Abstract
Stress contributes to major depressive disorder (MDD) and chronic pain, which affect a significant portion of the global population, but researchers have not clearly determined how these conditions are initiated or amplified by stress. The chronic social defeat stress (CSDS) model is a mouse model of psychosocial stress that exhibits depressive-like behavior and chronic pain. We hypothesized that metabotropic glutamate receptor 5 (mGluR5) expressed in the nucleus accumbens (NAc) normalizes the depressive-like behaviors and pain following CSDS. Here, we show that CSDS induced both pain and social avoidance and that the level of mGluR5 decreased in susceptible mice. Overexpression of mGluR5 in the NAc shell and core prevented the development of depressive-like behaviors and pain in susceptible mice, respectively. Conversely, depression-like behaviors and pain were exacerbated in mice with mGluR5 knockdown in the NAc shell and core, respectively, compared to control mice subjected to 3 days of social defeat stress. Furthermore, (RS)-2-chloro-5-hydroxyphenylglycine (CHPG), an mGluR5 agonist, reversed the reduction in the level of the endocannabinoid (eCB) 2-arachidonoylglycerol (2-AG) in the NAc of susceptible mice, an effect that was blocked by 3-((2-methyl-1, 3-thiazol-4-yl) ethynyl) pyridine hydrochloride (MTEP), an mGluR5 antagonist. In addition, the injection of CHPG into the NAc shell and core normalized depressive-like behaviors and pain, respectively, and these effects were inhibited by AM251, a cannabinoid type 1 receptor (CB1R) antagonist. Based on these results, mGluR5-mediated eCB production in the NAc relieves stress-induced depressive-like behaviors and pain.
Graphical abstract
Similar content being viewed by others
Avoid common mistakes on your manuscript.
Introduction
Major depressive disorder (MDD) and related pain disorders affect a significant portion of the global population. These conditions lead to reduced productivity, personal suffering and significant healthcare costs [1]. Chronic pain is usually accompanied by increased anxiety and depression that influence quality of life [2]. Conversely, patients with depressive disorder are vulnerable to pain [3]. Studies exploring the relationships between MDD and chronic pain are helpful for developing effective clinical interventions for these syndromes.
The chronic social defeat stress (CSDS) procedure, which is used to establish an animal model of psychosocial stress, consists of two independent phases: the induction phase and testing phase [4]. This stress-based model of depressive-like behaviors has been widely used to simulate the symptomatology of human depression [5, 6]. In this model, stress evokes behavioral and physiological changes, such as social avoidance, anhedonia, and altered corticosterone levels [7]. Interestingly, some mice subjected to the CSDS model are resilient and do not exhibit the depressive-like behavior of social avoidance [5]. Similarly, not every individual exposed to chronic stress develops psychopathological conditions [8]. In addition, the CSDS model was recently reported to induce hyperalgesia, which is not associated with the depressive-like behavior of social avoidance [9]. Based on these results, stress is an important factor triggering both depressive-like behaviors and related chronic pain.
The clinical manifestations of comorbid depression and pain indicate that common or interacting neural circuits, neuroanatomical structures, and neurotransmitter systems underlie the persistence of negative mood and physical pain [10, 11]. Previous studies involving human subjects and animal models have revealed the critical role of the nucleus accumbens (NAc) in concurrent pain and depression [12, 13]. Metabotropic glutamate receptor (mGluR)-mediated neuronal changes are of particular interest. mGluR5, a G protein-coupled receptor, is important for modulating plastic changes in neural circuits [14, 15]. mGluR5 is also well known to be involved in various neurological disorders, including chronic pain and mood disorders such as depression and drug addiction [16,17,18]. mGluR5 expressed in the NAc is critical for promoting resilience to chronic stress [19]. According to more recent reports, however, mGluR5 is expressed in the medial prefrontal cortex (mPFC), and its expression levels in the mPFC are changed in response to chronic pain and depressive disorders [20]. For instance, prominent upregulation of mGluR5 was detected in the mPFC of animals with chronic neuropathic pain [21]. Therefore, the elucidation of the contribution of mGluR5 to MDD and related pain will help us understand the underlying mechanisms.
Endogenous cannabinoids (eCBs) are lipid mediators with essential modulatory functions in the brain [22] that play important roles in a wide variety of physiological processes, including affective and nociceptive responses [23]. The two major eCBs, anandamide (AEA) and 2-arachidonoyl glycerol (2-AG), are produced in the postsynapse and signal in a retrograde direction to modulate synaptic strength via the activation of presynaptic cannabinoid type 1 receptor (CB1R) [24]. Activation of CB1R leads to acute depression of synaptic transmission that induces endocannabinoid-mediated long-term depression (LTD), which was originally discovered in the NAc [25], in combination with enhanced eCB signaling. The restoration of eCB signaling in the NAc protects against CSDS-induced anxiety-like behaviors by enhancing 2-AG signaling. The production of eCB is associated with the activity of mGluR5 [26,27,28]. The phosphorylation of mGluR5 at tyrosine residues is critical for maximal signaling [29], illustrating that a reduction in phosphorylation might subsequently alter eCB production and signaling.
Based on these findings, we designed a series of experiments to ask whether mGluR5 in the NAc plays an important role in stress-induced pain threshold alterations and depressive-like behaviors through eCB signaling. The elucidation of the mechanism that mediates depression and nociceptive symptoms in the context of psychological stress may lead to the development of novel therapeutic strategies for the management of chronic pain states and depressive-like behaviors in patients.
Materials and Methods
Animals
All experiments and procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (Eighth Edition) published by the National Research Council (USA) and were approved by the Institutional Animal Care and Use Committee of Sixth People’s Hospital Affiliated with Shanghai Jiao Tong University. Specific pathogen-free (SPF) male C57BL/6J mice (12–20 weeks old; Fudan University Medical Animal Center, Shanghai, China) were used. The mice were housed in a temperature- and humidity-controlled environment (lights on 07:00–19:00) and provided a standard rodent diet and water ad libitum.
