Introduction

Major depressive disorder, simply known as depression, is a common severe and recurrent disorder worldwide characterized by lowered mood and social activity and cognitive impairment [1,2,3]. WHO projected major depression will rank first cause of burden of disease by 2030 [4]. However, limited drugs exist in clinic due to the uncharacterized molecular mechanisms of major depression. There are many factors causing depression including genetic and environmental factors [5]. A substantial body of researches have shown that stress is the main cause contributes to the development of depression [2]. Many types of stress models such as chronic social defeat stress, chronic restraint stress, and chronic unpredictable stress are applied to detect the underlying mechanism of depression [6]. Chronic social defeat stress has proven to be useful in detecting the mechanism of psychiatric disorders induced by stress including depression, anxiety and post-traumatic stress disorder [7,8,9]. So, in the present study, we utilized chronic social defeat stress depression model to investigate the mechanism of depression.

Dynorphin, an endogenous opioid receptor peptide, and its receptors, the κ opioid receptors are widely expressed in brain regions that are related to emotion regulation [10,11,12]. Accumulating evidences indicate that κ opioid receptors are critically involved in the regulation of behavioral deficits induced by stress including anxiety, aversion, and dysphoria [13,14,15,16,17,18,19]. Previous studies showing that dysregulated serotonergic transmission contributes to major depressive disorder based on the findings that selective serotonin reuptake inhibitors are effective in the treatment of depression in the clinic [20,21,22]. Recent studies showing that κ opioid receptors decrease serotonin transmission through enhancing the trafficking of SERT from intracellular part to the cell membrane [23,24,25]. However, it is unresolved if plastic change of dynorphin/κ opioid receptor system occurs and contributes to the development of depression. The key brain regions and molecular mechanisms that are related to κ opioid receptor’s regulation of depression are remain to be detected.

Although Bruchas et al. have found that κ opioid receptors in the dorsal raphe nucleus were critically involved in stress induced behavioral adaptions in mice by enhancing serotonin transmission [23], it is still unknown which brain regions projecting from dorsal raphe nucleus played a key role in depression following stress. The serotoninergic system originates from dorsal raphe nucleus projects to the amygdala and hippocampus [26, 27], so it is possible that κ opioid receptor distributed in the amygdala, hippocampus take part in the process of depression. Amygdala is an important brain region involved in the regulation of mood including positive emotions such as reward and aversive emotions such as fear, anxiety, and depression [28,29,30,31]. Psychiatric disorders have often been associated with enhanced aversive processing and hyperactive of amygdala signaling [32,33,34,35,36]. Dynorphin/κ opioid receptor system is widely distributed in the amygdala [11], it has been previously demonstrated that stress altered the function of κ opioid receptor in the amygdala [37, 38]. However, whether dynorphin/κ opioid receptor system in amygdala takes part in the process of depression induced by chronic social defeat stress is still unknown.

In the present study, by use of pharmacological, biochemical, and genetic techniques, we investigated the role of κ opioid receptors and the key brain regions in the regulation of depression following chronic stress. We first established a stable depression model of chronic social defeat stress to examine the exact role of dynorphin/κ opioid receptor in depression, the results showed that social avoidance was induced by chronic social defeat stress and could be prevented by systemic κ opioid receptor antagonism and deletion of κ opioid receptor. We further found that amygdala was critically involved in depression following chronic social defeat stress by showing that c-fos and ERK were activated and ERK activation could be blocked by systemic κ opioid receptor antagonism. We finally demonstrated that the expression of dynorphin A increased significantly in the amygdala, antagonism of κ opioid receptor in amygdala blocked depressive-like behavior induced by chronic social defeat stress.

Materials and methods

Materials

nor-Binaltorphimine dihydrochloride (norBNI, ab120078) and anti-dynorphin A antibody (ab82509) were purchased from Abcam (Cambridge, MA, USA). Phosphatase and protease inhibitor (04906837001 and 11836170001) were purchased from Roche (Basel, Switzerland). Anti-actin antibody (A1978) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibody of c-fos (sc-166940), p-ERK (sc-7383), HRP-goat anti-rabbit IgG (sc-2004), and HRP-goat anti-mouse IgG (sc-2005) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). ERK antibody (4695) was purchased from Cell Signaling Technology (Boston, MA, USA). Fluor 488 goat anti-rabbit (IgG) (A-11070) secondary antibody was purchased from Invitrogen (Carlsbad, CA, USA).

