Anxiolytic effects of GLYX-13 in animal models of posttraumatic stress disorder-like behavior

Abstract

In the present study, we investigated the effectiveness of GLYX-13, an NMDA receptor glycine site functional partial agonist, to alleviate the enhanced anxiety and fear response in both a mouse and rat model of stress-induced behavioral changes that might be relevant to posttraumatic stress disorder (PTSD). Studies over the last decades have suggested that the hyperactivity of hypothalamic–pituitary–adrenal (HPA) axis is one of the most consistent findings in stress-related disease. Herein, we used these animal models to further investigate the effect of GLYX-13 on the stress hormone levels and glucocorticoid receptor (GR) expression. We found that exposure to foot shock induced long-lasting behavioral deficiencies in mice, including freezing and anxiety-like behaviors, that were significantly ameliorated by the long-term administration of GLYX-13 (5 or 10 mg/kg). Our enzyme-linked immunosorbent assay results showed that long-term administration of GLYX-13 at behaviorally effective doses (5 or 10 mg/kg) significantly decreased the elevated serum levels of both corticosterone and its upstream stress hormone adrenocorticotropic hormone in rats subjected to the TDS procedure. These results suggest that GLYX-13 exerts a therapeutic effect on PTSD-like stress responding that is accompanied by (or associated with) modulation of the HPA axis, including inhibition of stress hormone levels and upregulation of hippocampal GR expression.

Keywords

Posttraumatic stress disorder electric foot shock HPA axis GLYX-13 glucocorticoid receptors

Introduction

Posttraumatic stress disorder (PTSD) has become a major neurological and psychiatric issue, manifesting a high co-morbidity with depression and panic and anxiety disorders (Fokkens et al., 2015; George et al., 2015; McFarlane and Forbes, 2015).The prevalence rate of PTSD has been reported to be 9.5–11.2% in the general population, emphasizing the need to identify potential risk factors (LeardMann et al., 2015; Muhie et al., 2015). To date, the exact etiology of PTSD, a complex disorder, is unclear, and research into the underlying neurobiology has implicated alterations of a myriad of neurotransmitter and neuroendocrine systems, including serotonin, norepinephrine, and GABA, as well as dysregulation of the hypothalamic–pituitary–adrenal (HPA) axis (Cohen et al., 2009; Simeon et al., 2007). The HPA axis, an important neuroendocrine system, plays a key role in the pathophysiology of PTSD. The stress hormone upstream of glucocorticoids, adrenocorticotropic hormone (ACTH), which is responsible for the release and regulation of glucocorticoids, is also elevated in patients with anxiety disorders (Zaba et al., 2015). In experimental animals, chronic stress paradigms can recapitulate not only many behavioral characteristics but also several of the biochemical changes associated with elevated corticosterone and ACTH levels (De Quervain and Margraf, 2008; Griffin et al., 2014). These alterations can be normalized by administration of most therapeutic agents currently used in the clinic to treat anxiety, which is consistent with their anxiolytic response.

Recently, many studies have reported that N-methyl-d-aspartate (NMDA) receptor activation is also associated with the formation of spontaneous intrusive memories and that high NMDA receptor activity may serve as a risk factor for developing PTSD (Yamamoto et al., 2008). The noncompetitive NMDA receptor antagonist ketamine has recently attracted attention for its anti-PTSD effects (Juven-Wetzler et al., 2014; Zhang et al., 2015). McGhee et al. reported that patients at a military medical center who received ketamine had significantly lower rates of PTSD than those who did not (McGhee et al., 2008). These preclinical and clinical findings support the idea that novel pharmacological tools targeting NMDA receptors maybe promising candidates for new anti-PTSD drugs. Unfortunately, ketamine causes unacceptable dissociative side effects and is a substance of abuse (Burgdorf et al., 2013). Recent studies have demonstrated that GLYX-13, an NMDA receptor glycine site functional partial agonist, produces rapid onset antidepressant-like effects without the psychotomimetic side effects of NMDA receptor antagonists (Lu et al., 2015; Zhang et al., 2008). Given all of the above-mentioned factors, it is reasonable to predict that GLYX-13 would be effective in treating PTSD. Therefore, the present study examined the effect of GLYX-13 in alleviating the enhanced anxiety and fear response induced in two animal models of possible relevance to PTSD.