Social Defeat Stress and Social Interaction
The social defeat stress procedure was conducted as described previously [30]. Briefly, each C57BL/6J male mouse was subjected to an unfamiliar aggressive resident CD1 male mouse for 5 min for physical defeat. After defeat, both the intruder mouse and the resident CD1 mouse were housed together, but separated by a perforated plastic divider that allows visual, olfactory, and auditory contact, but not physical interaction, for the remainder of the 24-h period. The same test mouse was subjected to social defeat from a different aggressive CD1 resident mouse each day for 3 consecutive days or 10 consecutive days (10-day CSDS procedure). Nondefeated control mice were housed opposite another C57BL/6 mouse instead of a CD1 mouse. Twenty-four hours after the last social defeat session, the social interaction test (defeated mice and nondefeated control mice) was conducted in an open-field arena (43.5 cm × 43.5 cm) equipped with a wire mesh cage (10 × 6.5 cm). Social avoidance behavior was recorded with a video tracking system and measured according to a two-stage interaction test. In the first “no target” stage, an empty wire mesh cage was placed in the interaction zone, and the behavior of the C57BL/6J mice was recorded for 10 min. For the second “target” stage, a novel CD1 aggressor was placed in the wire mesh cage. Both the test mouse and the target CD1 mouse maintained visual, olfactory, and auditory contacts but made no direct physical contact. The time spent by the mouse in the “interaction zone” (26.0 × 14.5 cm) and “corner zone” (43.5 × 8.0 cm) of the arena was recorded with video-tracking software (SMART 2.5, Panlab). The social interaction test was performed starting 24 h after the final day of the CSDS procedure. The social interaction ratio was defined as [time spent in the interaction zone / (time spent in the interaction zone + time in the corner zone) × 100 (%)] [31]. According to the social interaction ratio, defeated mice were divided into susceptible and resilient subpopulations. If the social interaction ratio was greater than the lower endpoint of the 95% confidence interval of that of control mice, a defeated mouse was considered resilient.
Tail Suspension Test (TST)
Mice were individually suspended by the tail from a metal hanger with adhesive tape in the tail suspension box approximately 20 cm above the ground. The immobility time during the last 4 min of the 6-min TST session was recorded for each mouse. Immobility was defined as only minor limb movements or no movement at all.
von Frey Filament Test
The nociceptive mechanical threshold of the right hind paw was measured with an electronic von Frey device (Bioseb, France). Briefly, each mouse was individually placed in a cage with a mesh floor and adapted to the environment before testing. After a 30-min habituation period, a series of von Frey filaments with increasing pressure (0.04, 0.07, 0.16, 0.4, 0.6, 1.0, 1.4, and 2.0 g) was applied to the plantar surface of the right hind paw until the mouse withdrew its paw. The filaments, starting with the 0.04-g filament, were perpendicularly applied from underneath the mesh floor to stimulate the plantar surface of the paw for 5–6 s in each trial. We recorded the force applied at the time of withdrawal, and responses are reported in grams. The cutoff threshold was set to 2 g. Stimulation was conducted via the up-down method, and the 50% withdrawal threshold was determined from the results. The threshold value was determined by averaging three consecutive trials at 5-min intervals.
Mass Spectrometry Detection of Anandamide (AEA) and 2-Arachidonoylglycerol (2-AG)
The NAc region was dissected from the brain. Tissue samples were weighed and placed in borosilicate glass culture tubes containing 2 ml of acetonitrile with 5 pmol of [d8] AEA and 5 nmol of [d8] 2-AG for extraction. The tissues were homogenized with a glass rod and sonicated for 30 min in an ice-cold water bath. The supernatants were transferred to new glass tubes and evaporated to dryness under N2 gas. The samples were resuspended in 300 μl of acetonitrile to recapture any lipids that had adhered to the glass tube and dried again under N2 gas. Finally, lipid extracts were suspended in 20 μl of acetonitrile and stored at – 80 °C until analysis. Tissue AEA and 2-AG levels were determined using liquid chromatography-mass spectrometry (LC-MS/MS) as described previously [32].
Virus Preparation
A chemically synthesized mGrm5 transcript, NM_001081414, was amplified by PCR using the following primer sets: mGrm5-F: 5’-AGGTCGACTCTAGAGGATCCCGCCACCATGGTCCTTCTGTTGATTCTGTCAGTC-3’ and mGrm5-R: 5’-TCCTTGTAGTCCATACCCAACGATGAAGAACTCTGCGTGTAATC-3’. The amplified fragment was inserted into the BamH I and Age I restriction enzyme sites of the GV314 plasmid (CMV-MCS-3FLAG-SV40-EGFP) (Genechem, Shanghai).
A chemically synthesized miR30-based shRNA designed to knock down mGluR5 expression was amplified by PCR using the following primer sets: miR-30 (mGlu5)-F: 5’-ACGAGCTGTACAAGGCTAGCTAAGCCTTGTTAAGTGCTCGCTTCG-3’ and miR-30 (mGlu5)-R: 5’-GTTGATTATCGATAACCGGTCGCGTCGCCGCGTGTTTAAACGC-3’. The amplified fragment was inserted into the Nhe I and Age I restriction enzyme sites of the GV412 plasmid (AAV9-CMV-EGFP-MCS) (Genechem, Shanghai). All viral vectors were stored in aliquots at – 80 °C.
Stereotaxic Injection and Cannula Implantation
Mice were anesthetized with pentobarbital sodium (45 mg·kg−1, i.p.) and then fixed in a stereotaxic frame [33]. Two hundred nanoliters of the virus were injected bilaterally into the NAc shell (from the bregma: + 1.6 mm anteroposterior; ± 0.5 mm lateral; − 4.1 mm dorsoventral) or into the NAc core (from the bregma: + 1.3 mm anteroposterior; ± 1.0 mm lateral; − 4.1 mm dorsoventral) at a slow rate (20 nl·min−1) using a syringe pump. The needle was withdrawn 10 min after the end of infusion. The location of the injection sites was confirmed in each mouse by observing coronal sections (50 μm) containing the NAc, and mice in which the virus was injected into the incorrect site were excluded from the experiments.
For microinjection of the drugs into the NAc shell and core, a guide cannula (26 gauge, 5-mm long; Plastics One) was chronically implanted + 1.6 mm anteroposterior, ± 0.5 mm lateral, and − 3.1 mm dorsoventral from the bregma and + 1.3 mm anteroposterior, ± 1.0 mm lateral, and – 3.1 mm dorsoventral from the bregma and secured with dental acrylic cement. Obturators were placed in the guide cannulas after implantation.
