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. 2022 Nov 28;21:100503. doi: 10.1016/j.ynstr.2022.100503

(2R,6R)-hydroxynorketamine acts through GluA1-induced synaptic plasticity to alleviate PTSD-like effects in rat models

Yu Li a,1, YaLin Du c,1, Chen Wang a, GuoHua Lu a, HongWei Sun a, YuJia Kong d, WeiWen Wang e, Bo Lian f, ChangJiang Li a, Ling Wang g, XianQiang Zhang b,∗∗,1, Lin Sun a,∗,1
PMCID: PMC9755068  PMID: 36532380

Abstract

Post-traumatic stress disorder (PTSD) is a debilitating mental disorder with high morbidity and great social and economic relevance. However, extant pharmacotherapies of PTSD require long-term use to maintain effectiveness and have enormous side effects. The glutamatergic system, especially the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR), is an important target of current research on the mechanism of PTSD. Postsynaptic AMPAR function and expression are known to be increased by (2R, 6R)-hydronorketamine (HNK), the primary metabolite of ketamine. However, whether (2R,6R)-HNK alleviates PTSD-like effects via AMPAR upregulation is yet to be known.

In the present study, rats were exposed to single prolonged stress and electric foot shock (SPS&S). Afterwards, gradient concentrations of (2R,6R)-HNK (20, 50, and 100 μM) were administered by intracerebroventricular (i.c.v.) injection. Open field, elevated plus maze, freezing behavior, and forced swimming tests were used to examine PTSD-like symptoms. In addition, the protein levels of GluA1, BDNF and PSD-95 were analyzed using western blotting and immunofluorescence, and the synaptic ultrastructure of the prefrontal cortex (PFC) was observed by transmission electron microscopy.

We found that (2R,6R)-HNK changed SPS&S-induced behavioral expression, such as increasing autonomous activity and residence time in the open arm and decreasing immobility time. Likewise, (2R,6R)-HNK (50 μM) increased GluA1, BDNF, and PSD-95 protein expression in the PFC. Changes in synaptic ultrastructure induced by SPS&S were reversed by administration of (2R,6R)-HNK. Overall, we find that (2R,6R)-HNK can ameliorate SPS&S-induced fear avoidance in rats, as well as rat cognates of anxiety and depression. This may be related to GluA1-mediated synaptic plasticity in the PFC.

Keywords: Post-traumatic stress disorder (PTSD); (2R,6R)-Hydroxynorketamine ((2R,6R)-HNK); Synaptic plasticity; Glutamatergic nervous system; GluA1

1. Introduction

Post-traumatic stress disorder (PTSD), characterized by increased arousal, negative emotions, re-experiencing, avoidance, and intrusive memories of trauma, is a serious and disabling disorder that occurs in an individual in response to witnessing or experiencing a major traumatic event. In the general population, the incidence of PTSD is estimated at 7.8% (Feder et al., 2014), but it can be as high as 25–35% in groups who often experience severe psychological trauma such as combat veterans and disaster victims (Kredlow et al., 2022). PTSD is significantly correlated with several comorbid psychiatric disorders, high rates of attempted suicide, and other adverse consequences (Hien et al., 2021). The US Food and Drug Administration have approved sertraline and paroxetine for the intervention of PTSD (Bryant, 2019; Mithoefer et al., 2019). These pharmacotherapies require long-term use to maintain effectiveness and have low response rates and numerous side effects such as sexual dysfunction, weight gain, and sleep disturbances. There is thus a dire need to identify novel drug targets and develop more effective pharmaceuticals therefor.

Clinical studies have indicated that ketamine, an N-methyl-D-aspartate (NMDA) antagonist, induces rapid and sustained antidepressant actions and is also used to treat the symptoms of PTSD (P. Zanos and Gould, 2018; Zhang et al., 2021). However, ketamine's dissociative properties and other side effects have limited its use as a treatment (Riggs et al., 2020). There may be a workaround for this, however. Ketamine is rapidly and stereoselectively metabolized into a number of metabolites, one of which is (2R,6R)-hydronorketamine (HNK). (2R,6R)-HNK is most prevalent in the plasma of humans and in the plasma and brain of rodents following ketamine administration ("Erratum to: "Relationship of Ketamine's Plasma Metabolites with Response, Diagnosis, and Side Effects in Major Depression" by Zarate (2012). (Biol Psychiatry 2012; 72(4): 331–338)," 2016; Highland et al., 2019; P. Zanos, 2017). While ketamine's side effects are the result of its antagonism of the NMDA receptor (NMDAR), some studies have proposed that NMDAR inhibition itself may not be what mediates ketamine's antidepressant effects, and, moreover, that (2R,6R)-HNK does not inhibit NMDAR (Carliss et al., 2007; Z. Zanos et al., 2018). (2R,6R)-HNK has been demonstrated to reverse chronic stress-induced negative affect by repairing damaged α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-dependent glutamatergic transmission. Therefore, (2R,6R)-HNK has the potential to positively treat PTSD symptoms. However the efficacy, side effects, neurobiological changes, and mechanism of (2R,6R)-HNK are unknown.

AMPAR is a tetrameric protein complex made up of various combinations of GluA1, -2, -3, and -4 (G. H. H. Diering and R, 2018). GluA1 is expressed in the majority of neurons in the nervous system (G. H. Diering and Huganir, 2018). Synaptic transmission efficacy can be bilaterally modified through the phosphorylation status of Ser831 and Ser845 sites at the GluA1 subunit, and this is considered a critical event for synaptic plasticity (Leal RB and Formolo, 2020a). Impairment of GluA1 palmitoylation causes PTSD-like symptoms (Oota-Ishigaki et al., 2022). Ergo, decreasing GluA1 might lower activity-dependent synaptic transmission as well as undermining synaptic plasticity and memory formation.