In addition, considering the crucial role of the HPA axis in the pathophysiology and therapy of PTSD, the present study compared the ability of GLYX-13 and sertraline (Ser), an a selective serotonin reuptake inhibitor (SSRI) serving here as a positive control, to alleviate the enhanced anxiety and fear responses induced in these two animal models; using inescapable electric foot shock and also time-dependent sensitization (TDS), a paradigm causing prolonged HPA axis hyperactivity, to investigate whether chronic administration of behaviorally effective doses of GLYX-13 (0.5,5,10 mg/kg) could decrease the elevated corticosterone and ACTH levels observed in these animal models. We also assessed the effect of GLYX-13 on hippocampal GR expression in the stressed rats.

Materials and methods

Animals

Both male ICR mice (18±2 g) and male Sprague-Dawley rats (180±10 g) were purchased from Beijing Vital Laboratory Animal Technology Company (Beijing, China). The animals were maintained under standard conditions of controlled temperature (23±1°C), humidity (45%). All animals were housed in a 12-h light/dark cycle starting at least 5 days before the experiment and had access to water and food ad libitum. They were group-housed with the same mates throughout the acclimation and testing periods. Experiments were conducted in accordance with the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals. The experimental procedures were approved by the institutional committee on animal care and use, and all efforts were made to minimize animal suffering and reduce the number of animals used for the experiments.

Drugs and drug administration

Rabbit anti-GR (α+β) and rabbit anti-β-actin antibodies were purchased from Santa Cruz Biotechnology (CA, USA). A horseradish peroxidase-conjugated goat anti-rabbit IgG antibody was purchased from Zhongshan Technology (Beijing, China). Rat enzyme-linked immunosorbent assay (ELISA) kits for corticosterone and ACTH were purchased from TPI Inc. (WA, USA). Sertraline (Ser), purchased from Sigma-Aldrich (St. Louis, MO, USA), was administered as a positive control in all behavioral tests at a dose (15 mg/kg, i.g.), which is based on the results of a previous study (Zhang et al., 2015). The doses of GLYX-13 used here were based on those previously published with minor adjustments (Lu et al., 2015). To test the effects of repeated treatments on behavior, Ser (15 mg/kg, i.g.) or GLYX-13 (0.5, 5, 10 mg/kg, i.g.) was given once daily.

Electric foot shock with long-term drug administration in mice

The electric foot shock procedure was conducted as described previously (Qiu et al., 2013; Zhang et al., 2015). Briefly, for the training session, a Plexiglas chamber (20×10×10 cm) with a stainless steel grid floor (9-mm grid interval) was used. Electric foot shocks were delivered through the grid floor using an isolated shock generator (Med Associates Inc., USA). Each mouse was placed in the chamber, and after a 5-min adaptation period, a total of 15 intermittent inescapable foot shocks (intensity: 0.8 mA, interval: 10 s, and duration: 10 s) were delivered for 5 min. Control animals were placed in the same chamber for 10 min, without electric foot shocks. Beginning the day after the foot shock procedure (day 1), Ser (15 mg/kg, i.g.) or GLYX-13 (0.5, 5, 10 mg/kg, i.g.) was administered once daily between 8:00 and 9:00 a.m. The drug doses and the administration time were selected based on the results of our previous studies (Qiu et al., 2013). A schematic of the treatment and behavioral testing schedules is shown in Figure 1.

Contextual freezing

The contextual freezing test was performed as described previously with minor modifications (Qiu et al., 2013; Zhang et al., 2015). Briefly, all animals were exposed to the same chamber in which the foot shocks had been delivered 15 days after the initial exposure, but without foot shock this time, to measure the duration of contextual freezing behavior. Freezing was defined as the absence of all movement except that necessary for respiration (Fanselow, 1980). Total cumulative freezing time (total time spent freezing during each assessment period) was measured and analyzed automatically using automated computer software (Video Freeze SOF-843, Med Associates Inc., USA). The percent of time spent freezing was calculated by dividing the freezing time by the total time.

