was used to examine enrichment in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, specifically focusing on long-term potentiation (LTP) and long-term depression (LTD) pathways. All behavioral and electrophysiological data underwent statistical analysis using ANOVA followed by Fisher’s PLSD post hoc tests [1].

2) This study performed by the research team of Zhang et al investigated the effects of GLYX-13, a novel NMDA receptor glycine site partial agonist, on long-term synaptic plasticity determined by LTP and LTD, as well as NMDAR transmission in rat hippocampal Schaffer collateral-CA1 synapses. For electrophysiological studies, Sprague-Dawley rats, aged 12-18 days old, were utilized. Animals were deeply anesthetized with diethyl ether and then decapitated. The brains were rapidly excised and submerged in ice-cold artificial cerebrospinal fluid (ACSF) to preserve tissue integrity. This ACSF solution contained: 124 NaCl mM, 4 KCl mM, 2 MgSO4 mM, 2 CaCl2 mM, 1.25 NaH2PO4 mM, 26 NaHCO3 mM, 10 glucose mM, adjusted to pH 7.4, and continuously gassed with 95% O2/5% CO2. The brain was hemisected, frontal lobes removed, and individual hemispheres were glued to a stage for slicing. Coronal hippocampal slices, 300 µm thick, were prepared. To prevent seizure activity and maintain a controlled experimental environment, a cut was made between the hippocampal CA1 and CA3 regions. Slices were then transferred to an interface holding chamber for at least one hour of incubation at room temperature before recordings commenced [2].

Extracellular recordings were performed by transferring slices to an interface recording chamber, continuously perfused at 3 ml/min with oxygenated ACSF. Low-resistance recording electrodes were positioned in the apical dendritic region of the Schaffer collateral termination field in stratum radiatum of the CA1 region to record field excitatory postsynaptic potentials (fEPSPs). A bipolar stainless steel stimulating electrode was placed on Schaffer collateral-commissural fibers in the CA3 stratum radiatum. Constant current stimulus intensity was adjusted to evoke approximately half-maximal fEPSPs once every 30 seconds (50-100 pA, 100 µs duration). fEPSP slope was measured by linear interpolation from 20-80% of the maximum negative deflection. Only slices exhibiting stable baseline slopes (within ± 10% for at least 10 minutes) were included in experiments. LTP was induced by high-frequency stimulation (HFS) consisting of three 100 Hz trains, each 500 ms in duration, separated by 60-second intervals. LTD was induced by low-frequency stimulation (LFS) of 2 Hz for 10 minutes.. GLYX-13 was applied in various concentrations (100 nM to 100 µM) during HFS or LFS protocols, both alone and in combination with the glycine site agonist D-serine [2].

Intracellular whole-cell patch clamp recordings were obtained from CA1 pyramidal neurons in slices submerged in a recording chamber at room temperature, continuously perfused with oxygenated ACSF. Patch pipettes, pulled from borosilicate glass with a flaming/brown micropipette puller, had tip resistances of 5-6 MΩ when filled with intracellular solution containing: 135 CsMeSO3 mM, 8 NaCl mM, 10 HEPES mM, 0.2 EGTA mM, 2 Mg-ATP mM, 0.3 Na-GTP mM, 1 QX-314 mM, adjusted to pH 7.25 and 280 ± 10 mOsm. Neurons were voltage-clamped at -60mV to record excitatory postsynaptic currents (EPSCs) and at -40mV to isolate NMDAR currents, relieving voltage-dependent magnesium block. Pharmacological isolation of NMDAR currents involved perfusing slices with ACSF containing 0 mM Mg2+, 3 mM Ca2+, 10 µM picrotoxin, and 10 µM CNQX. Single electrical pulses were delivered at a duration of 80 µs every 30 seconds. Paired-pulse facilitation (PPF) was assessed by measuring the ratio of second to first EPSC amplitudes with a 100 ms interstimulus interval. 50 µM of the NMDAR-specific antagonist D-AP5 was used to confirm NMDAR dependence. 10 µM D-serine was used to test the occlusion of GLYX-13 effects at the glycine site.

