BDNF-TrkB signaling-mediated upregulation of Narp is involved in the antidepressant-like effects of (2R,6R)-hydroxynorketamine in a chronic restraint stress mouse model

Background Preclinical studies have indicated that the ketamine metabolite (2R,6R)-hydroxynorketamine (HNK) is a rapid-acting antidepressant drug with limited dissociation properties and low abuse potential. However, its effects and molecular mechanisms remain unclear. In this work, we examined the involvement of brain-derived neurotrophic factor (BDNF), tropomyosin receptor kinase B (TrkB) and Narp in the antidepressant-like actions of (2R,6R)-HNK in a chronic restraint stress (CRS) mouse model. Methods C57BL/6 male mice were subjected to CRS for 8 h per day for 14 consecutive days. Open field, forced swimming, novelty suppressed feeding, and tail suspension tests were performed after administering (2R,6R)-HNK (10 mg/kg), a combination of (2R,6R)-HNK and NBQX (an alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor antagonist; 10 mg/kg), or a combination of (2R,6R)-HNK and ANA-12 (a TrkB receptor antagonist; 0.5 mg/kg). The mRNA levels of Bdnf and Narp in the hippocampus were determined by quantitative reverse transcription-PCR (qRT–PCR). Western blotting was used to determine the hippocampal protein levels of GluA1, GluA2, BDNF, Narp, PSD95, and synaptophysin, as well as the p-TrkB/TrkB protein ratio. Results (2R,6R)-HNK had rapid antidepressant-like effects in CRS mice. Furthermore, (2R,6R)-HNK significantly ameliorated CRS-induced downregulation of GluA1, GluA2, BDNF, Narp, PSD95, and the p-TrkB/TrkB protein ratio in the hippocampus. The effects of (2R,6R)-HNK were blocked by combinations with NBQX or ANA-12. Conclusion BDNF-TrkB signaling-mediated upregulation of Narp in the hippocampus may play a key role in the antidepressant-like effect of (2R,6R)-HNK in the CRS model of depression. Supplementary Information The online version contains supplementary material available at 10.1186/s12888-022-03838-x.


Introduction
Major depressive disorder (MDD) is a chronic and debilitating mental disorder that affects over 264 million people worldwide and causes serious health and socioeconomic consequences [1]. Current monoaminergic-based pharmacotherapies often take several weeks or months to alleviate clinical symptoms [2]. In addition, treatment resistance and nonresponse rates of up to 30% have made these current treatments less reliable [2]. Laboratory and clinical studies have provided strong evidence for the rapid-acting (within hours) and sustained (lasting up to 7 days) antidepressant-like actions of (R,S)-ketamine (ketamine), an N-methyl-D-aspartate (NMDA) receptor antagonist, in treatment-resistant patients with MDD [3][4][5][6]. Although ketamine is a promising alternative to standard clinically prescribed drugs and is regarded as one of the most significant advances in psychiatry in recent decades, its dissociative properties, changes in sensory perception, and abuse liability [7] have prompted a search for alternative compounds that trigger robust antidepressant-like effects without inducing psychotomimetic side effects.
Recently, one of the ketamine metabolites, (2R,6R)hydroxynorketamine (HNK), has been proposed as an ideal next-generation agent, as it has strikingly rapid and robust antidepressant-like effects without the adverse effects of ketamine [8][9][10][11][12][13]. This interesting metabolite has been reported to function as an antidepressant in animal models by enhancing the expression and function of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in the hippocampus [8,10,13], and it has piqued the interest of researchers to determine its clinical efficacy in depressed patients and to investigate its underlying mechanisms.
The activity-dependent release of brain-derived neurotrophic factor (BDNF) and the activation of downstream tropomyosin receptor kinase B (TrkB) receptors in the hippocampus play critical roles in the antidepressantlike effects of ketamine and its metabolites [8,14,15]. The stimulation of BDNF-TrkB signaling promotes the transcription of many synaptic genes and increases the number and function of synapses [16]. Neuronal activityregulated pentraxin 2 (Narp) is highly expressed in the hippocampus and cortex and is associated with excitatory synaptogenesis and AMPA receptor aggregation [17,18]. There is some evidence that BDNF expression and Narp expression are related [5,19,20]. Mariga et al. demonstrated that BDNF directly regulates Narp, mediating glutamatergic transmission and mossy fiber plasticity in the hippocampus [5].
In this work, we sought to investigate whether the ketamine metabolite (2R,6R)-HNK rescues chronic restraint stress (CRS)-induced depression-like behavior through upregulation of AMPA receptors expression mechanisms. We also investigated the role of BDNF-mediated Narp expression in the antidepressant-like effects of (2R,6R)-HNK.