Open field test in mice

To investigated whether any GLYX-13-induced reversion of contextual freezing could be attributed to alterations of locomotor activity, we conducted the locomotor activity test as described previously with minor modifications (Chen et al., 2013; Jin et al., 2015; Xue et al., 2013). Briefly, 16 days after the animals were subjected to foot shock, mice were placed in the corner of a plastic box (36 × 29 × 23 cm³) having a base divided into 16 equal sectors for a 5-min acclimation period. The number of crossings (all four paws placed in a new square) and the number of times the animals reared (both front paws raised from the floor) were recorded during in the next 5 min.

Staircase test in mice

Staircase test is a relatively simple and efficient experiment to screen anxiolytic agents and can also be applied to assess PTSD-like anxiogenic responses in this animal model. The staircase was constructed from polyvinylchloride and consisted of five identical steps (2.5 cm high × 10 cm wide × 7.5 cm deep). The height of the wall remained consistently 12.5 cm above the stairs along the entire length of the staircase. On day 18 after foot shock, each mouse was individually placed on the floor of the box with its back toward the staircase. Each mouse was placed individually onto the staircase. The number of times the animal reared and the number of steps it climbed were recorded for 3 min. A step was considered climbed only when the mouse placed all four paws on the stair. The number of steps descended was not counted. At the end of 3 min, the mouse was removed, and the staircase was cleaned with an alcohol-soaked sponge to eliminate any residual odors. The drug treatments were randomized, and the observer was blind to the grouping. All studies were conducted between 8:00 and 11:00 a.m.

TDS procedure with long-term drug administration in rats

The TDS procedure was performed as described previously (Zhang et al., 2014a, 2014b). Briefly, after a 1-week acclimatization period, the rats were restrained for 2 h, and then each rat was immobilized inside a disposable, clear polyethylene rodent restraint cone (day 1). The large end of the cone was closed with tape. The bag size was adjusted according to the size of the animal to achieve complete immobilization. A hole in the small end of the cone allowed the rats to breathe freely. The rats were then individually placed in a clear acrylic cylinder (24-cm diameter, 50-cm height), filled with water (24°C) to 2/3 the height of the cylinder, and forced to swim for 20 min. Following a 15-min recuperation period, rats were exposed to diethyl ether until they lost consciousness. The rats were allowed to recover for 1week and were then subjected to a brief re-stress on day 7 (20-min swim stress). Starting on the first day after the TDS procedure (day 2), GLYX-13 (0. 5, 5, or 10 mg/kg) was given once daily between 8:00 and 9:00 a.m. To minimize the effects of multiple testing, tests were ordered from least to most stressful, and cage mates were tested simultaneously or on consecutive days. The originality of this protocol is that the same cohort of animals was tested in different behavioral paradigms, according to the procedure described previously (Zhang et al., 2014b). The treatment schedule and behavioral test design is shown in Figure 2.

Locomotor activity test in rats

Thirteen days after the TDS procedure, locomotor activity was measured by placing each rat individually in a clear open field apparatus (36 × 36 × 36 cm³) having a black rubber floor. Each animal was allowed to habituate to the environment for 5 min. In the subsequent 5 min, the distance traveled and the time of ambulation for all spontaneous movements were automatically recorded using a VIDEO-MEX-V image analytic system (Columbus Instruments, USA).

Contextual fear paradigm in rats

The contextual fear paradigm was conducted after the 14-day drug treatments were completed. On the first day of the paradigm (14 days after the TDS procedure), each rat was exposed to the conditioning context for 180 s in the conditioning chamber (60 × 21 × 30 cm³) without any additional stimulation. Immediately after that, a foot shock (0.8 mA, 4 s) was given through a stainless steel grid floor (Med Associates Inc. USA). Twenty-four hours after the initial foot shock (15 days after the TDS procedure), the rat was placed in the same conditioning chamber in which the previous foot shock had been administered, and the contextual fear response was evaluated by measuring the duration of freezing behavior during a 5-min interval. Freezing behavior was defined as a total absence of body or head movement except for that associated with breathing and was rated by observers blinded to the treatment group. The data were recorded as the average time (in seconds) spent freezing (Zhang et al., 2014b).