Calcium imaging utilized a customized two-photon laser-scanning Olympus BX61WI microscope with a 60x/1.1 nA objective. Green and red fluorescence were separated by a 565 nm dichroic mirror and filtered before detection by photomultiplier tubes. Neurons were loaded with Fluor-594 for dendritic structure visualization and Calcium Green for [Ca2+] dynamics via the patch pipette. Baseline fluorescence (F0) was averaged from control images, and ΔF/F was calculated. No EGTA was added to the internal solution for calcium imaging [2].

 

Discussion

1) The results of the study conducted by Burgdorf et al found that administration of a single intravenous dose of rapastinel (3 mg/kg) elicited a rapid and enduring reversal of depressive- and PTSD-like behaviors induced by chronic unpredictable stress (CUS), alongside significant changes in electrophysiological and transcriptomic markers in the medial prefrontal cortex (MPFC). In the Porsolt forced swim test, CUS exposure led to an increase in floating time, indicative of behavioral despair. However, rats treated with rapastinel demonstrated a significant reduction in floating time across all tested time points—1 hour, 1 day, 7 days, and 14 days post-dosing—compared to CUS-exposed rats receiving vehicle, underscoring rapastinel’s robust and sustained antidepressant-like efficacy [1].

Rapastinel also ameliorated CUS-induced anhedonia, as evidenced by its ability to restore sucrose preference, which was significantly diminished in CUS+vehicle rats compared to No CUS controls. This positive effect was observed 4 days post-dosing. Furthermore, rapastinel significantly decreased feeding latency in a novel environment during the novelty-induced hypophagia (NIH) test at 2 days post-dosing, reversing the prolonged latencies seen in CUS+vehicle animals. Importantly, these effects on anxiety-like behavior were specific, as rapastinel did not alter total food consumption in the home cage or general locomotor activity (line crosses) in the novel environment. Beyond behavioral and anhedonic measures, rapastinel treatment also resulted in increased body weight gain at 2 weeks post-dosing compared to CUS+vehicle rats, which exhibited reduced weight gain relative to unstressed controls [1].

 

Figure 1: Changes in B) forced swim test scores, C) novelty induced hypophagia, D) sucrose preference, and E) weight gain across the experimental treatment groups.

In the positive emotional learning (PEL) test, rapastinel significantly enhanced hedonic ultrasonic vocalizations (USVs) in response to a conditioned stimulus at both 3 hours and 2 weeks post-dosing in CUS-treated rats. This reversed the CUS-induced reduction in hedonic USVs and modulated the balance away from aversive 20-kHz USVs observed in CUS+vehicle animals. Complementary to these findings, CUS+rapastinel rats also showed increased running speed and center crosses during heterospecific play, contrasting with the deficits observed in CUS+vehicle rats, further supporting the compound’s pro-social and reward-related benefits without affecting overall locomotor activity.

 

Figure 2: Changes in A) positive emotional learning, B) contextual fear extinction, and C) contextual fear consolidation across the experimental treatment groups.

Rapastinel demonstrated a profound impact on CFE, a critical measure for PTSD-like memory. CUS exposure led to elevated freezing levels and impaired both fear extinction learning and consolidation. In contrast, CUS+rapastinel treated rats exhibited reduced freezing levels and accelerated fear extinction over days 3-14 post-dosing compared to CUS+vehicle animals. Crucially, rapastinel also prevented fear reconsolidation, indicating a therapeutic potential for mitigating persistent fear memories [1].

Electrophysiological studies in MPFC slices revealed that rapastinel profoundly influenced synaptic plasticity. Acute bath application of 100 nM rapastinel in vitro significantly increased the magnitude of LTP induced by submaximal theta-burst stimulation. More remarkably, a single intravenous dose of rapastinel in vivo resulted in a persistent increase in LTP magnitude in MPFC slices measured 24 hours later *ex vivo*. While CUS significantly impaired LTP induction in MPFC, rapastinel not only reversed this impairment but also enhanced LTP magnitude to levels significantly greater than those observed in No CUS control rats, pointing to a metaplasticity-like effect. The N-methyl-D-aspartate receptor (NMDAR) antagonist D-AP5 completely abolished LTP in these synapses, confirming the NMDAR-dependent nature of these plasticity mechanisms. Furthermore, CUS-induced increases in the NR2B/NR2A NMDAR current ratio in layer V pyramidal cells of the MPFC were normalized by rapastinel treatment, while basal synaptic transmission, assessed by input-output relations and paired-pulse facilitation, remained unaltered.