A large amount of animal studies suggest that the prefrontal cortex (PFC), which is involved in the formation of negative mood and fear memory, is the key brain region associated with PTSD (Yabuki and Fukunaga, 2019). The PFC regulates the more basic emotional processes occurring in subcortical and brainstem regions (Dixon et al., 2017). Damaged glutamatergic neurotransmission is associated with impaired synaptic signaling within the PFC neurons and defective contextual fear memory (Blaze et al., 2021). In agreement with animal studies, functional magnetic resonance imaging (MRI) has established that there is reduced blood flow and function in the PFCs of patients with PTSD when exposed to fear stimuli (Rauch and Phelps, 2006; Shin et al., 2005). Previous animal research has revealed that modulation of AMPARs in the PFC mediates the antidepressant action of (2R,6R)-HNK (N. Yao et al., 2018). Therefore, exploring the neurobiological effects after injecting of (2R,6R)-HNK on PFC have become of particular interest in the field of post-traumatic stress disorder.

Aside from GluA1, there are a few other proteins of interest concerning (2R,6R)-HNK's mechanism (Dell'Osso et al., 2009). demonstrated that patients with PTSD had lower brain-derived neurotropic factor (BDNF) levels than healthy controls and recent evidence suggests that stress directly decreases BDNF expression (Krystal et al., 2017). Other studies have reported that BDNF is required for (2R,6R)-HNK-mediated actions (Chou, 2020). BDNF is closely related to the expression of GluA1 and its transmembrane transport during the process of synaptic function regulation (Xiao et al., 2019). There is also evidence that suggests that synaptic plasticity is impaired in PTSD (Citri and Malenka, 2008). BDNF is known to be essential in learning, memory, and synaptic plasticity (J. Yu et al., 2019). Synaptic plasticity can be measured by determining postsynaptic density (PSD). The best studied of the PSD proteins, PSD-95, is mainly concentrated in mature excitatory glutamate synapses, and is particularly important for controlling the synaptic concentration of AMPAR. PSD-95 and GluA1 both affect postsynaptic functional plasticity (Zhou et al., 2018). Additionally, pharmacological treatments known to upregulate PSD-95 is shown to improve spatial and fear memory (Avila et al., 2017; Irvine et al., 2011). As such, PSD-95 might serve as a novel target for disorders such as PTSD (Citri and Malenka, 2008).

In PTSD patients, glutamatergic nervous system function is impaired when synaptic AMPAR concentration was reduced. However, (2R,6R)-HNK is able to increase presynaptic glutamate release, activate postsynaptic AMPAR, and enhance synaptic transmission (Chou et al., 2018). Thus, it was hypothesized that (2R,6R)-HNK activates the downstream GluA1-BDNF signaling pathway by increasing glutamate release from nerve ending in the PFC, both directly and indirectly enhancing synaptic plasticity and ameliorating PTSD symptoms, like negative emotion and fear memory. To test our hypotheses, we used a single prolonged stress and electric foot shock (SPS&S) procedure developed in our lab to stimulate chronic stress in rats. Following this, (2R,6R)-HNK was injected intracerebroventricularly (i.c.v.) in the lateral ventricle. Behavioral and biochemical tests were then performed. Finally, synaptic ultrastructure was examined by transmission electron microscopy (TEM) to monitor changes in synaptic plasticity in the PFC.

2. Materials and methods

2.1. Animals

A total of sixty adult male 8-week-old Sprague Dawley rats, provided by the Animal Center of Wei Fang Medical University, were divided into six groups of ten, each with an average weight of 230 ± 20g. Five of the six groups were randomly designated for the SPS&S procedure, with the remaining group serving as a control. The rats were allowed to acclimate to their new environment, where the ambient temperature was 22–26 °C, the humidity was 40%–60%, the light/dark cycle was 12 h on/12 h off (light on from 08:00 to 20:00), and they were provided food and water ab libitum. All experiments were conducted in accordance with the guidelines of the National Institutes of Health and were approved by the Institutional Animal Care and Use Committee of Wei Fang Medical University.

2.2. SPS&S procedure

After the adaptation period, the rats were exposed to the SPS&S procedure, which is an adaptation of a previously described procedure (L. L. Ni et al., 2020; W. Wang et al., 2008). First, the animals were immobilized in restraint tubes for 2 h, then immediately forced to swim for 20 min in a plastic cylinder, 46 cm deep and 20 cm in diameter, filled with room temperature water. Rats were provided a 15 min recovery period, then exposed to anhydrous diethyl ether until completely anesthetized. While anesthetized, the rats were placed in electric shock cages. After awaking, a 1 mA current was applied to the floor of the cage for 6 s. This repeated 10 times at random intervals within a 15 min period. Thereafter, all animals were returned to their cages with fresh bedding and left undisturbed for 14 days. An overview of the experimental schedule is provided in Fig. 1.

Fig. 1.

Fig. 1

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Experimental schedule of the SPS&S procedure

Before the experiment, all rats were allowed to acclimate to their new environment for 7 days. Then, 14 days after SPS&S, the designated groups were treated with drugs injection in their lateral ventricles.

2.3. Drugs

After the 14 day recuperation period, the SPS&S-treated rats were randomly assigned to one of five groups. Four of these five groups were to receive an i.c.v. injection of a different treatment in their lateral ventricles; the other group was not injected. One group received only 0.9% saline (SPS&S-Sal); one group received (2R,6R)-HNK at a concentration of 20 μM (SPS&S-20); another 50 μM (SPS&S-50); and another 100 μM (SPS&S-100). The complete control group had the same experience as the SPS&S group except that the SPS&S procedure was not performed. Stereotaxic coordinates for the injection site were as follows: 0.8 mm anterior, 1.6 mm lateral, and 3.5 mm from the bregma (Hamidi et al., 2019). After a recovery period of 7 days, drugs or saline were administered in a volume of 5 μL.