Elevated plus maze (EPM) task in rats

The EPM task is one of the most widely used tests for assessing anxiety responses of rodents that might be relevant to PTSD (Zhang et al., 2014a, 2015). The apparatus consisted of four branching arms (50 × 10 cm²), with two arms open and the other two enclosed by walls (14 cm high). The arms were connected by a center platform (10 × 10 cm²), and the maze was located 50 cm above the ground. Eighteen days after the TDS procedure (day 18), individual rats were placed in the central platform, facing the closed arms. For the purpose of the analysis, open-arm activity was quantified as (1) the time spent in the open arms relative to that in the open arms plus the closed arms, and (2) the total number of entries into the open arms relative to the number of entries into the open arms plus the closed arms. Rats were scored as entering an open or closed arm only when all four paws passed over the dividing line. The maze was cleaned with a 5% ethanol/water solution after each test to remove any confounding olfactory cues and dried thoroughly between sessions.

Enzyme-linked immunosorbent assay

Rats used in the TDS test were sacrificed after completing last test procedure. Their blood was sampled, centrifuged (860 g, 20 min) at 4°C and then stored at −20°C until used in the analyses. Serum corticosterone and ACTH levels were detected using ELISA kits (magnetic solid phase; TPI Inc., WA, USA) according to the manufacturer’s instructions. A sample (or standard) and conjugate was added to each well, and the plate was incubated for 1 h at room temperature without a blocking agent. After several washes and proper color development, the optical density value was determined at a wavelength of 450 nm using an ELISA plate reader. The assay sensitivities for corticosterone and ACTH were 0.7 nmol/L and 1 pg/mL, respectively.

Western blotting

Following decapitation, rat hippocampus were isolated rapidly and stored at −80°C for western blotting detection. The tissues were weighed, sonicated in RIPA lyses buffer supplemented with fresh protease and phosphatase inhibitors, and centrifuged at 13,800 g for 25 min. Then the protein concentration was determined by bicinchoninic acid (BCA) assay. After denaturation and electrophoresis, the proteins were transferred onto a NC membrane in semi-dry conditions. Following blocking in 1% BSA for 2 h, the protein membrane was incubated in primary antibody rabbit anti GR (1:1000) at 4°C overnight and then in secondary antibody HRP-conjugated goat anti-rabbit IgG (1:5000) for 1 h. ECL + kit (APPLYGEN Company, Beijing, China) was used for the detection of protein band and the chemiluminescence signal was transformed into a digital image using Kodak films for sequent analysis with software. Band intensities were quantified using the ImageQuant software package (GE Healthcare).

Statistical analysis

All data are expressed as the mean ± SEM. One-way analysis of variance (ANOVA) were used to compare the differences between two groups (i.e., control non-shocked mice vs. shocked mice; shocked mice vs. shocked + Ser-treated mice; non-TDS rats vs. post-TDS rats; post-TDS rats vs. TDS + Ser-treated rats), with Bonferroni corrections performed as needed to control for Type I errors. The behavioral effects of long-term GLYX-13 treatment after electric foot shock administered to mice or the TDS procedure conducted in rats were analyzed using one-way ANOVA followed by Dunnett’s test. For all tests, differences of p < 0.05 were considered statistically significant.

Results

Long-term behavioral effects following GLYX-13 administration after electric foot shock in mice

There were no significant differences between the control non-shocked mice and the shocked mice in the number of line crossings (Figure 3(a)) or number of times the mice reared (Figure 3(b)) (one-way ANOVA, p > 0.05). Daily oral administration of either Ser (15 mg/kg) or GLYX-13 also did not significantly affect the number of line crossings (Ser: one-way ANOVA, p > 0.05; GLYX-13: one-way ANOVA, F_(5,60) = 1.874, p > 0.05) or rearing (Ser: one-way ANOVA, p > 0.05; GLYX-13: one-way ANOVA, F_(5,60) = 1.547, p > 0.05). These results indicate that neither foot shock nor repeated GLYX-13 administration affected locomotor activity in this animal model.

By contrast, exposure to electric foot shock caused a significant contextual freezing response (one-way ANOVA, p < 0.001), with the time spent freezing increased in the shocked compared with the non-shocked mice, indicating that the electric foot shock model are successfully developed. Administration of Ser (15 mg/kg), the positive control, significantly reduced the freezing behavior (one-way ANOVA, p < 0.01), demonstrating the predictive validity of this model. Chronic treatment with GLYX-13 showed a significant main effect on the contextual freezing response in mice exposed to foot shock (one-way ANOVA, F_(5,60) = 0.524, p < 0.05). Further post hoc analysis revealed that GLYX-13 at doses of both 5 and 10 mg/kg significantly decreased the contextual freezing response (Dunnett’s test, P< 0.01, compared with the foot shock vehicle-treated group) on day 15 (Figure 3(c)). These results demonstrated a persistent fear response in mice to a context associated with a traumatic event and indicated that repeated treatment with GLYX-13 (5 and 10 mg/kg) alleviated the contextual freezing behavior in the stressed mice.