Intracranial Microinjection of Drugs
An internal cannula (33 gauge; Plastics One) connected to a 1-μl syringe (Hamilton) via polyethylene (PE)-20 tubing was inserted into the guide.
3-((2-Methyl-1,3-thiazol-4-yl)ethynyl) pyridine hydrochloride (MTEP hydrochloride, Tocris Bioscience) (10 μg per side) was unilaterally microinjected into the NAc of susceptible mice immediately before CHPG (10 μg per side) treatment. AM251, vehicle control or (RS)-2-chloro-5-hydroxyphenylglycine (CHPG, Tocris Bioscience) was unilaterally microinjected into the NAc core or shell of susceptible mice in a volume of 0.5 μl per side over 30 s. After 1 min, the internal cannula was withdrawn and the obturator was replaced. The mice received a microinjection of AM251 (MedChem Express, 0.8 μg per side), vehicle control or CHPG (10 μg per side) once a day for 3 consecutive days. The dose and treatment time of drug administration, alone or in combination, were chosen based on previous studies [19, 34].
Immunohistochemistry
Mice were transcardially perfused with 4% paraformaldehyde (wt/vol) in 0.1 M PBS (pH 7.4). The brains of the mice were removed, fixed overnight with the paraformaldehyde solution, and then stored in 30% sucrose in PBS. All brains were then frozen in O.C.T. (Sakura Finetek, Inc.) and cut into 30-μm coronal sections using a cryostat (Leica Biosystems). For mGluR5 immunofluorescence staining, the sections were blocked with PBS containing 3% normal donkey serum and 0.3% Triton X-100 for 1 h at room temperature. Thereafter, the sections were incubated with an anti-mGluR5 antibody (1:200; 2237-1, Epitomics) in PBS containing 0.5% Triton X-100 (vol/vol) at 18–20 °C overnight. Sections were then washed with PBS three times and incubated with a secondary antibody conjugated to Alexa Fluor in 0.5% Triton X-100 in PBS for 2 h at room temperature. Images were captured using a Leica TCS SP5II confocal microscope with LAS AF Lite software (Leica Microsystems).
Immunoblotting
The brains were placed in a coronal brain matrix (1-mm slice interval; ASI-instrument). Single-edged blades were inserted into the appropriate slits. Coronal slices with a 1–2-mm thickness were placed in a dish containing ice-cold PBS for NAc dissection. The NAc was dissected rapidly and immediately stored at – 80 °C. Individual tissues were thawed on ice as needed and placed in RIPA buffer (Beyotime Institute of Biotechnology, Haimen, China) containing a protease inhibitor (Calbiochem, Schwalbach, Germany). The tissue lysates were centrifuged at 12000×g for 15 min at 4 °C, and the supernatant was collected to measure the protein concentration using a BCA kit (Beyotime Institute of Biotechnology, Haimen, China). The samples were then resolved on SDS-polyacrylamide gels and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% nonfat dry milk plus 0.05% Tween 20 (vol/vol) in PBS for 1 h at room temperature and then incubated with the appropriate primary antibodies against mGluR5 (1:1000; ab76316, Abcam) and β-actin (1:2000; catalog no. 4970, Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C. The membranes were washed three times with 1× TBST and then incubated with an HRP-conjugated secondary antibody (1:2000; catalog no. 7074, Cell Signaling Technology, Danvers, MA, USA) for 1 h. The bands were detected using ImageQuant LAS 4000 mini (GE Healthcare Life Sciences, USA) with an enhanced chemiluminescence (ECL) detection kit (Millipore, USA), and the band densities were quantified using ImageJ software (version 4.0.0, USA).
Statistical Analysis
All data are presented as the means ± SEM. The statistical analysis was performed with GraphPad Prism 7.00 software (GraphPad Software Inc., San Diego, CA, USA). Differences between the means were analyzed by one-way analysis of variance (ANOVA). One-way ANOVA followed by Bonferroni’s post hoc test was performed to compare multiple independent groups. Unpaired Student’s t tests were used to compare data from two groups. P < 0.05 was considered statistically significant.
Results
Stress Induces Depressive-Like Behaviors and Pain
Male mouse were exposed to an aggressive retired breeder CD1 mouse as a social defeat stressor for 10 days to explore the effect of stress on depression and pain. Twenty-four hours after the last defeat stress exposure, the social interaction behaviors of mice were tested in an open-field arena with an interaction zone (Fig. 1A). After the last defeat episode, the mice were separated into two groups based on social interaction ratios: the susceptible group and resilient group (Fig. 1B). The time spent in the interaction zone in the absence of a target mouse was similar among the different groups (Fig. 1C). The time spent in the interaction zone in the presence of a target mouse was shorter for the susceptible group than for the control and resilient groups (Fig. 1D). Based on this result, susceptible mice exhibited a strong social avoidance behavior. We also detected the depressive-like phenotypes of mice using the TST. The immobility time in the TST was longer for susceptible mice than for control and resilient mice (Fig. 1E). The electronic von Frey test was used to evaluate mechanical nociception and the effect of stress on pain. The mechanical thresholds were decreased in susceptible and resilient mice compared with control mice. However, the mechanical thresholds were not different between susceptible and resilient mice (Fig. 1F). Thus, mice that underwent CSDS (resilient and susceptible) exhibit a higher sensitivity to pain in the mechanical nociceptive test.
Stress Induces Changes in mGluR5 Expression in the NAc
A Western blot assay was performed to measure the level of mGluR5 in the NAc of mice and to determine whether the mGluR5 level in the NAc changed after 10 days of CSDS. Lower expression of the mGluR5 protein was observed in the NAc of susceptible mice than in the NAc of control and resilient mice (Fig. 2A and B).