2.4. Behavioral tests

Rats were subjected to behavioral tests 24 h post-injection. The tests were conducted in a quiet environment, with sufficient time between each one. All the rats were tested in the same sequence, which consisted of: 1) open field test; 2) elevated plus maze test; 3) freezing behavior test; and 4) forced swim test. These tests were arranged from least to most stressful in order to minimize the impact of residual stress from one test on the next (Hu et al., 2017; Swiergiel et al., 2007). All experiments and data acquisition were carried out during adulthood, because results can vary depending on the age at which animals were exposed to stress.

2.4.1. Open field test (OFT)

The OFT is used to assess exploration activity and anxiety level in rats (Schmitt U, 1998). The chamber is composed of an unlidded box (100 cm × 100 cm × 50 cm) with a black floor and four walls. When the experiment begins, rats are placed lightly in the middle of the floor facing a particular direction, with each rat oriented the same way. During the 5 min test session, the total distance traveled, time in the center area, number of outer and inner squares entered, and number of times the rats stand upright are recorded. At the end of each experiment, 70% ethanol is used erase the scent cues left by the last rat. The Smart 3.0 video tracking system (Panlab SL, Barcelona, Spain), was used to record and quantify the trail of the animals.

2.4.2. Elevated plus maze test (EPMT)

The EPMT is used to assess anxiety levels in rodents (Slupski et al., 2017). The elevated plus maze consists of a cross- or plus-shaped platform. Two arms of the plus are enclosed by walls, while the two intersecting arms are open. Each arm is 50 cm long and 10 cm wide, with the central nexus consisting of an area of 10 cm × 10 cm. The platform is located 100 cm from the ground. A rat is placed on central platform facing one of the enclosed arms in each experiment and left to wander the maze for 5 min. The amount of time the animal spends in the open vs. enclosed arms is recorded, with more time spent in the enclosed spaces denoting higher anxiety. Once again, the maze is wiped clean with ethanol after each test in order to remove the scent cues of the previous rat, and, once again, the Smart 3.0 video tracking system was used to monitor and quantify the movements of test animals.

2.4.3. Freezing behavior test (FBT)

The FBT was developed to measure the fear response in rodents (L. H. L. H. Ni et al., 2020). Rats are placed into a neutral test chamber (50 cm × 50 cm × 25 cm) for 3 min and then returned to their home cage. Freezing time is defined as complete inactivity of the body or head except breathing. Behavior was monitored via overhead cameras and freezing time was observed manually by experimenters who were blinded to conditions.

2.4.4. Forced swim test (FST)

The FST is designed to evaluate a rodent's response to depression (Haj-Mirzaian et al., 2014). During the FST, rats are placed in a cylindrical (46 cm × 23 cm diameter) glass fiber-reinforced plastic (GFRP) containing 36 cm high room temperature water for 15 min. After 24h, the rats were allowed to swim under the same conditions for 6 min. During the last 4 min of the test, rats are monitored for how much time they spend mobile vs. immobile. Immobility is defined as periods when the mouse floats passively and does nothing but keep its head above water. Immobility time was counted by an experimenter who was blinded to the groups being tested.

2.5. Biochemical tests

2.5.1. Western blot (WB) assay

The anesthetized rats (n = 3 per group) were sacrificed and their PFCs isolated (H. N. Wang et al., 2009). PFCs were manually dissociated in ice cold RIPA buffer containing protease and phosphatase inhibitors. Protein concentration was measured by bicinchoninic acid (BCA) assay. Electrophoresis was then performed on 12% sodium dodecyl sulfate polyacrylamide gel electrophopresis (SDS-PAGE) with 40–50 mg of total protein added to each lane. The resulting gel was then transferred to a polyvinylidene fluoride (PVDF) film. After blocking with 5% non-fat milk for 2 h, the films were incubated with anti-GluA1 (1:1000, ab31232, Abcam), anti-BDNF (1:1000, ab205067, Abcam), anti-PSD-95 (1:1000, ab18258, Abcam), and anti-β-actin (1:2000, HRP-66009, Proteintech) in antibody diluent (A1800, Solarbio, China) at 4 °C for 24 h. After washing with Tris-buffered saline +0.2% Tween 20 (TBST) buffer three times for 15 min, the films were incubated in secondary antibody, which consisted of anti-PSD-95 and anti-GluA1 (peroxidase Affinipure goat anti-rabbit IgG (H + L), each diluted 1:200, ZB-2305, ZSGB-BIO). The film was then washed again three times in TBST. Finally, we activated the blot using the Western Chemiluminescent HRP-substrate (WBKLS0050, Millipore, Billerica, MA, USA). The blot was scanned and then analyzed via densitometry using ImageJ software (NIH, Bethesda, MD)\.

2.5.2. Immunofluorescence

The immunofluorescence assay was carried out similarly to the method described by Xu et al. (Xu et al., 2010). Rats (n = 3 per group) were anesthetized and infused with 0.9% normal saline and 4% paraformaldehyde through the heart. The brain was rapidly removed after perfusion and fixed overnight in 4% paraformaldehyde. The brain was then immersed in progressively higher concentrations of sucrose solution until it lost buoyancy and sank in solution. Following the location described by Paxinos and Watson, 20 μm slices of the PFC were cut using a freezing cryotome. Frozen slices were incubated a 37 °C for 30 min, then washed with phosphate-buffered saline (PBS). After drying, the sections were blocked in 10% normal sheep serum and incubated once again at 37 °C for 1–2 h, whereafter mouse primary antibody (BDNF 1:200, ab108319, Abcam; PSD-95 1:100, ab18258, Abcam; GluA1 1:100, ab31232, Abcam) was added. The negative control group was added with equal volume PBS solution, and the sections were incubated in a camera-obscuring box at 37 °C for 1 h, at room temperature for 1 h, and then overnight at 4 °C in the dark. The next day, the sections were removed from the dark box, incubated again at 37 °C for 1.5 h, then washed with PBS buffer. Secondary antibody was then added according to the species of the first antibody (anti-mouse, Proteintech SA00006-3; anti-rabbit, Proteintech SA00006-2) and incubated at 37 °C for 1.5 h. Afterwards, the core was dyed with DAPI, the anti-fluorescence attenuation sealing tablet was used to seal tissue, and the section observed under confocal microscope. The brightness and contrast of the digital image were adjusted with Photoshop software.