In the staircase test, as shown in Figure 3(d) and (e), mice that had been previously exposed to foot shock exhibited an increase in the number of times they reared (one-way ANOVA, p < 0.05) but failed to demonstrate a significant change in the number of steps (one-way ANOVA, p > 0.05) compared with non-shocked mice. These results indicated that the shocked animals continued to avoid an aversive-like compartment and exhibited a fear response to a context associated with a traumatic event. However, repeated administration of either GLYX-13 or Ser to shocked mice decreased the number of times the mice reared (GLYX-13, 5 and 10 mg/kg, one-way ANOVA, F_(5,60) = 2.456, p < 0.05; Ser, 15 mg/kg, one-way ANOVA, p < 0.05) without affecting their climbing behavior (GLYX-13, one-way ANOVA, F_(5,60) = 1.023, p > 0.05; Ser, 15 mg/kg, one-way ANOVA, p > 0.05) in the staircase test compared with shocked mice administered vehicle (Figure 3(d) and (e)), suggesting that GLYX-13 significantly ameliorated PTSD-like anxiogenic behavior.

To examine the possibility that TDS and/or drug treatments influenced baseline locomotor activity in fear-conditioned rats (subjected to fear conditioning 24 h before the measurement), we investigated the level of spontaneous locomotor activity for each group. The results showed that there was no significant difference among groups, indicating that neither the TDS procedure nor chronic drug treatments affected spontaneous activity in rats (Figure 4(a)).

By contrast, compared with control rats, rats exposed to the TDS procedure demonstrated a significantly increased contextual freezing response (one-way ANOVA, p < 0.05). The results of one-way ANOVA analyses revealed that exposure to the TDS procedure, followed by a 14-day undisturbed period, significantly increased the contextual freezing response, compared with that in controls (F_(5,60) = 3.875, p < 0.01). The administration of Ser or GLYX-13 (5 and 10 mg/kg) for 14 days alleviated the enhanced contextual freezing in rats that had experienced the TDS procedure (p < 0.01; Figure 4(b)).

As shown in Figure 4(c), one-way ANOVA analyses revealed that TDS-exposed animals showed significant reductions in the percent of time spent in the open arms (F_(5,60)= 4.785, p < 0.01), and in the amount of entries into open arms (F_(5,60) = 2.985, p < 0.01) in the elevated plus maze task compared with those for control rats. However, post hoc comparisons indicated that chronic administration with Ser (15 mg/kg) or GLYX-13 (5 and 10mg/kg) significantly increased both the reduced percent of time spent and entries into open arms compared with those in control animals (Figure 4(c)).

GLYX-13 reduces elevated serum corticosterone and ACTH levels in rats exposed to TDS

The results of our ELISA analysis demonstrated that TDS exposure significantly increased serum corticosterone levels in rats by 105.35% (p < 0.01, versus control-vehicle). However, as shown in Figure 5(a), chronic administration of GLYX-13 at doses of 5 and 10 mg/ kg or Ser at 15 mg/kg attenuated this increase, reducing the elevated serum corticosterone levels by 58.36%, 60.12%, and 50.12%,respectively (F_(5,60) = 8.523, p < 0.05 versus stress-vehicle).

As shown in Figure 5(b), serum ACTH levels were also significantly increased in TDS-exposed rats by 162.32% compared with those in control rats (p < 0.001). Consistent with its effect on serum corticosterone levels, chronic GLYX-13 administration at both 5 and 10 mg/kg ameliorated this increase, reducing serum ACTH levels by 53.38% and 51.01%, respectively (p < 0.05 and p < 0.01 versus stress-vehicle, respectively). A similar result was observed in the Ser-treated group (p < 0.05 versus stress-vehicle). Together, these results indicated that chronic GLYX-13 treatment significantly decreased stress hormone (corticosterone and ACTH) level.