Overexpression of mGluR5 in the NAc Shell or Core Alleviates Stress-Induced Depression-Like Behaviors and Pain, Respectively
We showed that mGluR5 expression was decreased in the NAc of susceptible mice after CSDS, consistent with a previous study [19]. Moreover, this study revealed that virus-induced mGluR5 expression in the NAc shell of mGluR5−/− mice plays an important role in protecting against stress-induced depression-like behaviors [19]. We therefore tested whether viral rescue of mGluR5 expression in the NAc shell is critical for preventing stress-induced depression-like and pain behaviors in susceptible mice (Fig. 3A). An in vitro experiment showed an increased level of mGluR5 in cells transfected with mGluR5-AV compared with cells transfected with control-AV (Fig. 3B). After the CSDS procedure, an adenovirus expressing mGluR5 was stereotaxically injected into the NAc shell of susceptible mice, and these adenoviruses were expressed effectively in vivo (Fig. 3C). Western blot results showed an increased expression level of mGluR5 in the NAc shell of susceptible mice 5 days after the injection (Fig. 3D). Thus, all subsequent behavioral procedures were performed 5 days after the injection. The time spent in the interaction zone in the absence of a target mouse was not different between the two groups (Fig. 3E). Moreover, compared with control-AV-injected susceptible mice, upregulation of mGluR5 expression in the NAc shell of susceptible mice significantly increased the interaction time in the presence of a target mouse (Fig. 3F). Compared with control-AAV-injected susceptible mice, rescue of mGluR5 expression in the NAc shell of susceptible mice decreased the immobility time in the TST (Fig. 3G). However, compared with control-AV-injected susceptible mice, rescue of mGluR5 in the NAc shell of susceptible mice did not change the mechanical thresholds (Fig. 3H). These results show that mGluR5 expression in the NAc shell in susceptible mice contributes to resilience to depression-like behaviors and does not influence pain sensitivity.
Furthermore, we determined whether viral rescue of mGluR5 expression in the NAc core plays a critical role in preventing stress-induced depression-like and pain behaviors in susceptible mice (Fig. 4A). After the CSDS procedure, an adenovirus expressing mGluR5 was stereotaxically injected the NAc core of susceptible mice, and the adenoviruses were expressed effectively (Fig. 4B). All behavioral procedures were performed 5 days after the injection. Compared with control-AV-injected susceptible mice, upregulation of mGluR5 expression in the NAc core of susceptible mice did not change the interaction time (Fig. 4 C and D). Similarly, compared with control-AV-injected susceptible mice, rescue of mGluR5 expression in the NAc core of susceptible mice did not alter the immobility time in the TST (Fig. 4E). However, compared with control-AV-injected susceptible mice, rescue of mGluR5 expression in the NAc core of susceptible mice increased the mechanical thresholds (Fig. 4F). Based on these data, mGluR5 expression in the NAc core of susceptible mice reduces pain sensitivity but does not change depression-like behaviors.
Conditional Deletion of mGluR5 in the NAc Shell or Core Induces Depressive-Like Behaviors and Increased Pain Sensitivity, Respectively
We next evaluated the level of mGluR5 in brain regions that may be responsible for the depressive-like and pain phenotypes of mice exposed to social defeat stress. We designed a series of experiments (Fig. 5A) and stereotaxically injected an AAV containing miR30-mGluR5-shRNA or control-shRNA into the bilateral NAc shell of naïve mice. These adenoviruses were expressed effectively in vivo (Fig. 5B). Western blot results showed decreased expression levels of mGluR5 in the NAc shell of mice 2 weeks after the injection (Fig. 5C). Thus, all subsequent procedures were performed 2 weeks after the injection. We subjected animals to 3 d of social defeat stress 2 weeks after the injection. The time spent in the interaction zone in the absence of a target mouse was not different between the mGluR5-shRNA and control-shRNA groups (Fig. 5D). Notably, mGluR5-shRNA mice spent less time in the interaction zone in the presence of a target mouse and exhibited a longer immobility time in the TST than control-shRNA mice (Fig. 5E–F). However, the mechanical thresholds were similar between the mGluR5-shRNA-treated mice and control-shRNA-treated mice (Fig. 5G). Therefore, conditional knockdown of mGluR5 in the NAc shell of mice increases both stress-induced social avoidance and depression-like behaviors but does not change pain sensitivity.
Furthermore, we wanted to determine whether conditional knockdown of mGluR5 expression in the NAc core contributes to stress-induced depressive-like behaviors and pain (Fig. 6A). We stereotaxically injected an AAV containing miR30-mGluR5-shRNA or control-shRNA into the bilateral NAc core of naïve mice (Fig. 6B). The animals were subjected to 3 days of social defeat stress 2 weeks after the injection. The time spent in the interaction zone in the absence of a target mouse was not different between the mGluR5-shRNA and control-shRNA groups (Fig. 6C). The time spent in the interaction zone in the presence of a target mouse and immobility time in the TST were similar between the mGluR5-shRNA-treated mice and control-shRNA-treated mice (Fig. 6D–E). However, the mechanical thresholds increased in mGluR5-shRNA-treated mice compared with control-shRNA-treated mice (Fig. 6F). These results indicated that conditional knockdown of mGluR5 in the NAc core of mice increases stress-induced pain sensitivity but does not change either stress-induced social avoidance.
mGluR5-Mediated Production of eCB Within the NAc Is Impaired in Susceptible Mice
The eCB system is composed of CB1R and cannabinoid receptor type 2 (CB2R) [35,36,37]. Two major endogenous ligands, AEA and 2-AG, bind to CB1R [35, 38]. According to previous reports, sustained disruption of the levels of the eCB 2-AG within the amygdala following CSDS results in pathological states of anxiety, while an increase in 2-AG levels in the NAc increases behavioral resiliency to chronic stress in mice [32, 34]. Together, these studies indicate that eCB signaling is an important regulator of depression and anxiety. Thus, we postulated that the susceptible mice might exhibit impaired eCB induction. We measured eCB levels in the NAc of susceptible mice to test this hypothesis. The level of 2-AG in the NAc was decreased in susceptible mice compared with control mice (Fig. 7A). Furthermore, activation of mGluR5 is known to increase the induction of eCB signaling [26]. We therefore determined whether pharmacological activation of mGluR5 reversed the induction of eCB signaling within the NAc of susceptible mice. We injected either vehicle or CHPG in the presence or absence of MPEP into the NAc of susceptible mice once per day for 3 days and quantified eCB levels 24 h after the final injection. As expected, CHPG restored 2-AG induction in susceptible mice, but the effect was blocked by MPEP, confirming that 2-AG induction is mediated by mGluR5 in the NAc after CSDS (Fig. 7A). However, the level of AEA in susceptible mice was similar among different groups (Fig. 7B).