κ Opioid receptor antagonist, norBNI, with a very long duration of action in vivo, is detected in mouse brain over 21 days after a single intraperitoneal injection [39]. norBNI was commonly pretreated for 24 h–14 days [40, 41]. Thus, in the present work, norBNI was injected at time points of 24 h before stress and 24 h before behavioral test (immediately after the last social defeat).

Animals

Male C57BL/6 mice weighing 20–25 g (8–10 weeks) were purchased from Shanghai Lingchang Biotechnology Co., Ltd. The κ opioid receptor knockout mice, a gift from Dr. Xin Xie’s lab at Shanghai Institute of Materia Medica, were originally bought from Jackson Laboratory. All mice were housed in a temperature controlled room (24 ± 2 °C) on a 12 h light/12 h dark cycle (lights on at 7:00 a.m.). C57BL/6 mice were housed in groups with 4–5 mice per cage and were maintained under standard laboratory conditions. Immediately after the last social defeat stress, the mice were housed singly through the behavioral test. Mice were allowed free access to food and water in their home cages throughout the experiments.

Male CD-1 mice (the aggressor mice) aged 4–6 months were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. CD-1 mice were housed singly with free access to food and water, and the mice were allowed to habituate to their cages for a minimum of 7 days prior to screening.

Social defeat stress

Social defeat stress was performed according to a previous study with minor modification [7, 42, 43]. Briefly, CD-1 mice was screened with strict standard, that is CD-1 mice must attack the C57BL/6 mouse for at least five consecutive sessions, and the latency to initial aggression must be <60 s through the 3 days screening process. Then, CD-1 mice were housed in cages that were separated into two compartments by perforated plastic separators. For each stress session (10 min per day), defeated mice were placed into the same compartment as the CD-1 aggressor. Following each 10 min defeat session, the defeated mice were housed for 24 h in the compartment adjacent to their respective CD-1 aggressor. It should be ensured that the CD-1 aggressor must be aggressive and was novel to defeated mice each day for 14 consecutive days. Non-stressed (control) mice were handled daily and housed in similar cages, one on each side of the separator. Immediately after the last stress episode, all C57BL/6 mice were single housed, 24 h later, separate groups of experimental mice were tested in social interaction.

Social interaction test

The social interaction open-field arenas (40 cm × 40 cm) are applied to test the social interaction of mice. The interaction zone of the arena contains a rectangular area (14 cm × 24 cm) projecting 8 cm around the enclosure that was applied to display the target CD-1 mice. The corner zones refer to two areas (9 cm × 9 cm) projecting from both corner joints opposing the enclosure.

Each social interaction test was composed of two 150-s phases, either with or without the target CD-1 mouse present in the interaction zone. During the first phase, when the target CD-1 aggressor was absent, the C57 mouse was taken from its home cage and placed directly into the center of the open-field opposite the empty enclosure, allowing for exploration of the open-field arena. And 150 s later, the C57 mouse was removed from the arena and returned to its home cage and a CD-1 mouse was placed into the enclosure. Then the C57 mouse was placed into the rear center of the open-field opposite the enclosure for another 150 s. The social interaction ratios in the interaction zone (SI ratio-interaction zone) and the corner zone (SI ratio-corner zone) were obtained by dividing the time spent in the interaction and corner zone when the target was present by the time spent in the interaction zone and corner zone when the target was absent.

Tail suspension test

The tail suspension test was performed according to protocol that was used by Porsolt et al. [44]. In brief, mice were suspended on a bar with surgical tape for 6 min. The immobility time characterized by remaining immobile was recorded for the last 4 min. When assessing behaviors and performing data analysis, investigators were blinded to the treatment conditions.

Locomotor activity test

C57 mice were placed in the locomotor chambers equipped with infrared video recorders. Mice activities were monitored for 20 min. DigBehav Animal Behavior Analysis System (Shanghai Jiliang Software Technology) was applied to analyze the distance moved of mice.

Light–dark transition test

This test was performed as previously described [40]. The chamber used to performing light–dark transition test (45 cm × 27 cm × 30 cm) was divided into two compartments, with one-third for the dark (red light: 4 lux) and two-thirds for the light (300 lux). Mice were placed in the dark compartment and allowed free exploration of the chamber for 5 min. An infrared video recorder was applied to record the movement path of mice. The duration of time spent in the light and dark compartments, latency to enter the light compartment, and the number of crossing of mice were assessed.

Cannula implantation

Briefly, animals were anesthetized with Zoletil (50 mg/kg, subcutaneous injection) under aseptic conditions. By use of stereotaxic apparatus (RWD Life Science), the 26-gauge guide cannulae were bilaterally implanted into the amygdala (anteroposterior: −1.0 mm; mediolateral: ±3.0 mm; dorsoventral: −3.0 mm). The stainless-steel screws, dental cement, and tissue adhesive were applied to make sure the cannulae were tightly anchored to the skull of mice. The plugs made of acupuncture needle were inserted into the cannulae for the prevention of blockage.