2.5.3. Transmission electron microscopy (TEM)

Our TEM methodology was based on the work of Tizro et al. (Tizro et al., 2019). Rats (n = 4 per group) were injected with 0.9% normal saline and 0.1 M PBS and 4% paraformaldehyde under anesthesia. Their PFCs were sectioned into blocks (1 mm × 1 mm × 1 mm) on ice and placed in 2.5% glutaraldehyde at 4 °C overnight. After cleaning with 0.1 M PBS solution, the blocks were fixed in 1% osmium tetroxide for 2 h and then washed with PBS. After dehydration with gradient ethanol and acetone, the tissues were stored in epoxy resin and stained with uranyl acetate and citric. Their ultrastructural changes were then observed by TEM.

2.6. Statistical analyses

All data were expressed as the mean ± standard error about the mean (SEM). Subjective evaluations were made by two independent evaluators who were blinded to the groups being judged. The normality and variance of the data distribution of each group were analyzed by Shapiro-Wilk or Anderson-Darling and Levene's tests. Mann-Whitney U test and Kruskal-Wallis test were carried out to compare groups with non-normal value distribution. Data for control and SPS&S groups were analyzed by Student's t-test. One-way analysis of variance (ANOVA) was used to analyze the drug groups results and followed by Dunnett's test using SPS&S statistical software (Version 22.0, SPS&S, Inc.; Chicago, IL, USA). The bar charts was performed using GraphPad Prism 9 software(GraphPad Software Inc.). Pearson's correlation coefficient was calculated to determine the specific connection among GluA1, BDNF, and PSD-95 proteins in the PFC. Stepwise linear regression analysis was also used to analyze the relationship between molecular and behavioral. Statistical significance was set at P < 0.05.

3. Results

3.1. Effects of SPS&S procedure

In the OFT, the SPS&S group exhibited less exploratory activity, which was manifested in reduced activity distance, time spent in the chamber's center, number of upright rearings, and number of crossings (Fig. 2A and B, E-F). In the EMPT, animals in the SPS&S group had fewer entries into the open arms and spent less time therein than the control group (Fig. 2C and D). Freezing and immobility times of SPS&S animals were both significant increased compared to the control group (Fig. 2G and H). Rat paths are illustrated in Fig. 2I-L.

Fig. 2.

Fig. 2

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Effect of SPS&S procedure on behaviors. A) Distance traveled by group in the center square (Mann-Whitney U test: z = −2.948, P = 0.002) in the OFT. B) Numbers of crossings (t = 3.781, P = 0.001) C, D) Number of entries into the open arms (t = 3.005, P = 0.008) and time spent in the open arms (t = 5.158, P = 0.000). E) The time spent in center area (t = 3.496, P = 0.003). F) Up-right numbers (t = 2.640, P = 0.017). G) Passive immobility time in FST (t = 2.944, P = 0.009). H) Freezing time in FBT (t = 2.829, P < 0.011). I, J, K, L) Representative video tracking images of OFT and EPMT. I, K) control group, J, L) SPS&S group. Data are represented as mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001compared with control group.

The protein expression of GluA1, BDNF, and PSD-95 in the SPS&S group was significantly lower than the control group, as measured by both WB and IF (Fig. 3A–I). From the results of TEM, obvious differences in synaptic ultrastructure were found between the control and SPS&S groups in the PFC. In contrast to the control group, the SPS&S group presented with increased in synaptic cleft width, with decreased PSD thickness and synaptic interface curvature (Fig. 3J-N).

Fig. 3.

Fig. 3

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Effect of SPS&S procedure on synaptic ultrastructure and expression of GluA1 and BDNF in the PFC. A, B, C) Relative expression of GluA1 (t = 3.486, P = 0.025), BDNF (t = 3.091, P = 0.037), and PSD-95 (t = 3.108, P = 0.036). D, E, F) Localization and distribution of GluA1, BDNF, and PSD-95. Bar = 20 μm. G) GluA1 in PFC (t = 7.073, P = 0.002). H) BDNF in PFC (t = 5.431, P = 0.007). I) PSD-95 in the PFC (t = 11.79, P = 0.000). J, K) PFC synaptic ultrastructure. L, M, N) Synaptic cleft width (L,t = −3.563, P = 0.012), postsynaptic density (M, z = -2.309, P = 0.021), and synaptic interface curvature (N,t = 2.708, P = 0.035). Data are represented as mean ± SEM.

*P < 0.05, **P < 0.01, and ***P < 0.001 compared with control group.

3.2. Effects of (2R,6R)-HNK on PTSD-like behaviors with SPS&S procedure and dose-effect relationship

At both the 50 μM and 100 μM doses, (2R,6R)-HNK-treated rats reared up-right significantly more times than vehicle control rats (P = 0.010 for both) in the OFT. Moreover, rats treated with 100 μM (2R,6R)-HNK displayed more chamber crossings (P = 0.016). Rats in the SPS&S-50 group exhibited more time in the center area than did the SPS&S-Sal group (P = 0.003). In the EMPT, no groups showed any statistically significance differences in time spent in center area or in the number of entries into the open arms. When compared with the SPS&S-Sal group, time spent in the open arms of the SPS&S-50 (P = 0.000) and SPS&S-100 (P = 0.028) groups was longer. Post hoc analysis showed that there was an obvious decrease in freezing time in rats treated with 50 μM (P = 0.001) and 100 μM (P = 0.001) (2R,6R)-HNK compared with the SPS&S-Sal group. After (2R,6R)-HNK treatment, the rats had a reduction in immobility time compared to saline treatment. There was also significant difference between the SPS&S-50 (P = 0.013) and SPS&S-100 (P = 0.004) groups compared to the SPS&S-Sal in the FST (Fig. 4A–H).