Enhancement of eCB Signaling in the NAc Shell or Core by Targeted Pharmacological Activation of mGluR5 in the NAc Shell or Core Alleviates Depressive-Like Behaviors and Relieves Pain in Mice Exposed to CSDS, Respectively
We next asked whether pharmacological modulation of mGluR5 within the NAc shell counteracted stress-induced depressive-like behaviors and pain though mGluR5-mediated eCB signaling (Fig. 8A). We injected either the vehicle or CHPG in the presence or absence of AM251 into the NAc shell of susceptible mice once per day for 3 days (Fig. 8B). Mice in the different groups spent a similar amount of time spent in the interaction zone in the absence of a target mouse (Fig. 8C). Susceptible mice that received an injection of CHPG into the NAc shell spent more time in the interaction zone in the presence of a target mouse and exhibited a decreased immobility time in the TST compared with susceptible mice in the vehicle group. Moreover, the effect of CHPG on stress-induced depressive-like behaviors in susceptible mice was blocked by an injection of AM251 into the NAc shell (Fig. 8D-E). However, the mechanical thresholds were similar between susceptible mice from different groups (Fig. 8F).
Furthermore, we explored whether pharmacological modulation of mGluR5 within the NAc core would counteract stress-induced depressive-like behaviors and pain though mGluR5-mediated eCB signaling (Fig. 9A). We injected either vehicle or CHPG in the presence or absence of AM251 into the NAc core of susceptible mice once per day for 3 days (Fig. 9B). Mice in the different groups spent similar amounts of time in the interaction zone in the absence of a target mouse (Fig. 9C). The time spent in the interaction zone in the presence of a target mouse and immobility time in the TST were not different between the CHPG group and vehicle group after CHPG was injected into the NAc core of susceptible mice (Fig. 9D–E). However, the mechanical thresholds increased in susceptible mice treated with CHPG compared to susceptible mice treated with the vehicle (Fig. 9F). Moreover, the inhibitory effect of the CHPG injection into the NAc core of susceptible mice on stress-induced pain behaviors was blocked by the injection of AM251 (Fig. 9F). These results suggest that social avoidance behavior and pain sensitivity are altered after exposure to CSDS. Collectively, these data indicate that mGluR5 signaling in the NAc regulates stress-induced depressive-like and pain behaviors through an eCB-dependent mechanism.
Discussion
CSDS induced a decrease in mGluR5 protein levels in the NAc in susceptible mice, which exhibited depressive-like and pain behaviors that were reversed by overexpression of mGluR5 in the shell and core of the NAc, respectively. Mice with a conditional deletion of mGluR5 in the shell and core of the NAc that were exposed to 3 days of social defeat stress were susceptible to depressive-like and pain behaviors, respectively. Furthermore, CSDS induced a decrease in eCB levels in the NAc of susceptible mice. This decrease was reversed by an injection of CHPG, and the effect of CHPG was blocked by MTEP. In addition, an injection of CHPG into the shell and core of the NAc ameliorated stress-induced depressive-like behaviors and pain, respectively. The effect of CHPG on CSDS-induced depressive-like behaviors and pain was blocked by AM251. These data support the hypothesis that mGluR5 mitigates stress-induced depressive-like behaviors and pain through eCB signaling in the NAc.
Chronic pain is well known to induce depressive-like behaviors. Depression also affects the prognosis and treatment of pain [39]. According to a previous study, a longer duration of depression can lead to greater severity of pain at baseline, and a long-term course of pain significantly increases the risk of developing depression after 2 years [40]. This finding implies the existence of shared underlying supraspinal brain mechanisms that modulate chronic pain and depression or anxiety. Several recent studies have elucidated that stressed mice (resilient and susceptible) exhibit a higher sensitivity to pain than unstressed mice in mechanical and chemical tests [9]. However, mice subjected to 30- and 60-min restraint stress show analgesia in both the hot plate and plantar tests of thermal pain [41]. This difference may be attributed to the intensity, duration, or quality of the stressor. In the present study, although CSDS was shown to lead to pain and depression, these two conditions may present independently, since resilient mice that did not manifest depressive-like behaviors displayed higher sensitivity to pain in the mechanical pain assay after exposure to CSDS.
One possible explanation for this behavioral phenotype is the alteration of mGluR5 expression in the brain. mGluR5 plays an important role in modulating neuronal excitability and is involved in the pathophysiology of various psychiatric and neurological disorders. Changes in mGluR5 expression and the functional impacts of these changes in different brain regions have been documented in studies of negative mood disorders and chronic pain [42,43,44,45,46,47]. However, the antidepressant phenotype in mice with mGluR5 loss-of-function is controversial among studies.
The expression of mGluR5 in the NAc was reported to be significantly downregulated in susceptible mice. The role of mGluR5 in the NAc is critical for the development of stress resilience via the modulation of cellular resilience mechanisms such as ΔFosB expression in response to stress [19]. Conversely, susceptible mice show a significant increase in mGluR5 protein levels in the hippocampus [7], confirming the regional specificity of CSDS-induced changes in mGluR5 expression. Consistent with this study [19], we observed an antidepressant phenotype in mice with mGluR5 gain-in-function in the NAc shell in our study, illustrating that mGluR5 expressed in different brain regions has a distinct role in modulating depressive-like behaviors. The PFC provides top-down control of affective and sensory processes. Animal and human imaging studies have shown that activation of the mPFC inhibits pain behaviors. A key projection target for the PFC is the NAc, which was also reported to be involved in pain regulation [48]. mGluR5 expressed in other brain regions has become the focus of studies exploring the mechanisms by which depression and pain are encoded. For instance, in neuropathic pain models, a PET analysis showed the downregulation of mGluR5 expression in the NAc and insular cortex [49]. The present study also focused on the NAc. In our study, the level of mGluR5 was decreased in the NAc of susceptible mice. Our behavioral data suggest that rescue or activation of mGluR5 expression in the shell of the NAc ameliorates stress-induced depressive-like behaviors and that rescue or activation of mGluR5 expression in the core of the NAc mitigates stress-induced pain. This study revealed the distinct roles of mGluR5 expressed in the core and shell of the NAc in stress-induced depressive-like behaviors and pain. Furthermore, we found that resilient mice exhibit pain, which may be attributed to the recovery of mGluR5 levels in the shell, but not in the core of the NAc.