Microinjection

Briefly, 33-gauge internal cannulae (Plastics One) were connected to a 10 μL microsyringe mounted on the microinfusion pump (Harvard Apparatus) and the infusion rate was 200 nL/min. The internal cannulae should extend 2 mm beyond the tips of the guide cannulae to prevent blockage. After injection, each mouse was given an additional 2 min for drug diffusion.

Immunoblotting

Mice were decapitated, the tissues of amygdala were dissected with 18 gauge syringe needle (0.8 mm inner diameter). Amygdala tissues were homogenated in protein lysis buffer mixed with phosphatase and protease inhibitor and then centrifuged with 10,000 × g for 5 min at 4 °C. The lower sediments were discarded, the upper clarified liquids were mixed with 4× loading buffer and then boiled in 90 °C hot water for 10 min. Equal amount of protein was electrophoresed on 12% SDS-polyacrylamide gels and transferred to nitrocellulose membranes. Then the membranes were blocked with 5% non-fat milk dilution in TBST for 1 h at room temperature and incubated with primary antibodies against dynorphin A (1:500), ERK (1:1000), p-ERK (1:200), and actin (1:5000) dilutions kept overnight at 4 °C. The next day, the nitrocellulose membranes were incubated with HRP conjugated goat anti-rabbit IgG (the secondary antibody of ERK, 1:10,000), goat anti-mouse IgG (the secondary antibody of p-ERK, 1:20,000) for 2 h at room temperature. At last, bands were developed with a chemiluminescent substrate (GE Healthcare). The immunopositive signals were quantified by quantity analysis software (Bio-Rad).

Immunofluorescence

One hour after social interaction testing, mice were perfused under deep anesthesia with 37 °C 0.9% saline followed by perfusion with 4% paraformaldehyde (PFA) in PBS. The brains were removed and fixed in 4% PFA at 4 °C overnight and dehydrated with 30% sucrose for 3 days. Coronal sections (30 μm in thickness) were cut on a cryostat (Leica, Germany) and brain slices were picked for immunofluorescence staining. Briefly, brain sections (two sections per sample) containing amygdala (mostly BLA), dorsal hippocampus (mostly CA3 subregion), hypothalamus (mostly dorsomedial hypothalamic nucleus) were incubated overnight at 4 °C with c-fos antibody. The sections were then incubated with corresponding secondary antibodies (Goat anti-Rabbit IgG Antibody, Alexa Fluor 555, 1:500, Invitrogen) at room temperature for 2 h followed by visualization using a fluorescence microscope (Olympus).

Histology

After behavior test, the mice were deeply anesthetized and perfused with 4% PFA, the brains were removed and kept in a 30% sucrose solution for 3 days to dehydration. Coronal sections (50 μm in thickness) were cut on cryostat (Leica) and Nissl staining was applied to detect the injection sites. In all experiments, data were only kept for analysis when the cannulae were placed in the right sites.

Statistical analysis

All data was expressed as mean ± SEM. Statistical analysis was performed using one- or two-way analysis of variance (ANOVA) with independent or repeated measures, or two-tailed unpaired t-test in accordance with the experimental design. Statistical significance was defined as P < 0.05.

Results

Chronic social defeat stress induced significant social avoidance in mice

The experimental design was shown in Fig. 1a, briefly, the experimental mice underwent 10-min social defeat episodes with an aggressive CD-1 and then separated with a separator each day for 14 days, on day 15, mice were assayed in a social interaction test. The results showed that mice displayed decreased time interacting with a novel social target (Fig. 1c). Two-tailed t-test revealed that the time spent in the corner zone significantly increased after chronic social defeat stress (Fig. 1d). Fig. 1b illustrated the paths of mice during social interaction test. The data obtained demonstrated that social defeat stress induced significant social avoidance in mice.

Fig. 1: Chronic social defeat stress induced significant social avoidance in mice.
figure 1

a Timeline for the process of chronic social defeat stress and the social interaction test in mice. b Representative traces of mouse locomotion (black lines) in unstressed and social defeat stressed mice. c, d Chronic social defeat stress induced significant social avoidance in mice. Chronic social defeat stress decreased SI ratio in the interaction zone (c) (n = 11–12, t (21) = 4.307, ***P < 0.001, t-test) and increased SI ratio in the corner zone (d) (n = 9–10, t (17) = 2.654, *P < 0.05, t-test). Values are expressed as mean ± SEM.