GLYX-13 increases the reduced expression of hippocampal GRs in rats exposed to TDS

The results of our western blotting analysis (Figure 6) showed that total GR expression in the hippocampus was decreased 28 days after rats were subjected to the TDS regimen (p < 0.001 versus control-vehicle). However, chronic GLYX-13 (5, 10 mg/kg) or Ser (15mg/kg) administration significantly increased hippocampal GR expression (F_(5,60) = 8.214, p < 0.01 versus stress-vehicle).

Discussion

In the present study, our results demonstrated that electric foot shock delivered to mice elicited the acquisition of conditioned fear, with mice showing an innate freezing behavior in the same context 15 days after their initial exposure to the foot shock. However, the present study demonstrated that chronic treatment with GLYX-13 caused significant suppression of enhanced anxiety and contextual fear induced by inescapable electric foot shocks in mice and TDS in rats, which is similar to the effects of the first line anti-PTSD drug sertraline. Our findings are consistent with those of Pynoos et al. (1996), who showed that foot shock associated with situational reminders did not affect the motor activity of male mice in an open field test performed 3–6 weeks after the first foot shock. The present study indicated that aversive foot shock followed by repeated reminders could be an animal model for PTSD like stress responding, and GLYX-13 with dose of 5 and 10 mg/kg are therapeutic in this animal model.

Accumulating evidence indicates that rats exposed to a TDS regimen exhibit symptoms of increased arousal, such as exaggerated fear responses to trauma-related and trauma-unrelated stimuli (Iwamoto et al., 2007), which is consistent with observations in patients with PTSD who show enhanced anxiety and fear in response to stimuli unrelated to trauma. The TDS procedure has been found to enhance contextual fear conditioning, and freezing behavior may serve as a good assessment for the severity of anxiety due to hippocampal dysfunction (Kohda et al., 2007; Ji and Maren, 2007).

The EPM and staircase tasks have also been used to evaluate anxiety-like behavior in rodents that might be relevant to PTSD (Zhang et al., 2015). The results of our EPM assay are consistent with previous studies that found that foot-shocked mice significantly decrease the number of entries into and the time spent in the open arms of the EPM (Li et al., 2006; Steenbergen et al., 1991). Moreover, the stress-affected animals showed an increase in the number of times they reared, without a change in the number of steps they climbed in the staircase task, an indication of anxiety-like behavior in animals that might be relevant to PTSD (Zhang et al., 2012). It has been suggested that fear and anxiety levels increase in animals after exposure to traumatic events (Rosen and Schulkin, 1998). These results resemble the situation in PTSD patients where repeated traumatization or re-experiencing aspects of a traumatic event may have stress-induced anxiety-like effects (Rau et al., 2005).The data presented here demonstrated that contextual freezing was significantly enhanced in rats exposed to the TDS regimen, and chronic administration of GLYX-13 at doses of 5 and 10 mg/kg successfully blocked these aversive effects. The present study also showed that TDS exposure produced representative anxiety-like behavior, as evidenced by the significantly decreased percent of time spent in and number of entries into the open arms of the EPM in TDS-exposed animals. GLYX-13 ameliorated these behavioral changes and alleviated the anxiety produced in rats after TDS exposure. However, PTSD is associated with cognitive impairments involving memory and attention in both stressed animals and patients (Wang et al., 2010). Moreover, the glutamate N-methyl-d-aspartate receptor (NMDAR) plays a critical role in learning and memory. Therefore, whether GLYX-13 alters long-term memory need to further study.

We also found that the TDS regimen used in the present study in rats significantly increased serum levels of the stress hormones corticosterone and ACTH and decreased hippocampal GR expression. Similar to the behavioral effects, the chronic administration of GLYX-13 at doses of 5 or 10 mg/kg to TDS-exposed rats significantly decreased the elevated serum hormones levels and increased the reduced hippocampal GR expression. Based on these data, we conclude that the effects of GLYX-13 exerted on the TDS-induced serum corticosterone and ACTH levels may be an important mechanism underlying its anxiolytic effects.