Transcriptomic analyses of MPFC tissue provided molecular insights correlating with the observed behavioral and electrophysiological changes. CUS exposure induced widespread alterations in cortical gene expression, characterized by the upregulation of genes involved in protein, carbohydrate, and lipid metabolism, and a notable downregulation of genes associated with synaptic plasticity, synapse structure/function, glutamate receptor signaling, and cytoskeletal components. Ontological analysis confirmed significant enrichment of these affected pathways within synaptic and post-synaptic compartments. Rapastinel treatment effectively reversed many of these CUS-induced transcriptomic deficits. Specifically, it led to the downregulation of protein and nucleic acid metabolism, and an upregulation of cytoskeletal-associated processes, including intracellular vesicular transport [1].

The most prominent effects of rapastinel were observed in biological processes linked to the positive regulation of synaptic transmission, ionotropic glutamate receptor activity, intracellular signaling, kinase-mediated changes, and synaptic remodeling, all indicating a significant restoration of synaptic function. Through KEGG pathway analysis, CUS treatment showed a modest, but significant, downregulation of LTD-associated transcripts. Critically, rapastinel treatment in CUS-exposed rats resulted in a marked enrichment, a 2.94-fold upregulation of LTP-associated, and a 2.70-fold upregulation of LTD-associated transcripts compared to CUS+vehicle rats. In conclusion, these multifaceted results strongly suggest that rapastinel exerts its rapid and long-lasting antidepressant and pro-cognitive effects by modulating NMDAR-mediated synaptic plasticity, particularly through a metaplasticity process within the medial prefrontal cortex [1].

2) The results of the study conducted Zhang et al demonstrated that GLYX-13, a glycine site partial agonist of the NMDAR, simultaneously enhanced LTP and reduced LTD at Schaffer collateral-CA1 synapses in rat hippocampal slices, suggesting a unique therapeutic potential as a cognitive enhancer. In dose-response experiments on HFS-induced LTP, GLYX-13 significantly enhanced the magnitude of LTP at concentrations ranging from 100 nM to 10 µM. Specifically, 100 nM GLYX-13 increased fEPSP slope by 195%, 1 µM by 275%, and 10 µM by 252% compared to control, indicating a robust pro-LTP effect. However, a higher concentration of 100 µM GLYX-13 led to a significant reduction in LTP magnitude to 126% of baseline, highlighting a complex, concentration-dependent modulation [2].

Regarding LTD, LFS typically reduced fEPSP slope to 58% of baseline in control slices. When slices were perfused with 100 nM GLYX-13, no significant alteration in LTD magnitude was observed. In contrast, 1 µM GLYX-13 significantly reduced LTD, resulting in a mean fEPSP slope of 91.7%. Further increasing the concentration to 10 µM or 100 µM led to a loss of GLYX-13’s modulatory effect on LTD, indicating that its suppressive action on LTD was also concentration-dependent and lost at higher doses. These findings imply that at low concentrations, GLYX-13 acts as both a promoter of LTP and a suppressor of LTD, while at higher concentrations, its LTP-enhancing effects diminish and its LTD-modulating properties cease.

To confirm that GLYX-13’s effects were mediated via the glycine site of the NMDAR, experiments involving the full glycine site agonist D-serine were conducted. Co-application of 1 µM GLYX-13 with a saturating concentration of 100 µM D-serine completely occluded the enhancement of LTP typically produced by GLYX-13 alone. A lower concentration of 10 µM D-serine produced a partial occlusion of LTP enhancement by GLYX-13. Conversely, 10 µM D-serine fully occluded the reduction of LTD caused by 1 µM GLYX-13. These results strongly support the conclusion that both the LTP enhancement and LTD suppression by GLYX-13 are mediated through its binding to the NMDAR glycine site [2].