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κ Opioid receptors play an important role in the development of social avoidance induced by chronic social defeat stress

The κ opioid receptor antagonist norBNI was applied to examine the effect of κ opioid receptor inhibition on the development of social avoidance induced by social defeat stress. The experimental design was shown in Fig. 2a, briefly, the norBNI was injected (intraperitoneal administration) into mice 24 h before social defeat stress, then the mice were subjected to social defeat stress for 14 days followed by social interaction test. Two-way ANOVA revealed that norBNI significantly prevented decreased interaction time (Fig. 2c) and increased time duration in corner zone (Fig. 2d) induced by social defeat stress. Fig. 2b showed the representative traces of mouse locomotion (black lines) in unstressed and social defeat stressed mice subjected to saline or norBNI.

Fig. 2: Systemic norBNI injection before social defeat stress prevented the development of social avoidance induced by chronic social defeat stress.
figure 2

a The timeline of the experiment. b–d Social avoidance induced by chronic social defeat stress could be blocked by norBNI administration systemically (i.p.) before social defeat stress. b Representative traces of mouse locomotion (black lines) in unstressed and social defeat stressed mice that were treated with systemic injection of norBNI. c The decreased SI ratio in the interaction zone induced by chronic social defeat stress was blocked by systemic norBNI injection (n = 14–19). F (1, 63) = 39.01, P < 0.0001 (factor of social defeat stress treatment), F (1, 63) = 9.822, P = 0.0026 (factor of norBNI treatment), F (1, 63) = 3.097, P = 0.0833 (interaction between factors), two-way ANOVA, ****P < 0.0001 compared with the control mice, **P < 0.01 compared with the chronic social defeat stress mice, Bonferroni’s post hoc test. d The increased SI ratio in corner zone induced by chronic social defeat stress was blocked by norBNI injection prior to stress (n = 14–18). F (1, 64) = 15.9, P = 0.0002 (factor of social defeat stress treatment), F (1, 64) = 7.661, P = 0.0074 (factor of norBNI treatment), F (1, 64) = 3.307, P = 0.0737 (interaction between factors), two-way ANOVA, ***P < 0.001 compared with the control mice, **P < 0.01 compared with chronic social defeat stress mice, Bonferroni’s post hoc test. Values are expressed as mean ± SEM.

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We then injected norBNI systematically into mice after social defeat stress to test the role of κ opioid receptors in the social avoidance. The experimental design was shown in Fig. 3a. The results were shown in Fig. 3b–d. Compared to the vehicle control group, norBNI injection immediately after stress tended to improve the social avoidance but the difference is not statistically significant . The paths of mice during social interaction test were shown in Fig. 3b.

Fig. 3: Systemic norBNI injection after stress partly blocked social avoidance induced by chronic social defeat stress.
figure 3

a The procedure for norBNI injection and chronic social defeat stress. b–d Chronic social defeat stress induced social avoidance was partly blocked by norBNI injection after stress. b Representative traces of mouse locomotion (black lines) in unstressed and social defeat stressed mice that were treated with systemic injection (i.p.) of saline or norBNI. c The decreased SI ratio in the interaction zone induced by chronic social defeat stress was partly blocked by systemic norBNI injection after exposure to stress (n = 8–11). F (1, 37) = 10.27, P = 0.0028 (factor of social defeat stress treatment), F (1, 37) = 0.2855, P = 0.5963 (factor of norBNI treatment), F (1, 37) = 1.680, P = 0.2029 (interaction between factors), two-way ANOVA, *P < 0.05 compared with control mice, Bonferroni’s post hoc test. d The increased SI ratio in corner zone induced by chronic social defeat stress was partly blocked by norBNI injection after stress (n = 10–11). F (1, 38) = 4.624, P = 0.0380 (factor of social defeat stress), F (1, 38) = 0.7548, P = 03904 (factor of norBNI treatment), F (1, 38) = 3.821, P = 0.0580 (interaction between factors), two-way ANOVA, *P < 0.05 compared with control mice, Bonferroni’s post hoc test. Values are expressed as mean ± SEM.

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We further confirmed whether κ opioid receptors were critically involved in the development of depression following chronic social defeat stress by use of κ opioid receptor knockout mice. Fig. 4a illustrated the breeding scheme to generate κ opioid receptor knockout mice. Briefly, we bred κ opioid receptor knockout mice (JAX Stock No. 007558) with wild-type mice to generate the F1 generation of heterozygote and then the heterozygote was hybridized with each other to obtain the F2 generation of κ opioid receptor knockout mice (with DNA strip in 580 bp) and wild-type mice (with DNA strip in 296 bp, Fig. 4b). Then the appropriate F2 generation mice were subjected to chronic social defeat stress. Fig. 4d, e showed the social interaction ratios in interaction zone and corner zone. Two-way ANOVA revealed that κ opioid receptor deletion significantly abolished decreased interaction time and increased corner time induced by chronic social defeat stress. Fig. 4c showed the representative traces of mouse locomotion (black lines) in unstressed and social defeat stressed wild type and knockout mice.