However, little is known about the mechanism by which mGluR5 mediates antinociceptive and antidepressant effects after CSDS. eCB signaling is an important modulator of synaptic transmission and plasticity in the brain and interacts with mGluR5 through the phospholipase C-diacylglycerol lipase α (DAGLα) pathway, which leads to the formation of 2-AG and CB1-mediated homo- or heterosynaptic inhibition of transmitter release [50,51,52]. Chronic unpredictable stress (CUS) induces depression- and anxiety-like behaviors in rodents [53], and the CUS-induced increase in pain sensitivity is due to an impairment in eCB signaling in the brain [54]. Importantly, URB597, an inhibitor of the AEA-degrading enzyme fatty acid amide hydrolase (FAAH), and JZL184, an inhibitor of the 2-AG-degrading enzyme monoacylglycerol lipase (MAGL), increase eCB levels in the brain and periphery and are both effective at alleviating CUS-induced depressive-like behaviors and thermal hyperalgesia [55]. Based on these results, eCB signaling plays an important role in stress-induced depressive-like behaviors and thermal hyperalgesia. Serum 2-AG levels are decreased in rodents exposed to chronic stress and individuals exposed to traumatic stress [56]. An increase in 2-AG levels in the NAc reverses anxiety-like behaviors and synaptic plasticity following CSDS [34]. In the present study, CHPG reversed the decrease in the level of 2-AG in the NAc of susceptible mice, an effect that was blocked by MPEP. This result confirmed that 2-AG production was mediated by mGluR5. We also demonstrated that an injection of CHPG into the NAc shell of susceptible mice inhibited stress-induced depressive-like behaviors. However, the effect of CHPG on stress-induced depressive-like behaviors was blocked by a reverse agonist of eCBs, AM251. In addition, an injection of CHPG into the NAc core of susceptible mice decreased stress-induced pain sensitivity, and this effect was also blocked by AM251. These results suggest that mGluR5 in the NAc alleviates CSDS-induced depressive-like behaviors and pain through eCB signaling.
The current study has some limitations in terms of understanding social stress induced depressive-like behaviors and pain. First, animal models do not actually mimic patients with chronic pain and depression in clinical settings. Second, the present study explored the relationship between depression and chronic pain using a social stress context. We only investigated whether depression would predispose an animal to pain chronification, but did not assess whether chronic pain would predispose mice to depressive-like behaviors. One important limitation of the study is that these studies are restricted to male animals due to the requirement for aggressive resident-intruder interactions. Chronic social stress is mechanistically different from chronic stresses without social components. Future studies should investigate changes in mGluR5 levels in the NAc in response to additional chronic stress procedures, such as chronic variable stress or newly established repeated social stress models that have enabled inclusion of female mice.
Conclusions
Overall, these data demonstrate that impairment in mGluR5 signaling in the NAc is a synaptic signature for behavioral adaptability following social stress. Restoration of mGluR5 signaling protects against CSDS-induced depressive-like behaviors and pain. However, knockdown of mGluR5 exacerbates CSDS-induced depressive-like behaviors and pain. Finally, activation of mGluR5 in the NAc alleviates CSDS-induced depressive-like behaviors and pain through eCB signaling. Thus, the relationship between mGluR5 and eCB signaling may prove to be relevant to our understanding of the relationship between stress, pain, and emotional behavior.
Data Availability
All the data supporting the findings of this study are available within the article and its Supplementary Information files and from the corresponding author upon reasonable request.
Abbreviations
- MDD:
-
Major depressive disorder
- CSDS:
-
Chronic social defeat stress
- NAc:
-
Nucleus accumbens
- eCB:
-
Endocannabinoid
- 2-AG:
-
2-Arachidonoylglycerol
- CHPG:
-
(RS)-2-chloro-5-hydroxyphenylglycine
- MTEP:
-
3-((2-Methyl-1,3-thiazol-4-yl)ethynyl) pyridine hydrochloride
- CB1R :
-
Cannabinoid type 1 receptor
- mGluR5:
-
Metabotropic glutamate receptor 5
- mPFC:
-
Medial prefrontal cortex
- AEA :
-
Anandamide
- LTD:
-
Long-term depression
- SPT:
-
Sucrose preference test
- TST:
-
Tail suspension test
References
Pagliusi M Jr, Bonet IJM, Brandao AF, Magalhaes SF, Tambeli CH, Parada CA, Sartori CR (2020) Therapeutic and preventive effect of voluntary running wheel Exercise on social defeat stress (SDS)-induced depressive-like behavior and chronic pain in mice. Neuroscience 428:165–177
Aaron RV, Fisher EA, de la Vega R, Lumley MA, Palermo TM (2019) Alexithymia in individuals with chronic pain and its relation to pain intensity, physical interference, depression, and anxiety: a systematic review and meta-analysis. Pain 160(5):994–1006
de Waal MW, Hegeman JM, Gussekloo J, Verhaak PF, van der Mast RC, Comijs HC (2016) The effect of pain on presence and severity of depressive disorders in older persons: the role of perceived control as mediator. J Affect Disord 197:239–244
Kudryavtseva NN, Bakshtanovskaya IV, Koryakina LA (1991) Social model of depression in mice of C57BL/6J strain. Pharmacol Biochem Behav 38(2):315–320
Krishnan V, Han MH, Graham DL, Berton O, Renthal W, Russo SJ, Laplant Q, Graham A et al (2007) Molecular adaptations underlying susceptibility and resistance to social defeat in brain reward regions. Cell 131(2):391–404
Lim BK, Huang KW, Grueter BA, Rothwell PE, Malenka RC (2012) Anhedonia requires MC4R-mediated synaptic adaptations in nucleus accumbens. Nature 487(7406):183–189