Fig. 4.

Fig. 4

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Effect of (2R,6R)-HNK on PTSD-like behaviors under the SPS&S procedure. A) Time spent in the center area in the OFT (P = 0.024, Kruskal-Wallis test). B) Up-right numbers (F (3, 36) = 4.753, P = 0.007) C, D) Time spent in the open arms (F (3, 36) = 6.233, P = 0.002) and number of entries into the open arms (P = 0.057 at Kruskal-Wallis test). E) Distance traveled in the center square by group (F (3, 36) = 0.5714, P = 0.638). F) Number of crossings (F (3, 36) = 3.109, P = 0.038). G) Passive immobility time in FST (F (3, 36) = 4.647, P = 0.008). H) Freezing time in FBT (F (3, 36) = 7.795, P = 0.000). I–P) Representative video tracking images of OFT and EPMT. I, M) SPS&S-Sal group; J, N) SPS&S-20 group; K, O) SPS&S-50 group; L, P) SPS&S-100 group. Data are represented as mean ± SEM.

*P < 0.05, **P < 0.01, and ***P < 0.001 compared with control group.

In order to refine the estimate of the differences among behavior-type and (2R,6R)-HNK, polynomial curve fittings were performed. The data revealed a U-shaped dose response following infusion of (2R,6R)-HNK (Fig. 5). The fitted curve peaked beyond 50 μM, indicating that a 50 μM dose of (2R,6R)-HNK is suboptimal, but that the optimal concentration is nearer to 50 μM than 20 or 100 μM.

Fig. 5.

Fig. 5

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Polynomial fitting between dose of medicine and behavior.

3.3. Effects of (2R,6R)-HNK on the expression of GluA1, BDNF, and PSD-95 with SPS&S procedure

The expression of GluA1, BDNF, and PSD-95 following treatment with (2R,6R)-HNK was compared with that in rats from the SPS&S-Sal group. WB results showed that the SPS&S-100 group trended toward had higher GluA1 expression relative to the SPS&S-Sal group (P = 0.022). There was a reduction in BDNF protein expression in the SPS&S-Sal group compared with the SPS&S-50 group (P = 0.014). Changes in the level of the PSD-95 protein responses were significantly higher in the SPS&S-50 (P = 0.031) and SPS&S-100 (P = 0.04) groups than in the SPS&S-Sal group (Fig. 6A–C).

Fig. 6.

Fig. 6

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Effect of (2R,6R)-HNK on the expression of GluA1 and BDNF in the PFC under the SPS&S procedure and results of correlation analysis. A, B, C) Relative expression of GluA1 (F (3, 8) = 7.780, P = 0.009), BDNF (F (3, 8) = 4.886, P = 0.033) and PSD-95 (F (3, 8) = 4.448, P = 0.041). D, E, F) Correlations between PSD-95 and BDNF (D), GluA1 and BDNF €, and GluA1 and PSD-95 (F) across all rats (n = 12). Correlations were computed using Pearson's R.

F (3, 8) = 7.253, P = 0.011.

Immunofluorescence showed that the levels of GluA1 (P = 0.031, P = 0.013), BDNF (P = 0.003, P = 0.002), and PSD-95 (P = 0.015, P = 0.002) were all increased with (2R,6R)-HNK administration in the PFC, especially in the SPS&S-50 and SPS&S-100 groups. (Fig. 7A–H).

Fig. 7.

Fig. 7

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Effect of (2R,6R)-HNK on the expression of GluA1, BDNF in the PFC under the SPS&S procedure. A) PFC image capture location diagram. B) Low magnification microscopic image of PFC. Bar = 200 μm. C, D, E) Localization and distribution of GluA1, BDNF, and PSD-95. Bar = 20 μm. F) GluA1 in PFC (P = 0.033, Kruskal-Wallis test). G) BDNF in PFC (F (3, 8) = 12.941, P = 0.002). H) PSD-95 in PFC (F (3, 8) = 16.1, P = 0.001). Data are represented as mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with control group.

Pearson's correlation analysis demonstrated that there were two-object cross-correlations among the three parts (Fig. 6). The results of pairwise correlational analysis and further regression analysis supported a model by which increased GluA1 protein expression leads to increased expression of the BDNF and PSD-95 protein (Table 1).

Table 1.

Results of regression analysis.

Independent variable Dependent variable R R2 F β T
GluA1 PSD-95 0.768 0.590 14.383 0.768 3.792**
GluA1 BDNF 0.750 0.562 12.845 0.750 3.584**
BDNF PSD-95 0.745 0.555 12.472 0.745 3.532**
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*P < 0.05, and**P < 0.01.

An exploratory factor analysis was conducted by principal component analysis. Kaiser-Meyer-Olkin measure of sampling adequacy (coefficient = 0.754) and Bartlett's test of sphericity (P = 0.001) showed that the structure of data was suitable for further analysis. One factor with eigenvalues greater than 1 was retained from the variables of GluA1, PSD-95, and BDNF protein expression only, which accounted for 83.621% of the total variance. Three variables were included in the multivariate regression analysis, and stepwise linear regression analysis was also used to analyze the relationship be-tween molecular and behavioral results (Table 2). The results of these statistical analyses showed that changes in GluA1 was the single strongest predictor of time spent in center area, up-right numbers, and freezing time, suggesting that GluA1 level was an effective predictor of anxiety-like behavior and contextual fear responses. BDNF and PSD-95 protein levels were strongly correlated with immobility time, suggesting that expression of these proteins is a predictor of depression-like behavior. PSD-95 was also a strong predictor of numbers of crossings and the amount of time spent in the open arms, indicating that PSD-95 plays a pivotal role in exploratory activity and anxiety-like behavior.