Fig. 4: Deletion of κ opioid receptor abolished social avoidance induced by chronic social defeat stress.
figure 4

a The breeding scheme to generate κ opioid receptor knockout mice. b The representative gel photographed on top of a UV box. c–e Systemic deletion of κ opioid receptor abolished social avoidance induced by chronic social defeat stress. c The representative traces of wild type and κ opioid receptor knockout mouse locomotion in unstressed and social defeat stressed mice. d Deletion of κ opioid receptor blocked chronic social defeat stress-induced decrease of SI ratio in interaction zone (n = 3–8). F (1, 17) = 5.553, P = 0.0028 (factor of social defeat stress), F (1, 17) = 8.281, P = 0.0104 (factor of KOR knockout), F (1, 17) = 8.103, P = 0.0115 (interaction between factors), two-way ANOVA, *P < 0.05 compared with control mice, ***P < 0.001 compared with chronic social defeat stress mice, Bonferroni’s post hoc test. Increase of SI ratio in corner zone (e) (n = 3–8). F (1, 17) = 2.266, P = 0.1506 (factor of social defeat stress), F (1, 17) = 2.705, P = 0.1184 (factor of KOR knockout), F (1, 17) = 4.895, P = 0.0409 (interaction between factors), two-way ANOVA, *P < 0.05 compared with chronic social defeat stress group, Bonferroni’s post hoc test. Values are expressed as mean ± SEM.

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Amygdala is critically involved in social avoidance following chronic social defeat stress

To determine the potential neuroanatomical sites in depressive-like behaviors following chronic social defeat stress, c-fos staining was applied to screen brain regions related to mood disorders. As shown in Fig. 5, Student’s t test revealed that chronic social defeat stress induced significant activation of c-fos in the amygdala but not in hippocampus and hypothalamus, strongly suggesting that amygdala is an important neural substrate mediating social avoidance following chronic social defeat stress.

Fig. 5: C-fos staining in amygdala, hippocampus, and hypothamalus following chronic social defeat stress.
figure 5

a Representative images of c-fos in amygdala, hippocampus, and hypothamalus of control (left part) and chronic social defeat stress (right part). b Chronic social defeat stress significantly increased c-fos expression in the amygdala (n = 6–7. t (11) = 3.309, **P < 0.01, t-test) without influencing its expression in hippocampus (c) and hypothamalus (d). Values are expressed as mean ± SEM. Scale bar = 100 μm.

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It has been previously demonstrated that ERK is an important molecule in the downstream of κ opioid receptor signaling pathway [45, 46]. To determine if κ opioid receptors in the amygdala were involved in depressive-like behaviors, we measured ERK activation determined by the level of phosphorylated ERK (p-ERK) in different brain regions including amygdala, NAc, hippocampus, and hypothalamus. The results showed that the expression of p-ERK was significantly increased in the amygdala and the increase could be blocked by systemic κ opioid receptor antagonism (Fig. 6a). Both social defeat stress and norBNI injection did not influence ERK activation in NAc, hippocampus, and hypothalamus (Fig. 6b–d).

Fig. 6: Chronic social defeat stress activated ERK in amygdala through acting on κ opioid receptor.
figure 6

a The level of phosphorylated ERK was increased in the amygdala by chronic social defeat stress and could be blocked by norBNI pretreatment (n = 5). F (1, 16) = 4.408, P = 0.0520 (factor of social defeat stress), F (1, 16) = 1.935, P = 0.1832 (factor of norBNI treatment), F (1, 16) = 9.487, P = 0.0072 (interaction between factors), two-way ANOVA, *P < 0.05 compared with the control group, *P < 0.05 compared with chronic social defeat stress group, Bonferroni’s post hoc test. b–d Chronic social defeat stress and κ opioid receptor antagonism did not influence ERK activation in the NAc, hippocampus, and hypothalamus. Top: representative immunoblots of p-ERK (from left to right: control/vehicle group, social defeat stress/vehicle group, control/norBNI group, social defeat stress/norBNI group). Bottom: relative amounts of p-ERK quantified by densitometry. Values are expressed as mean ± SEM.