Wagner KV, Hartmann J, Labermaier C, Hausl AS, Zhao G, Harbich D, Schmid B, Wang XD et al (2015) Homer1/mGluR5 activity moderates vulnerability to chronic social stress. Neuropsychopharmacology 40(5):1222–1233
Strain JJ (2018) The psychobiology of stress, depression, adjustment disorders and resilience. World J Biol Psychiatry 19(sup1):S14–S20
Pagliusi MOF Jr, Bonet IJM, Dias EV, Vieira AS, Tambeli CH, Parada CA, Sartori CR (2018) Social defeat stress induces hyperalgesia and increases truncated BDNF isoforms in the nucleus accumbens regardless of the depressive-like behavior induction in mice. Eur J Neurosci 48:1635–1646
Robinson MJ, Edwards SE, Iyengar S, Bymaster F, Clark M, Katon W (2009) Depression and pain. Front Biosci (Landmark Ed) 14:5031–5051
Goldenberg DL (2010) Pain/depression dyad: a key to a better understanding and treatment of functional somatic syndromes. Am J Med 123(8):675–682
Massaly N, Copits BA, Wilson-Poe AR, Hipolito L, Markovic T, Yoon HJ, Liu S, Walicki MC et al (2019) Pain-induced negative affect is mediated via recruitment of the nucleus accumbens kappa opioid system. Neuron 102(3):564–573 e6
Heshmati M, Aleyasin H, Menard C, Christoffel DJ, Flanigan ME, Pfau ML, Hodes GE, Lepack AE et al (2018) Cell-type-specific role for nucleus accumbens neuroligin-2 in depression and stress susceptibility. Proc Natl Acad Sci U S A 115(5):1111–1116
Niswender CM, Conn PJ (2010) Metabotropic glutamate receptors: physiology, pharmacology, and disease. Annu Rev Pharmacol Toxicol 50:295–322
Kim CH, Lee J, Lee JY, Roche KW (2008) Metabotropic glutamate receptors: phosphorylation and receptor signaling. J Neurosci Res 86(1):1–10
Guo B, Wang J, Yao H, Ren K, Chen J, Yang J, Cai G, Liu H et al (2018) Chronic inflammatory pain impairs mGluR5-mediated depolarization-induced suppression of excitation in the anterior cingulate cortex. Cereb Cortex 28(6):2118–2130
Smith ACW, Scofield MD, Heinsbroek JA, Gipson CD, Neuhofer D, Roberts-Wolfe DJ, Spencer S, Garcia-Keller C et al (2017) Accumbens nNOS interneurons regulate cocaine relapse. J Neurosci 37(4):742–756
Jiang X, Lin W, Cheng Y, Wang D (2020) mGluR5 facilitates long-term synaptic depression in a stress-induced depressive mouse model. Can J Psychiatr 65(5):347–355
Shin S, Kwon O, Kang JI, Kwon S, Oh S, Choi J, Kim CH, Kim DG (2015) mGluR5 in the nucleus accumbens is critical for promoting resilience to chronic stress. Nat Neurosci 18(7):1017–1024
Chung G, Kim SJ, Kim SK (2018) Metabotropic glutamate receptor 5 in the medial prefrontal cortex as a molecular determinant of pain and ensuing depression. Front Mol Neurosci 11:376
Chung G, Kim CY, Yun YC, Yoon SH, Kim MH, Kim YK, Kim SJ (2017) Upregulation of prefrontal metabotropic glutamate receptor 5 mediates neuropathic pain and negative mood symptoms after spinal nerve injury in rats. Sci Rep 7(1):9743
Bisogno T, Berrendero F, Ambrosino G, Cebeira M, Ramos JA, Fernandez-Ruiz JJ, Di Marzo V (1999) Brain regional distribution of endocannabinoids: implications for their biosynthesis and biological function. Biochem Biophys Res Commun 256(2):377–380
Fitzgibbon M, Kerr DM, Henry RJ, Finn DP, Roche M (2019) Endocannabinoid modulation of inflammatory hyperalgesia in the IFN-alpha mouse model of depression. Brain Behav Immun 82:372–381
Wilson RI, Nicoll RA (2001) Endogenous cannabinoids mediate retrograde signalling at hippocampal synapses. Nature 410(6828):588–592
Robbe D, Kopf M, Remaury A, Bockaert J, Manzoni OJ (2002) Endogenous cannabinoids mediate long-term synaptic depression in the nucleus accumbens. Proc Natl Acad Sci U S A 99(12):8384–8388
Maccarrone M, Rossi S, Bari M, De Chiara V, Fezza F, Musella A, Gasperi V, Prosperetti C et al (2008) Anandamide inhibits metabolism and physiological actions of 2-arachidonoylglycerol in the striatum. Nat Neurosci 11(2):152–159
Maejima T, Oka S, Hashimotodani Y, Ohno-Shosaku T, Aiba A, Wu D, Waku K, Sugiura T et al (2005) Synaptically driven endocannabinoid release requires Ca2+-assisted metabotropic glutamate receptor subtype 1 to phospholipase Cbeta4 signaling cascade in the cerebellum. J Neurosci 25(29):6826–6835
Zhu PJ, Lovinger DM (2005) Retrograde endocannabinoid signaling in a postsynaptic neuron/synaptic bouton preparation from basolateral amygdala. J Neurosci 25(26):6199–6207
Orlando LR, Dunah AW, Standaert DG, Young AB (2002) Tyrosine phosphorylation of the metabotropic glutamate receptor mGluR5 in striatal neurons. Neuropharmacology 43(2):161–173
Golden SA, Covington HE 3rd, Berton O, Russo SJ (2011) A standardized protocol for repeated social defeat stress in mice. Nat Protoc 6(8):1183–1191
Guo M, Lu Y, Garza JC, Li Y, Chua SC, Zhang W, Lu B, Lu XY (2012) Forebrain glutamatergic neurons mediate leptin action on depression-like behaviors and synaptic depression. Transl Psychiatry 2:e83
Qin Z, Zhou X, Pandey NR, Vecchiarelli HA, Stewart CA, Zhang X, Lagace DC, Brunel JM et al (2015) Chronic stress induces anxiety via an amygdalar intracellular cascade that impairs endocannabinoid signaling. Neuron 85(6):1319–1331
Li MX, Zheng HL, Luo Y, He JG, Wang W, Han J, Zhang L, Wang X et al (2018) Gene deficiency and pharmacological inhibition of caspase-1 confers resilience to chronic social defeat stress via regulating the stability of surface AMPARs. Mol Psychiatry 23(3):556–568
Bosch-Bouju C, Larrieu T, Linders L, Manzoni OJ, Laye S (2016) Endocannabinoid-mediated plasticity in nucleus accumbens controls vulnerability to anxiety after social defeat stress. Cell Rep 16(5):1237–1242
Devane WA, Hanus L, Breuer A, Pertwee RG, Stevenson LA, Griffin G, Gibson D, Mandelbaum A et al (1992) Isolation and structure of a brain constituent that binds to the cannabinoid receptor. Science 258(5090):1946–1949