Table 2.

Results of regression analysis between expression levels of GluA1, BDNF and PSD-95 proteins to various behavioral indexes.

Independent variable Dependent variable R R2 F β T
GluA1 Freezing time 0.798 0.637 17.555 −0.798 −4.190**
Up-right number 0.970 0.941 160.939 0.970 12.686***
Time spent in center area 0.961 0.923 119.862 0.961 10.948***
PSD-95 Crossing numbers 0.731 0.534 11.477 0.731 3.388**
Time spent in the open arms 0.812 0.660 19.416 0.812 4.406**
PSD-95 Immobility time 0.947 0.897 39.297 −0.586 −3.660**
BDNF −0.426 −2.659*
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*P < 0.05, **P < 0.01, and ***P < 0.001.

3.4. Effects of (2R,6R)-HNK on the synaptic ultrastructure with SPS&S procedure

The results measured by TEM showed that, following treatment by (2R,6R)-HNK, the synaptic ultrastructure in the PFC had obvious differences. Compared with the SPS&S-Sal group, the width of the synaptic cleft in the SPS&S-50 group was narrower (P = 0.024) and the curvature of synaptic interface was increased (P = 0.038). There was no difference in synaptic cleft width between the SPS&S-20 group or SPS&S-100 group and the SPS&S-Sal group. Increased PSD thickness was observed in the SPS&S-50 group (P = 0.007) and SPS&S-100 group (P = 0.010) compared with the SPS&S-Sal group in the PFC, whereas the SPS&S-20 group was not statistically different therefrom (Fig. 8A–D).

Fig. 8.

Fig. 8

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Effect of (2R,6R)-HNK on the synaptic ultrastructure in the PFC under the SPS&S procedure. A) Synaptic ultrastructure. B) Curvature of synaptic interface (F (3, 12) = 5.633, P = 0.012) C) Postsynaptic density (F (3, 12) = 6.021 P = 0.010) D) Synaptic cleft width (F (3, 12) = 3.705, P = 0.043). Data were represented as mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with control group.

4. Discussion

PTSD is the most frequent psychiatric disorder in individuals who have experienced a traumatic incident. To date, most therapeutic strategies for PTSD have been limited to treating its symptoms. For example, selective serotonin reuptake inhibitors are effective antidepressants, methylphenidate is used for reducing mood instability, donepezil can improve cognition, and antioxidants or anticonvulsants serve to relieve excitotoxicity (Kaplan et al., 2018; Wheaton et al., 2011). The present study was designed to assess the effectiveness of (2R,6R)-HNK in alleviating PTSD-like symptoms in a rat model, to assess the underlying molecular mechanisms, and to explore the dose-effect relationship between (2R,6R)-HNK and PTSD. Animals subjected to the SPS&S procedure developed PTSD-like behaviors; however, injection of (2R,6R)-HNK was able to significantly suppress these symptoms. In addition, (2R,6R)-HNK was able to upregulate the expression of BDNF, GluA1, and PSD-95 in the PFCs of SPS&S rats. Meanwhile, synaptic structure was significantly restored, hinting that behavioral effects might be potentially based on the modulation of BDNF, GluA1, and PSD-95 expression within the PFC.

Understanding the pathophysiology of PTSD is critical to developing novel treatment options therefor. To this end, it is necessary to use dependable animal models that faithfully mirror symptoms in humans (Torok et al., 2019; Xi et al., 2021). We modified the classic SPS protocol by adding a single inescapable electric foot shock to enhance the fear responses (Hou et al., 2018). The SPS&S rats exhibited significant PTSD-like symptoms, including enhanced conditioned fear responses, amplified acoustic startle responses, and anxiety and depression-like behaviors, which was consistent with previous studies (Hou et al., 2018; Xi et al., 2021). Rats in this model also displayed significantly reduced GluA1, BDNF, and PSD-95 within their PFCs, which was partly consistent with the results of another study (J. Wang et al., 2022). Rats subjected to the SPS&S procedure also had decreased synaptic interface curvature and postsynaptic density and increased synaptic cleft width in their PFCs, as assessed by TEM. These findings further confirmed the validity of the PTSD model.

Expression of the AMPAR subunit GluA1 in the PFC was decreased in the SPS&S group. SPS&S model rats were likely to exhibit severe damage in synaptic transmission in glutamatergic neurons, with subsequent functional inhibition in the PFC. Notably, some studies have reported that GluA1 expression levels were increased in a restraint-stressed model (Novaes, Bueno-de-Camargo, & Munhoz, 2021). Yet another study using shock and predator odor showed that GluA1 will aggregates in the PFC (Leal RB and Formolo, 2020b). These discrepant findings may arise from differences in the stress model used, indicating that different stimuli produce different effects for the PFC. Furthermore, timing differences may partially explain these divergent results.

Due to the high complexity and diversity of symptoms in PTSD, therapy is limited to treating its symptoms. Current treatments carry high side-effect profiles and limited efficacy. As an NMDA receptor antagonist, ketamine plays a vital role in rapidly alleviating symptoms of PTSD (P. Zanos, 2017). Its mechanism of action seems to be through increasing glutamate receptors signaling in key AMPA-to-NMDA neural circuits (Tizabi et al., 2012). Previous studies have shown that (2R,6R)-HNK, one of the metabolites of ketamine, can initiate and sustain rapid antidepressant effects, with established efficacy against a myriad of symptoms and no apparent adverse effects (P. Zanos et al., 2019). As such, we speculated that administration of (2R,6R)-HNK might rapidly rescue stress-induced PTSD-like behavior in our SPS&S paradigm, particularly in reversing negative emotion behavior deficits after traumatic stress exposure.