As demonstrated by many studies, hyperactivity of the HPA axis, which is commonly seen in patients with PTSD, is reversed during clinically effective therapy with antidepressant drugs (Kohda et al., 2007). Among a multitude of molecular events, altering stress hormone levels and hippocampal GR density are two important mechanisms by which anxiolytic drugs may exert their clinical effects (Fenchel et al., 2015; Santa Ana et al., 2006). The HPA axis, as the name implies, consists of a feedback loop composed of the hypothalamus and pituitary and adrenal glands. Briefly, the hypothalamus releases corticotrophin releasing hormone and arginine vasopressin in response to a stressor, which in turn activate the secretion of ACTH from the pituitary, which finally stimulates the secretion of cortisol (in humans) or corticosterone (in rodents) from the adrenal cortex (Mehta and Binder, 2012; Zaba et al., 2015). As observed in our study, TDS exposure significantly increased serum corticosterone and ACTH levels in rats, which was accompanied by anxiety-like behavioral alterations. However, GLYX-13 (5 or 10 mg/kg) administration significantly decreased the elevated serum corticosterone and ACTH levels, which may be a primary neuroendocrine mechanism underlying its behavioral effects. The hippocampus, known to contain a high density of GRs, is a key structure in the inhibition of stressed-induced HPA axis activation and is also sensitive to glucocorticoid-mediated impairment (Marshall et al., 2002; Zaba et al., 2015). Indeed, it has been suggested that prolonged overproduction of glucocorticoids, whether as a result of ongoing stress or a genetic predisposition to HPA axis hyperactivity, especially in the hippocampus, which is essential for HPA axis restraint. These desensitized GRs reduce GR-mediated negative feedback by endogenous glucocorticoids, which, on the other hand, promote the sustaining hypersecretion of glucocorticoids (this process is known as the glucocorticoid cascade hypothesis). In animals, TDS exposure paradigms have been shown to down regulate hippocampal GR number. In the present study, we found that the TDS regimen significantly reduced hippocampal GR expression, which was likely impaired by the over secretion of corticosterone. Chronic GLYX-13 treatment reversed this change, and this maybe the neuroendocrine mechanism underlying its behavioral effects. In mammals, the HPA axis and the serotonin (5-HT) system closely interact in the CNS (particularly in the hippocampus) and are greatly involved in stress-related disorders (Seckl and Meaney, 2006; Van Zuiden et al., 2015). Given that 5-HT in the hippocampus is highly sensitive to glucocorticoids, it is not surprising that chronic hypercortisolism could cause dysfunction of the 5-HT system, especially in the hippocampus (Gotovac et al., 2003; Takeda et al., 2000) . In our previous work, we found that chronic GLYX-13 administration significantly increased the levels of hippocampal 5-HT and its metabolite 5-hydroxyindoleacetic acid in chronically stressed rats. Therefore, one may speculate that the GLYX-13-induced amelioration of the monoaminergic system may be associated with its effect on HPA axis. In addition, many studies have shown that hyperactivity of the HPA axis or sustaining hypersecretion of glucocorticoids may result in hippocampal atrophy in PTSD, including dendritic shrinkage, impaired neurogenesis, and deficient neurotrophic factors (De Quervain and Margraf, 2008; Lehrner et al., 2014; Zoladz et al., 2012). Studies in Wang’s laboratory have shown that chronic GLYX-13 treatment increases hippocampal neurogenesis and hippocampal BDNF and pCREB expression in chronically stressed rats (Lu et al., 2015). Accordingly, it seems that the beneficial effects that GLYX-13 produced on the hippocampus may, at least partly, arise from its regulating the hyperactivity of HPA axis. Taken together, the aforementioned mechanisms through which the effects of GLYX-13 may be mediated are not contradictory, but connect with and support one another. Thus, GLYX-13 appears to play a significant role in modulating HPA axis function, which may be an important and essential mechanism underlying its anxiolytic effects.

In summary, our findings indicate that GLYX-13 demonstrated a therapeutic effect on PTSD-like stress responding that is accompanied by (or associated with) modulation of the HPA axis. Future studies are needed to clarify which receptor system is responsible for the anti-anxiety effects of GLYX-13 in animal models relevant to PTSD. The results of these investigations have the implications for the neural theory of PTSD-like stress behavior which may have clinical implications for the treatment of PTSD.

Footnotes

Acknowledgements

The authors thank Dr. Xue Ming for providing the reference data.

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Beijing Natural Science Foundation (grant number 7152019), The foundation of Beijingshi liuxue renyuan zeyou zizhu, and The foundation of Beijingshi youxiu rencai gugan xiangmu.

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