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Upregulation of dynorphin/κ opioid receptor in amygdala mediates social avoidance following chronic social defeat stress

Finally, we examined if dynorphin/κ opioid receptor system in the amygdala mediates depressive-like behaviors following chronic social defeat stress. We firstly tested the expression of dynorphin A by Western blot, and the results showed that the expression level of dynorphin A in the amygdala was significantly increased (Fig. 7a). To test if κ opioid receptor activation was essential for chronic social defeat stress-induced social avoidance, norBNI was injected into amygdala 24 h before social defeat stress followed by 14 days of social defeat stress (Fig. 7a). Fig. 7b–d showed that norBNI injection prevented the decreased interaction time and the increased corner time in mice subjected to social defeat stress. The representative traces of mouse locomotion (black lines) in unstressed and social defeat stressed norBNI treatment mice were shown in Fig. 7b. Fig. 7e, f showed the schematic representation of injection sites and Nissl staining.

Fig. 7: Dynorphin/κ opioid receptor activation in the amygdala mediates chronic social defeat stress-induced social avoidance.
figure 7

a The expression of dynorphin A increased following chronic social defeat stress (n = 16–17). t(31) = 2.12, *P < 0.05, t-test. b–d The social avoidance induced by chronic social defeat stress was blocked by κ opioid receptor antagonism in the amygdala. b Representative traces of mouse locomotion (black lines) in unstressed and social defeat stressed mice that were treated with vehicle or norBNI in the amygdala. c Microinjection of norBNI in the amygdala blocked chronic social defeat stress-induced decrease in SI ratio in interaction zone (c, n = 9–14). F (1, 41) = 10.39, P = 0.0025 (factor of social defeat stress treatment), F (1, 41) = 9.880, P = 0.0031 (factor of amygdala norBNI treatment), F (1, 41) = 0.9297, P = 0.3406 (interaction between factors), two-way ANOVA, *P < 0.05 compared with the control group or chronic social defeat stress group, Bonferroni’s post hoc test. And increased SI ratio in corner zone (d, n = 8–13). F (1, 39) = 5.825, P = 0.0206 (factor of social defeat stress treatment), F (1, 39) = 4.981, P = 0.0315 (factor of amygdala norBNI treatment), F (1, 39) = 3.934, P = 0.0544 (interaction between factors), two-way ANOVA, *P < 0.05 compared with the control or chronic social defeat stress group, Bonferroni’s post hoc test. Schematic representation of injection sites and Nissl staining were shown in e and f. ○ Control + vehicle, ● Chronic social defeat stress + vehicle, □ Control + norBNI, ■ Chronic social defeat stress + norBNI. Values are expressed as mean ± SEM.

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We have also conducted experiments including tail suspension test, light–dark transition test to evaluate the effect of antagonism of κ opioid receptor in amygdala on other depression related behaviors including behavioral despair and anxiety. Our data showed that chronic social defeat stress caused an increment of immobility time in the tail suspension test (Fig. 8a) without significant effect on distance traveled of mice (Fig. 8b). Intra-amygdala microinjection of norBNI (2.5 μg/0.5 μL per side) completely blocked chronic social defeat stress-induced increment of immobility time (Fig. 8a). In the light–dark transition test, chronic social defeat stress induced a decrease of time in light compartment (Fig. 8c), an increase of time in dark compartment (Fig. 8d). And the latency to enter the light compartment was also significantly increased (Fig. 8e) after social defeat stress, and chronic social defeat stress tended to decrease (but not significantly) the number of crossings (Fig. 8f). Intra-amygdala microinjection of norBNI (2.5 μg/0.5 μL per side) completely blocked chronic social defeat stress-induced decrease of time in light compartment (Fig. 8c) and increase in dark compartment (Fig. 8d) as well as the increased latency to light compartment (Fig. 8e).

Fig. 8: Antagonism of κ opioid receptor in the amygdala abolished depressive-like behaviors induced by chronic social defeat stress measured by tail suspension test and light–dark transition test.
figure 8