Despite being a metabolite of ketamine, (2R,6R)-HNK operates through a slightly different mechanism than ketamine, as (2R,6R)-HNK does not inhibit NMDA receptors directly (Lumsden EW and Myers, 2019). AMPARs mediate the majority of excitatory transmission in the central nervous system and enable the synaptic plasticity that underlies both physiological and pathological conditions. Of the constituent proteins that make up AMPARs, GluA1 has been shown to play the most prominent role in synaptic plasticity (Tzakis and Holahan, 2020). In the present study, the protein level of GluA1 in (2R,6R)-HNK-treated rats was remarkably higher than that in the vehicle-treated controls. We speculated that the therapeutic effect of (2R,6R)-HNK was mediated by increasing the expression of AMPAR within the PFC, which, notably, is different from the mechanism of ketamine. Congruent with this hypothesis, we observed an upregulation of GluA1 in the PFC following treatment of rats with (2R,6R)-HNK. Meanwhile, others have reported a concentration-dependent increase in GluA1 levels following (2R,6R)-HNK treatment of a hippocampal-cortical coculture (Shaffer et al., 2019).

In recent years, studies have found that, beyond (2R,6R)-HNK's antidepressant role, it may have prophylactic stress protection (Chen et al., 2020), analgesic (Kroin et al., 2019), and anti-aggression and pro-social properties (Chou, 2020). Nonetheless, its ability to treat PTSD and its underlying neurobiological mechanisms has not been confirmed. Our selection of dosage range for (2R,6R)-HNK is predicated on the extant literature thereon. Some studies have reported that 10 μM (2R,6R)-HNK produces an intrahippocampal concentration comparable to that generated by antidepressant metabolism (Lumsden et al., 2019; P. Zanos, 2017). Others reported that 50 μM of (2R,6R)-HNK resulted in a significant decrease in phosphorylation of eukaryotic elongation factor 2 levels in the hippocampus, which, in turn, lead to antidepressant action (Suzuki et al., 2017). Based on these reports, we selected a dosage range of 20 μM, 50 μM, and 100 μM. Overall, we observed the best efficacy from the 50 μM dose, which conferred improvements in spontaneous exploration, locomotor activity, and anxiety reduction. Improvements in the two former parameters, as well as in depression-like behaviors, were also noted in the 100 μM dose. The SPS&S rats' fear response declined significantly, demonstrating that (2R,6R)-HNK had positive outcomes on depression and despair in SPS&S group. However, there was no significant difference in behavior between SPS&S-Sal and SPS&S-20 rats. Thus, (2R,6R)-HNK alleviates PTSD-like symptoms in SPS&S rats in a concentration-dependent manner. Since most outcomes were similar between the 50 and 100 μM doses, polynomial curve fittings between dose and behavior for all data were performed. This statistical approach produced curves that tended to increase from 20 to 50 μM, peaking past 50 μM before falling off towards 100 μM), demonstrating that the optimal dosage of (2R,6R)-HNK was likely somewhere between 50 and 100 μM by extrapolation on curves. Improving the route of drug administration drugs and establishing the optimal dosage are the next steps to be determined in investigating the efficacy of (2R,6R)-HNK.

While the potential side effects of (2R,6R)-HNK have not been rigorously explored, stronger aggression behaviors after injecting higher doses of (2R,6R)-HNK have been observed (Ye et al., 2019). Our observations of higher anxiety levels in rats injected with 100 μM (2R,6R)-HNK (relative to those injected with 50 μM) indicate that aggression and anxiety share a common underlying mechanism. In support of this, a number of studies have been published that show increased levels of GluA1 in the PFC promote aggressive behavior in male rodents (Takahashi and Miczek, 2014; Wang et al., 2011).

BDNF is a widely expressed neurotrophic factor in the nervous system that plays a critical role during neurogenesis as well as the in the function of neurons in the brain. A reduction in BDNF is strongly associated with PTSD (Andero and Ressler, 2012; Chang et al., 2021). BDNF expression is closely correlated with transmembrane expression of GluA1 (Xiao et al., 2019). Meanwhile, alterations in BDNF levels may be involved in the synaptic deficits caused by stress (Zagrebelsky M, 2014). The expression of BDNF in SPS&S-50 μM-treated rats significantly increased, while there was no significant difference in BDNF expression between saline-treated rats and either those treated with SPS&S-20 μM or SPS&S-100μM, despite some visible increase in both. Previous studies have demonstrated that (2R,6R)-HNK administration increases BDNF protein levels in hippocampal synaptic neurons (Panos Zanos et al., 2016), which is consistent with our results, and indicative that BDNF may play a role in in (2R,6R)-HNK's anti-PTSD actions, specifically as a downstream mediator of its effects.

PSD-95 is thought to be involved in the regulation of synapses and neuronal plasticity as well as the synthesis of neurotransmitters and other neurotrophic factors. PSD-95 interacts indirectly with AMPAR through binding to its auxiliary subunit Stargazin (Guo et al., 2018). The synaptic targeting of GluA1 may be indirectly enhanced by the interaction between PSD-95 and Stargazin (Guo et al., 2014). Moreover, PSD-95 plays an important functional role in maintaining the stability of AMPA receptors, enhancing synaptic function, and promoting the growth of dendritic spines (Lisek et al., 2017). PSD-95 is likely to be regulated by the activation of BDNF and GluA1 (Leal et al., 2014). PSD-95 increased at the protein level within the PFC following treatment with (2R,6R)-HNK, suggesting that (2R,6R)-HNK might alter synaptic structure and function in our PTSD model rats, and its changes were closely related to relief of PTSD symptoms after (2R,6R)-HNK injection. These improved effects on synaptic plasticity work at least in part through GluA1-mediated BDNF. Regression analyses between GluA1, BDNF, and PSD-95 and the tested behaviors indicated that BDNF was closely related to anxiety-like and depression-like symptoms, while GluA1 was associated with fear responses. This is partially consistent with previous studies (H. Yao et al., 2021; H. J. Yu et al., 2018).