Animal groups
All experimental procedures were approved by the Ethics Committee of Zhongda Hospital, Medical School, Southeast University. All animal experiments were carried out in strict accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Eighty male 7-week-old C57BL/6J mice were purchased from the Animal Center of Southeast University, Nanjing, China. This initial study was carried out in male mice to control for hormonal variables, as ovarian hormones are necessary for the efficacy of (2R,6R)-HNK in females [21]. Animals were housed in groups of four per cage under controlled illumination (12 h light/dark cycle, lights on 07:00 to 19:00) and temperature (23 ± 1 °C) with free access to food and water. After one week of acclimation, the mice were randomly divided into five groups (n = 16): the control group, the CRS group, the CRS plus HNK (Sigma-Aldrich, St. Louis, MO, USA) group, the CRS plus HNK plus NBQX (an AMPA receptor antagonist; Tocris Bioscience, Bristol, UK) group, and the CRS plus HNK plus ANA-12 (a noncompetitive TrkB receptor antagonist; Maybridge Chemical Company, Tintagel, UK) group. Mice were decapitated after the behavioral tests, and the hippocampus was rapidly dissected, frozen, and stored at -80 °C for further use. The schematic timeline of the experimental procedure is summarized in Fig. 1.

Chronic restraint stress
For restraint stress, 8-week-old mice weighing 22-23 g were individually placed head-first into a well-ventilated 50 ml polypropylene conical tube, and their tails were removed through a 3 cm long tube and a small hole in the cap of the tube. Mice could not move forward or backward in this device. This restraint stress was administered to animals daily for 8 h, from 9 am to 5 pm, for 14 consecutive days [22,23]. The control mice remained undisturbed in their original cages in their home environment. The stressed animals were returned to their home environment following the session of restraint stress.

Open field test (OFT)
Exploration in response to a novel open field was measured 2 h after (2R,6R)-HNK administration. Animals were placed in the center of an arena (50 cm × 50 cm × 40 cm; length × width × height) in a dimly lit room and allowed to move freely for 5 min. A video camera positioned directly above the arena was used to track the movement of each animal with software (XR-XZ301, Shanghai Softmaze Information Technology Co., Ltd., Shanghai, China). The dependent measurements were the total distance traveled, the time spent in the center, and the number of entries into the center.

Forced swimming test (FST)
The test was performed 4 h after (2R,6R)-HNK administration to evaluate depression-like behavior. Mice were placed in a glass container (20 cm diameter × 30 cm height) filled to a depth of 15 cm with water (23-25 °C) and allowed to swim for 6 min. The immobility time during the last 4 min was measured by an observer blinded to animal treatment. The immobility time refers to the time when a mouse floated passively with no additional activity or movements other than those required to maintain balance in the water [25]. After the experiment, the mouse body was wiped dry with absorbent paper, and the mouse was returned to its the original cage. The water was replaced at the end of each test.

Novelty suppressed feeding test
The novelty suppressed feeding test (NSFT) was carried out according to previous studies [26,27]. The testing apparatus was a plastic box (50 cm × 50 cm × 40 cm; length × width × height), the floor of which was covered with approximately 2 cm of wooden bedding. The mice were housed alone in freshly made home cages and food deprived 24 h prior to behavioral testing. At the time of testing, a single food pellet was placed on a white paper platform in the center of the box. An animal was placed in a corner of the box, and the time needed for the mice to consume some food (the feeding latency) was recorded by a trained observer. Immediately afterward, the animal was returned to its home cage, which contained preweighed food pellets, and the amount of food consumed by the mouse during the next 5 min was measured. Each mouse was weighed before food deprivation and before testing to assess the percentage of body weight loss.

Tail suspension test (TST)
Mice were suspended by their tails and secured to a horizontal bar with tape. The immobility time was recorded for 6 min. Mice were considered immobile only when they hung passively and were completely motionless [26]. The behavioral apparatus was thoroughly cleaned with 70% ethanol between animals.

Quantitative mRNA measurements
We analyzed the mRNA levels of Bdnf and Narp in the hippocampus via quantitative reverse transcription-PCR (qRT-PCR) in a StepOnePlus ™ Real-Time PCR System (Applied Biosystems, Foster City, CA), as previously described [28]. We extracted RNA from the samples using an RNeasy Plus Kit (Qiagen, Valencia, CA), reverse-transcribed it with a high-capacity cDNA reverse transcription kit (Bio-Rad Laboratories, Hercules, CA), and then analyzed it with qRT-PCR. TaqMan probes for Bdnf (Mm04230607_s1) and Narp (Mm00479438_m1) were obtained from Applied Biosystems (Carlsbad, CA, USA). Data were normalized to glyceraldehyde-3-phosphate dehydrogenase (Gapdh) mRNA (Mm99999915_ g1). The gene expression was calculated using the ΔΔCT method [29], and data are presented as the relative fold change from control animals.