Further investigations into NMDAR currents revealed a concentration-dependent, bidirectional modulation. Single-shock evoked NMDAR EPSCs in CA1 pyramidal neurons were increased by 43% with 100 nM GLYX-13, but reduced by 28% with 1 µM GLYX-13. The inhibition by GLYX-13 at 1 µM was occluded by D-serine, consistent with GLYX-13 acting as a competitive antagonist at this modulatory site. Paired-pulse facilitation remained unaffected by GLYX-13, suggesting that its effects are primarily postsynaptic.

GLYX-13 also exhibited frequency-dependent modulation of NMDAR currents and Ca2+ influx. During high-frequency bursts (4 pulses/100 Hz), both 100 nM and 1 µM GLYX-13 significantly increased burst-activated NMDAR currents by 119% and 32.7% respectively, a stark contrast to its inhibitory effect on single-shock evoked currents at 1 µM. Two-photon microscopy confirmed this, showing that 1 µM GLYX-13 selectively enhanced burst-induced NMDAR-dependent spine Ca2+ influx in individual dendritic spines. The frequency-dependent actions of GLYX-13 were summarized such that low-frequency evoked NMDAR currents were reduced, while high-frequency evoked currents were enhanced, with these actions depending on extracellular D-serine concentration [2].

Finally, the study elucidated the role of NR2B-containing NMDARs in GLYX-13’s effects. Using the open-channel blocker MK-801, GLYX-13 was found to slow the block of synaptic NMDARs but enhance the block of extrasynaptic NMDARs during high-frequency bursts. This suggested that extrasynaptic NMDARs mediate the enhancement of burst-evoked currents and LTP, while synaptic NMDARs mediate the reduction of LTD. The NR2B-selective antagonist ifenprodil further clarified this: the reduction of single-shock evoked NMDAR EPSCs by 1 µM GLYX-13 was unaffected by ifenprodil, indicating an NR2B-independent mechanism. Conversely, ifenprodil converted GLYX-13’s enhancement of burst-evoked NMDAR EPSCs into a reduction, supporting the hypothesis that GLYX-13 potentiates NMDAR currents via selective action on NR2B-containing extrasynaptic receptors.

In contrast to GLYX-13, D-cycloserine (DCS) showed different effects; low concentrations of DCS of 1 µM enhanced LTP without affecting LTD, while higher concentrations of 10-100 µM impaired LTP and 100 µM DCS enhanced LTD. These differences suggest distinct mechanisms despite both being glycine site partial agonists. Overall, the findings indicate that GLYX-13’s unique ability to simultaneously enhance LTP and suppress LTD, coupled with its selective, frequency-dependent modulation of NMDARs, particularly extrasynaptic NR2B-containing receptors, positions it as a promising candidate for learning and memory enhancement [2].

Disclaimer

**LAB USE ONLY**
*This information is for educational purposes only and does not constitute medical advice. THE PRODUCTS DESCRIBED HEREIN ARE FOR RESEARCH USE ONLY. All clinical research must be conducted with oversight from the appropriate Institutional Review Board (IRB). All preclinical research must be conducted with oversight from the appropriate Institutional Animal Care and Use Committee (IACUC) following the guidelines of the Animal Welfare Act (AWA).

Citations

[1] Burgdorf J, Kroes RA, Zhang XL, et al. Rapastinel (GLYX-13) has therapeutic potential for the treatment of post-traumatic stress disorder: Characterization of a NMDA receptor-mediated metaplasticity process in the medial prefrontal cortex of rats. Behav Brain Res. 2015;294:177-185. doi:10.1016/j.bbr.2015.07.039

[2] Zhang XL, Sullivan JA, Moskal JR, Stanton PK. A NMDA receptor glycine site partial agonist, GLYX-13, simultaneously enhances LTP and reduces LTD at Schaffer collateral-CA1 synapses in hippocampus. Neuropharmacology. 2008;55(7):1238-1250. doi:10.1016/j.neuropharm.2008.08.018

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