a Microinjection of norBNI in the amygdala blocked chronic social defeat stress induced increment of immobility time (n = 9–10). F (1, 33) = 0.4761, P = 0.4950 (factor of social defeat stress), F (1, 33) = 7.803, P = 0.0086 (factor of amygdala norBNI treatment), F (1, 33) = 10.71, P = 0.0025 (interaction between factors), two-way ANOVA, *P < 0.05 compared with the control group, **P < 0.01 compared with chronic social defeat stress treated vehicle group, Bonferroni’s post hoc test. b Intra-amygdala injection of norBNI had no effect on distance traveled of mice in control and chronic social defeat stressed mice (n = 9–10). F (1, 33) = 0.3113, P = 0.05807 (factor of social defeat stress), F (1, 33) = 0.1607, P = 0.6911 (factor of amygdala norBNI treatment), F (1, 33) = 1.082, P = 0.3059 (interaction between factors), two-way ANOVA, Bonferroni’s post hoc test. c–f Microinjection of norBNI in the amygdala blocked anxiety-like behavior measured by light–dark transition test after chronic social defeat stress illustrated by the time spent in the light compartment (c, n = 9–10). F (1, 33) = 1.584, P = 0.217 (factor of social defeat stress treatment), F (1, 33) = 6.696, P = 0.01101 (factor of amygdala norBNI treatment), F (1, 33) = 9.262, P = 0.0046 (interaction between factors), two-way ANOVA, *P < 0.05 compared with the control group and chronic social defeat stress treated vehicle group, Bonferroni’s post hoc test. The time spent in the dark compartment (d, n = 9–10). F (1, 33) = 1.584, P = 0.217 (factor of social defeat stress), F (1, 33) = 6.696, P = 0.01101 (factor of amygdala norBNI treatment), F (1, 33) = 9.262, P = 0.0046 (interaction between factors), two-way ANOVA, *P < 0.05 compared with the control group and chronic social defeat stress treated vehicle group, Bonferroni’s post hoc test. And the time spent prior to the first entry to the light compartment (e, n = 9–10). F (1, 32) = 4.264, P = 0.0471 (factor of social defeat stress), F (1, 32) = 2.703, P = 0.1100 (factor of amygdala norBNI treatment), F (1, 32) = 7.092, P = 0.0120 (interaction between factors), two-way ANOVA, *P < 0.05 compared with the control group and chronic social defeat stress treated vehicle group, Bonferroni’s post hoc test. Without significant effect on number of crossing (f, P > 0.05). Values are expressed as mean ± SEM.

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Discussion

Stress is an important environmental factor causing depression. Previous study showed that κ opioid receptors and their endogenous ligand dynorphin are critically involved in the regulation of stress- induced behavioral deficits including aversion and dysphoria, so we speculate that dynorphin/κ opioid receptor system is critically involved in the development of depression. The present study reveals that κ opioid receptors are involved in the development of depressive-like behavior following chronic social defeat stress, and upregulation of dynorphin/κ opioid receptor in the amygdala and its activation of ERK modulate the depressive-like behaviors in mice subjected to social defeat stress. Therefore, our study provides evidence of the critical brain region involved in κ opioid receptor-modulated depressive-like behaviors as well as the potential mechanisms, thereby suggesting molecular targets for the prevention and treatment of depressive-like behaviors.

Important role of κ opioid receptor in the development of depressive-like behavior induced by chronic social defeat stress

In animal models, chronic social defeat stress could induce behavioral defeats including anxiety, behavioral despair, and social avoidance in mice, among which, social avoidance is the earliest and the most enduring behavioral phenotype appeared after exposure to social defeat stress [1, 47, 48]. In addition, social avoidance is a major risk factor for the development and maintenance of anxiety and depression [49, 50]. So we choose social avoidance as the main behavioral readout of our study.

By use of pharmacological methods, we found that systemic antagonism of κ opioid receptor before stress prevented social avoidance, strongly suggesting that κ opioid receptors were involved in the development of depression following social defeat stress. However, if norBNI was given after stress, when the depression model was established, norBNI only partly blocked depressive-like behavior. These results strongly suggest that κ opioid receptors are much more likely involved in the development of depression. κ Opioid receptor deletion abolished the development of social avoidance, further confirmed the conclusion that κ opioid receptors were involved in the development of depression.

It should be noted that there exists difference of opinion about the role of κ opioid receptor in social avoidance in the literature. Williams et al. found that inhibition of κ opioid receptors blocked social avoidance, while Donahue et al. found that antagonism of κ opioid receptors failed to affect social avoidance [51, 52]. Our findings provide an explanation of the divergence by showing that κ opioid receptor antagonist given before stress could prevent social avoidance induced by chronic social defeat stress. If the depression model has been established, the effect of κ opioid receptor antagonists on depression is limited.

Dynorphin/κ opioid receptor system in the amygdala mediate depression induced by chronic social defeat stress

The c-fos mRNA and protein are rapidly and transiently induced within 15 min of stimulation [53], and hence referred as an immediate early gene, so, c-fos activation was considered as a neuronal activation marker to screen brain regions that were related to behavioral phenotype. In order to find the key brain regions that were relevant to depression, the c-fos staining was applied to map the neuronal circuits that were activated in the process of chronic social defeat stress. The results showed that c-fos was significantly activated in the amygdala rather than in hippocampus and hypothamalus.