Western blotting analysis
The hippocampus was homogenized in an RIPA lysis buffer mixed with 1% protease inhibitor cocktail and 1% phenylmethanesulfonyl fluoride. After centrifugation at 13,000 g for 10 min at 4 °C, the supernatant was collected, The protein bands were detected by enhanced chemiluminescence, exposed to X-ray film, and quantitated with ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Statistical analysis
Statistical analyses were conducted on raw data using SigmaPlot 14.0 software (Systat Software Inc., San Jose, CA, USA), which automatically determined whether the dataset met test criteria (Shapiro-Wilk for the normality test and Brown-Forsythe for the equal variance test), and Prism V8.0 software (GraphPad, San Diego, CA, USA). Data are presented as the mean ± SEM. The differences between the groups were compared using one-way analysis of variance (ANOVA) followed by post hoc Holm-Sidak tests. P < 0.05 was considered significant.

Effects of (2R,6R)-HNK on CRS-induced depression-like behaviors
In the OFT, there was a significant difference between subjects from different groups in terms of the time they spent in the center (F (4,75) = 12.016, P < 0.001, Fig. 2B) and the number of entries into the center (F (4,75) = 13.148, P < 0.001, Fig. 2C), but not in terms of the distance traveled during the test (F (4,75) = 0.993, P = 0.417; Fig. 2A). Specifically, mice in the CRS group spent less time in the center and made fewer entries into the center than mice in the control group (time spent in the center: P < 0.001; entries into the center: P < 0.001) and mice in the CRS + HNK group (time spent in the center: P < 0.001; entries into the center: P = 0.001). Compared with mice administered (2R,6R)-HNK only, the time spent in the center and the number of entries into the center were decreased in mice pretreated with NBQX (time spent in the center: P = 0.001; entries into the center: P < 0.001) or cotreated with ANA-12 (time spent in the center: P = 0.002; entries into the center: P = 0.001).
In the FST, there was a significant difference in immobility time between treatment groups (F (4,75) = 12.810, P < 0.001, Fig. 2D). Multiple pairwise comparisons revealed that mice in the CRS group had longer immobility time than mice in the control group (P < 0.001) and mice in the CRS + HNK group (P < 0.001). The immobility times in the CRS + HNK + NBQX group (P = 0.002) and the CRS + HNK+ ANA-12 group (P = 0.011) were longer than that in the CRS + HNK group.
Mice in the control group lost more weight than mice in all other groups during the fast for the NSFT (F (4,75) = 23.082, P < 0.001, Fig. 2E). One-way ANOVA revealed that treatment had a significant effect on feeding latency (F (4,75) = 2.704, P = 0.037, Fig. 2F). However, the pairwise multiple comparison analysis did not show a significant difference between groups. The total food consumption was unaffected across all five groups (F (4,75) = 1.849, P = 0.128, Fig. 2G).
In the TST, there was a significant difference in immobility time between treatment groups (F (4,75) = 4.878, P = 0.001, Fig. 2H). Multiple pairwise comparisons revealed that the mice in the CRS group had significantly longer immobility time than the mice in the control (P = 0.015) and CRS + HNK (P = 0.020) groups. However, pretreatment with NBQX (P = 0.944 when compared to the CRS group) and coadministration with ANA-12 (P = 0.872 when compared to the CRS group) blocked the antidepressant-like action of (2R,6R)-HNK in the TST.

Roles of the mRNA levels of hippocampal Bdnf and Narp in the antidepressant-like activity of (2R,6R)-HNK
In the hippocampal gene transcription measurements, there was a statistically significant difference in the hippocampal mRNA levels of Bdnf between treatment groups (F (4,25) = 16.184, P < 0.001, Fig. 3A). The pairwise multiple comparison analysis revealed that, when compared with the CRS group, (2R,6R)-HNK ameliorated the CRS-induced decrease in Bdnf mRNA levels (P < 0.001), which was abolished by injection with NBQX (P < 0.001) but not ANA-12 (P = 0.876). Similarly, there was a statistically significant difference in the hippocampal Narp mRNA levels of subjects that received different treatments (F (4,25) = 37.831, P < 0.001, Fig. 3B). The pairwise multiple comparison analysis showed that, when compared with the CRS group, (2R,6R)-HNK ameliorated the CRS-induced decrease in Narp transcription (P < 0.001), which was abolished by both pretreatment with NBQX (P < 0.001) and cotreatment with ANA-12 (P < 0.001).