Herkenham M, Lynn AB, Little MD, Johnson MR, Melvin LS, de Costa BR, Rice KC (1990) Cannabinoid receptor localization in brain. Proc Natl Acad Sci U S A 87(5):1932–1936
Matsuda LA, Lolait SJ, Brownstein MJ, Young AC, Bonner TI (1990) Structure of a cannabinoid receptor and functional expression of the cloned cDNA. Nature 346(6284):561–564
Sugiura T, Kondo S, Sukagawa A, Nakane S, Shinoda A, Itoh K, Yamashita A, Waku K (1995) 2-Arachidonoylglycerol: a possible endogenous cannabinoid receptor ligand in brain. Biochem Biophys Res Commun 215(1):89–97
Bradley JJ (1963) Severe localized pain associated with the depressive syndrome. Br J Psychiatry 109:741–745
Gerrits MM, Vogelzangs N, van Oppen P, van Marwijk HW, van der Horst H, Penninx BW (2012) Impact of pain on the course of depressive and anxiety disorders. Pain 153(2):429–436
Atwal N, Winters BL, Vaughan CW (2020) Endogenous cannabinoid modulation of restraint stress-induced analgesia in thermal nociception. J Neurochem 152(1):92–102
Marsden WN (2013) Synaptic plasticity in depression: molecular, cellular and functional correlates. Prog Neuro-Psychopharmacol Biol Psychiatry 43:168–184
Duman RS, Aghajanian GK, Sanacora G, Krystal JH (2016) Synaptic plasticity and depression: new insights from stress and rapid-acting antidepressants. Nat Med 22(3):238–249
Bannerman DM, Sprengel R, Sanderson DJ, McHugh SB, Rawlins JN, Monyer H, Seeburg PH (2014) Hippocampal synaptic plasticity, spatial memory and anxiety. Nat Rev Neurosci 15(3):181–192
Kim SK, Hayashi H, Ishikawa T, Shibata K, Shigetomi E, Shinozaki Y, Inada H, Roh SE et al (2016) Cortical astrocytes rewire somatosensory cortical circuits for peripheral neuropathic pain. J Clin Invest 126(5):1983–1997
Cordeiro Matos S, Zhang Z, Seguela P (2015) Peripheral neuropathy induces HCN channel dysfunction in pyramidal neurons of the medial prefrontal cortex. J Neurosci 35(38):13244–13256
Kolber BJ (2015) mGluRs head to toe in pain. Prog Mol Biol Transl Sci 131:281–324
Martinez E, Lin HH, Zhou H, Dale J, Liu K, Wang J (2017) Corticostriatal regulation of acute pain. Front Cell Neurosci 11:146
de Laat B, Leurquin-Sterk G, Celen S, Bormans G, Koole M, Van Laere K, Casteels C (2015) Preclinical evaluation and quantification of 18F-FPEB as a radioligand for PET imaging of the metabotropic glutamate receptor 5. J Nucl Med 56(12):1954–1959
Guindon J, Hohmann AG (2009) The endocannabinoid system and pain. CNS Neurol Disord Drug Targets 8(6):403–421
Di Marzo V (2011) Endocannabinoid signaling in the brain: biosynthetic mechanisms in the limelight. Nat Neurosci 14(1):9–15
Kiritoshi T, Ji G, Neugebauer V (2016) Rescue of impaired mGluR5-driven endocannabinoid signaling restores prefrontal cortical output to inhibit pain in arthritic rats. J Neurosci 36(3):837–850
Mineur YS, Belzung C, Crusio WE (2006) Effects of unpredictable chronic mild stress on anxiety and depression-like behavior in mice. Behav Brain Res 175(1):43–50
Wang W, Sun D, Pan B, Roberts CJ, Sun X, Hillard CJ, Liu QS (2010) Deficiency in endocannabinoid signaling in the nucleus accumbens induced by chronic unpredictable stress. Neuropsychopharmacology 35(11):2249–2261
Lomazzo E, Bindila L, Remmers F, Lerner R, Schwitter C, Hoheisel U, Lutz B (2015) Therapeutic potential of inhibitors of endocannabinoid degradation for the treatment of stress-related hyperalgesia in an animal model of chronic pain. Neuropsychopharmacology 40(2):488–501
Hill MN, Miller GE, Carrier EJ, Gorzalka BB, Hillard CJ (2009) Circulating endocannabinoids and N-acyl ethanolamines are differentially regulated in major depression and following exposure to social stress. Psychoneuroendocrinology 34(8):1257–1262
Funding
This work was supported by a grant from the Natural Science Foundation of Shanghai to T.X. (21ZR1448400), the Interdisciplinary Program of Shanghai Jiao Tong University to T.X. (grant no. YG2021ZD23), a grant from the Natural Science Foundation of China to C.S. (81901141), and grants-in-aid from Shanghai Municipal Commission of Science and Technology (18DZ2260200).
Author information
Authors and Affiliations
Contributions
X.X., K.W., X.M., W.W., data curation, investigation, and methodology; H.W., M.H., methodology and software; H.S. and T.Y., formal analysis; S.C., visualization and funding acquisition; J.H., A.W. and T.X., writing and supervision.
Corresponding authors
Ethics declarations
Ethics Approval
All experiments and procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (Eighth Edition) published by the National Research Council (USA) and were approved by the Institutional Animal Care and Use Committee of Sixth People’s Hospital Affiliated with Shanghai Jiao Tong University.
Consent to Participate
Not applicable
Consent for Publication
Not applicable
Conflict of Interest
The authors declare no competing interests.
Additional information
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Highlights
• Chronic social defeat stress-induced depressive-like behaviors and pain by decreasing mGluR5 levels in the nucleus accumbens.
• Overexpression or activation of mGluR5 in the nucleus accumbens prevented the development of depressive-like behaviors and pain following stress.
• The enhancement of endocannabinoid signaling in the NAc by targeted pharmacological activation of mGluR5 in the NAc alleviates depressive-like behaviors and relieves pain in mice exposed to stress.
Supplementary Information
ESM 1
(PNG 5663 kb)
Rights and permissions
About this article
Cite this article
Xu, X., Wu, K., Ma, X. et al. mGluR5-Mediated eCB Signaling in the Nucleus Accumbens Controls Vulnerability to Depressive-Like Behaviors and Pain After Chronic Social Defeat Stress. Mol Neurobiol 58, 4944–4958 (2021). https://doi.org/10.1007/s12035-021-02469-9
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12035-021-02469-9