Emerging evidence suggests that GluA1 mediates diverse types of synaptic plasticity, thereby playing vital roles in brain function (Ge and Wang, 2021). Synaptic morphology and structural plasticity are the material bases of functional synaptic plasticity. Hence, changes in synaptic structure, such as in synaptic cleft width, synaptic interface curvature, and postsynaptic density, affect numerous facets of neurotransmission. In this study, we observed diminished synaptic interfaces and larger gaps in the synapses within the PFCs of SPS&S model rats. This corresponded with behaviors indicative of fear, anxiety, and depression. Treatment with (2R,6R)-HNK tended to reverse this phenotype, reducing synaptic cleft width and increasing synaptic interface curvature and postsynaptic density. This was particularly evident in the SPS&S-50 group. In addition, rats treated with (2R,6R)-HNK had increased neuronal number and synaptic enhancement. These parameters are involved in emotion and fear memory. These neuroregenerative and behavioral effects coincided with increases in the synaptic proteins GluA1, BDNF, and PSD-95. We hypothesize that the upregulation of these key neurological proteins were a core component of (2R,6R)-HNK's restorative effect in treating PTSD.

There are several limitations to the current study. First, while TEM gains in terms of examining minute neuronal structural detail, it loses in scope. As such, TEM does not inform as to the number of dendritic spines throughout the PFC, which Golgi staining to do it can. In so doing, there is the risk that our chosen method may miss the forest for the trees, so to speak. Work is needed to examine electrophysiological in vitro by patch clamp and to analyze whether they are related to GluA1 and BDNF in the future. Although the effects of (2R,6R)-HNK have recently been reported extensively, the emerging evidence that (2R,6R)-HNK may increase aggression needs to be evaluated, we will add several groups about injecting concentration of gradient (2R,6R)-HNK and saline into native rats in the next step to investigate drug safety effectiveness and see the consequences in plasticity and behaviour to native rats at the same time. Additionally, more work is needed to determine the extent of (2R,6R)-HNK's therapeutic effects and to avoid either short- or long-term relapse. Moreover, our future research will also focus on other brain regions such as hippocampus, amygdala, nucleus accumbens and hypothalamus to study whether the effect of (2R,6R)-HNK is related to the network between brain regions. Our study only explored negative emotions; other aspects of cognition, such as spatial memory (especially fear memory), were not assessed. In future studies, we will look at the effect of (2R,6R)-HNK on fear memory within different time windows using our SPS&S model. Finally, whether (2R,6R)-HNK is a better alternative to ketamine for treating PTSD remains to be directly examined.

5. Conclusion

In summary, we have established a rat model of PTSD using SPS&S. This model can induce a fear response with anxiety and depression-like behavior. Rats subjected to it exhibit a decline in GluA1 and BDNF expression along with damaged synaptic structures. (2R,6R)-HNK rapidly and effectively reverses these PTSD symptoms. We demonstrate for the first time that (2R,6R)-HNK might be involved in alleviating anxiety- and depression-like behaviors by stimulating GluA1-mediated synaptic plasticity in the PFC. These findings highlight the possibility of directly utilizing (2R,6R)-HNK for the treatment of PTSD.

Competing interests

The authors report no competing interests.

CRediT authorship contribution statement

Yu Li: Writing – original draft. YaLin Du: Writing – original draft. Chen Wang: Data curation. GuoHua Lu: Funding acquisition. HongWei Sun: Supervision. YuJia Kong: Formal analysis. WeiWen Wang: Funding acquisition. Bo Lian: Verification. ChangJiang Li: Resources. Ling Wang: Resources. XianQiang Zhang: Visualization, Conceptualization. Lin Sun: Writing – review & editing, Conceptualization, Funding acquisition.

Declaration of competing interest

The remaining authors have nothing to disclose.

Acknowledgments

This work was supported by National Natural Science Foundation of China (82101588; 82071517; 31771217), Ministry of Science and Technology of China (2017YFE0126500), Surface project of Natural Science Foundation of Shandong Province (ZR2020MC218), Institute of Psychology, Chinese Academy of Sciences (GJ202002), Medical Education Research project of Chinese Medical Association (20A1209), Education teaching reform and research found of Weifang Medical University (2019YB023), Science and Technology Development Program of Traditional Chinese Medicine in Shandong Province (2019WS594/202002010572), The Education and Teaching Reform Project of the Psychology and Education Reference Committee of the Ministry of Education, Curriculum Construction Class-13(20221013).

Contributor Information

Yu Li, Email: 473565867@qq.com.

YaLin Du, Email: 2889445907@qq.com.

Chen Wang, Email: 1532221547@qq.com.

GuoHua Lu, Email: ghluu1@163.com.

HongWei Sun, Email: sunhw@wfmc.edu.cn.

YuJia Kong, Email: yujia_kyj80@163.com.

WeiWen Wang, Email: wangww@psych.ac.cn.

Bo Lian, Email: lianbo@wfmc.edu.cn.

ChangJiang Li, Email: 391620@qq.com.

Ling Wang, Email: 2006ky@sina.com.

XianQiang Zhang, Email: xianqiang2018@163.com.

Lin Sun, Email: linsun2013@wfmc.edu.cn.

Data availability

No data was used for the research described in the article.

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