Discussion
The novel finding of this study is that increased AMPA receptors and BDNF expression, activation of downstream TrkB receptors, which resulted in increased Narp expression, are associated with the antidepressant-like effects of (2R,6R)-HNK. The antidepressant-like effects of (2R,6R)-HNK are blocked by the AMPA receptor antagonist NBQX and the TrkB antagonist ANA-12. Overall, these results suggest that the BDNF-TrkB signaling-mediated upregulation of Narp plays a key role in the antidepressant-like effects of (2R,6R)-HNK by influencing synaptic plasticity.
Chronic stress is a risk factor for psychiatric illnesses such as anxiety and depression [30,31]. For this reason, we established a chronic restraint stress animal model of depression, which has been well described in previous studies [22,23]. Kim et al. demonstrated that restraint treatment for 8 h per day for 14 days successfully produced anxiety-and depression-like behaviors, whereas restraint treatment for 2 h per day for 10 days was only marginally effective [22]. In our study, CRS-induced depression-like behaviors were reversed by (2R,6R)-HNK administration, confirming previous observations of the antidepressant-like effects of (2R,6R)-HNK [8,10,12,13,32]. However, (2R,6R)-HNK has also been reported to lack antidepressant effects or exert have poor antidepressant effects in chronic social-defeat stress (CSDS), lipopolysaccharide (LPS), chronic corticosterone, and learned helplessness (LH) models [33][34][35]. The reason for the discrepancy between these findings remains unclear, but it could partially be due to differences in the strain, species, animal models of depression, behavioral test procedures, or drug doses. A clinical trial of (2R,6R)-HNK for therapeutic efficacy in humans is ongoing at the United States National Institute for Mental Health [36]. It is of great interest to explore the antidepressant-like effects of (2R,6R)-HNK in MDD patients.
Currently, the precise mechanisms underlying the effects of (2R,6R)-HNK are still unknown. AMPA receptors play a role in the antidepressant-like activity of ketamine [37]. Ketamine-induced glutamate release activates AMPA receptors by acting on NMDA receptors, resulting in the synthesis and release of BDNF [38], which has been identified as an important mediator of synaptic plasticity [39]. Multiple studies have suggested that BDNF-TrkB signaling is important in the pathophysiology of depression and as a therapeutic target for antidepressants [8,14,15]. In this study, we found a marked increase in the AMPA receptor subunits GluA1 and GluA2 in the hippocampus after (2R,6R)-HNK administration in CRS mice, whereas pretreatment with NBQX significantly blocked the effects of (2R,6R)-HNK, which is consistent with the recent finding that (2R,6R)-HNK administration induces the upregulation of synaptic AMPA receptors [13,29]. The results provide evidence that (2R,6R)-HNK rescues chronic stressinduced depression-like behavior through increased AMPA receptors expression in the hippocampus. Fukumoto et al. found that the antidepressant-like actions of (2R,6R)-HNK were inhibited by knocking in the BDNF Val66Met allele (which blocks the processing and release of BDNF) or by injecting an anti-BDNF antibody into the medial prefrontal cortex (mPFC), demonstrating (2R,6R)-HNK induces long-lasting antidepressant behavioral responses via activity-dependent BDNF release [8]. A recent study also reported that the administration of a neutralizing BDNF antibody or inhibitors of the BDNF signaling pathway in the ventrolateral periaqueductal gray 30 min before the (2R,6R)-HNK treatment blocked the actions of (2R,6R)-HNK. However, the BDNF RNAi attenuated the actions of (2R,6R)-HNK [40]. As shown in the present study, the BDNF levels and the p-TrkB/TrkB ratio in the hippocampi of CRS mice both increased after (2R,6R)-HNK administration. The antidepressant-like effects of (2R,6R)-HNK were blocked by ANA-12, a TrkB receptor antagonist. Taken together, these findings suggest that AMPA receptor-driven BDNF-TrkB signaling plays a contributing role in mediating the antidepressantlike effects of (2R,6R)-HNK.
It has been reported that Narp is a direct transcriptional target of BDNF [5]. Acute BDNF withdrawal results in the downregulation of Narp, whereas BDNF greatly increases Narp transcription [5]. Furthermore, Narp knockout mice exhibit anxiety-and depressionlike behaviors [16,41]. The selective serotonin reuptake inhibitor (SSRI) fluoxetine increased hippocampal Narp mRNA expression in healthy control rats [42].