Numa et al. have reported that single stress (defined as acute stress model) increased c-Fos expression in distinct brain regions including hippocampal CA1 region, prelimbic cortex, NAc, hypothalamus, amygdala, BNST, DRN, VTA, and so on [54]. In our study, we try to investigate the mechanism of depression by using repeated social defeat stress model since the molecular and related brain regions may be different between acute and chronic social defeat stress. And also, it has been previously demonstrated that different c-fos activation was observed between acute and chronic social defeat stress [55]. Our results showed that the expression of c-fos was increased in the amygdala, so we inferred that activation of κ opioid receptor in the amygdala might lead to the emergency of depression following chronic social defeat stress.

We further demonstrated that dynorphin/κ opioid receptor in the amygdala play a key role in the development of depression by showing that the expression of dynorphin A in the amygdala increased by stress. We locally injected κ opioid receptor antagonist norBNI into amygdala to test if antagonism of κ opioid receptor could abolish depressive-like behaviors following chronic social defeat stress. The results showed that microinjection of norBNI into amygdala before social defeat stress prevented the development of depression (including social avoidance, behavioral despair, and anxiety-related behavior) induced by chronic social defeat stress, strongly suggesting that dynorphin/κ opioid receptor system distributed in the amygdala was critically involved in the development of depression. Our results are consistent with previous findings showing that activation of κ opioid receptors in the amygdala caused anxiogenic effects and antagonism of κ opioid receptors in the amygdala reduced anxiety-like responses [56,57,58].

ERK activation following social defeat stress may be the underlying mechanism of κ opioid receptor-modulated depressive-like behavior in mice

κ Opioid receptor is a member of the G-protein coupled receptor superfamily. κ Opioid receptor activation leads to the activation of G-protein coupled Receptor Kinases and members of the mitogen-activated protein kinase family including ERK1/2, p38, and JNK [59, 60]. It has been previously reported that ERK plays a critical role in the regulation of depression [61] and serves as an important mediator of the depressive-like behaviors [30, 62]. Combined with our previous study showing that ERK was critically involved in the modulation of aversive emotion induced by morphine withdrawal and κ opioid receptor activation [11, 40], we propose that ERK activation by κ opioid receptor may be the underlying mechanism of depressive-like behavior induced by chronic social defeat stress. Our results showed that the level of p-ERK was increased in the amygdala rather than in the hippocampus, hypothamalus, and nucleus accumbens, further confirmed previous results showing that amygdala is an important brain region involved in the development of depression. In addition, we found that social defeat stress-induced ERK activation could be blocked by systemic κ opioid receptor antagonism, strongly suggesting that chronic social defeat stress-induced activation of ERK was mediated by κ opioid receptor activation. Since ERK activation always occurred in depression and stress model [30, 31], we inferred that κ opioid receptor-induced ERK activation may be the underlying mechanism of depressive-like behaviors induced by social defeat stress.

κ Opioid receptors have been reported to decrease serotonin transmission through enhancing the trafficking of serotonin transporter from intracellular part to the cell membrane. And also it has been previously demonstrated that κ opioid receptor activation inhibited glutamate transmission and GABA transmission in vivo [58, 63,64,65]. Serotonin, GABA, and glutamate transmission are critically involved in depression [66,67,68,69,70,71]. So it is possible that κ opioid receptor activation induces depressive-like behaviors through regulation of serotonin, glutamate, and GABA transmission. It has been well documented that BLA is enriched in glutamate neurons, and ERK activation has been reported to inhibit glutamatergic activation [72]. So, we infer that KOR blockade prevents depression from chronic social defeat stress by inhibiting glutamate transmission.

Conclusion

The present study demonstrated that chronic social defeat stress induced depressive-like behaviors in mice. Pretreatment with κ opioid receptor antagonist norBNI blocked the development of depressive-like behaviors. After chronic social defeat stress, the expression level of dynorphin A significantly increased in the amygdala and κ opioid receptor antagonism in the amygdala completely blocked the development of depressive-like behaviors. Chronic social defeat stress activated ERK in the amygdala and the effect could be completely blocked by systemic κ opioid receptor antagonism. These findings suggested that depression following chronic social defeat stress was modulated by upregulation of dynorphin/κ opioid receptor system and ERK activation downstream of κ opioid receptor. More importantly, the results of the present study shed light on the κ opioid receptor antagonists as potential therapeutic agents for the treatment of mood disorders induced by chronic social defeat stress.