Major depressive disorder (MDD) is an intractable psychiatric disease. Approximately 17% of the world population is suffering from this disease [1,2]. The MDD age distribution study demonstrated that MDD is equally common for people from the full age span . The major MDD symptoms include long-term depressed mood, anhedonia, insomnia, and an increased risk of suicides . Despite numerous studies focusing on this debilitating disorder, the detailed MDD mechanism remains unclear. However, genetic factors and stress are considered to increase the risk of MDD development [5–8]. Over the recent decades, neuroimaging studies showed reduced volume of the mPFC and hippocampus in patients with depression  and postmortem studies revealed a significantly decreased number of synapses in the prefrontal cortex (PFC) of depressed patients . Rodent-model studies demonstrated that chronic stress decreases the number of neurons and glial cells in the hippocampus and PFC . Synaptogenesis is affected by multiple factors, including neurotrophic factors and inflammatory cytokines . Many studies demonstrated that the neurotrophic factor expression levels are important for defining the depression intensity [13,14]. For example, decreased expression levels of brain-derived neurotrophic factor (BDNF) are found in the PFC and hippocampus of patients with depression [6,15,16]. Stress-induced structural brain changes in depressed patients may be due to the reduced expression levels of neurotrophic and growth factors, as these factors are important for synaptic formation .
Neuregulin1 (NRG1) is an endogenous neurotrophic factor that exerts synaptic transmission and suppresses the long-term potentiation through the tyrosine kinase ErbB4 receptor [18,19]. NRG1 and ErbB4 are highly expressed in the cortex and hippocampus regions in the brain . Besides, NRG1-ErbB4 signaling has been reported to participate in synaptic plasticity and neuron survival [21–23]. Previous studies have demonstrated that NRG1 participates in the development of mental disorders . Moreover, the NRG1 and ErbB4 brain expression levels are significantly altered in subjects with affective disorders . Furthermore, recent studies demonstrated that the mRNA expression levels of the NRG1 are altered in the peripheral mononuclear cells of patients with depression . In a rat model of depression using a chronic unpredictable mild stress protocol in which rats went through a serial of stressors such as immobilization and food/water deprivation, the expression levels of NRG1 in the hippocampus region and plasma were shown to be changed . These data suggest that NRG1-ErbB4 signaling may be closely associated with depression, but the exact contribution of the NRG1-ErbB4 pathway in human depression is still unclear.
In the present study, we chose the chronic social defeat stress (CSDS) model to investigate the role of the NRG1-ErbB4 pathway in depression. The results showed a significant decrease in the NRG1 and ErbB4 protein expression levels in the mPFC and hippocampus of CSDS mice. NRG1 lateral ventricle administration was able to reverse depression behavior of the CSDS mice. Thus, the NRG1-ErbB4 signaling pathway might play a vital role in depression, and, therefore, become a potential target for the development of new antidepressants.
Materials and methods
C57BL/6J male mice (8 weeks old, 18–22 g) were purchased from the Animal Core Facility of Zhejiang University. CD1 mice (8–12 weeks old, 22–28 g) were purchased from the Charis River. All mice were maintained in the Animal Core Facility of Zhejiang University under pathogen-free conditions with ad libitum access to food and water and with a 12-h light-dark cycle (lights on at 8:00 a.m. and off at 8:00 p.m.). All animal experimental protocols were approved by the Institutional Animal Care and Use Committee of Zhejiang University and were in compliance with the institutional guidelines.
Reagents and antibodies
Rabbit anti-NRG1 (ab53104), rabbit anti-phospho-ErbB4 (ab109273), and rabbit anti-ErbB4 (ab32375) were purchased from Abcam (Cambridge, Massachusetts, USA). Rabbit anti-GAPDH (5174) was purchased from Cell Signaling Technology (Beverly, Massachusetts, USA). Recombinant Human Neuregulin-1/Heregulin-b1 (CYT-733) was purchased from ProSpec (Hamada, Israel).
Chronic social defeat stress paradigm
The CSDS paradigm was implemented as described previously . At the beginning of the experiment, CD1 male mice were monitored for 3 days to evaluate their aggressive characteristics. After that, male C57BL6/j mice from the experimental group were placed one by one into the cage to an aggressive CD1 mouse for 5–10 min, where they were attacked by the CD1 mouse. Then, the C57BL6/j and the CD1 mice were separated and maintained in two cages for 24 h with a perforated plexiglass barrier between the two cages. Mice from the control group were housed in the same conditions, with C57BL6/j and CD1 mice separated by a perforated plexiglass partition, which did not allow any physical or sensory contact. After completing 10-days CSDS, the mice performed behavioral tests.
Before all behavioral testing, mice were handled and acclimated to the testing room for 1 h. All behavioral tests were performed during the light period, avoiding the 1-h period right before and after the lights were turned on and off. After CSDS, mice performed the sucrose preference test, social contact test, and tail suspension test, with at least a 24-h interval between the tests.
Sucrose preference test
First, mice practiced with two 50-mL kettles filled with drinking water for 48 h. After that, one of the kettles was replaced with a kettle with 1% sucrose instead of drinking water. During the training, the positions of the kettles were changed every 12 h to avoid side preference development. After the training was completed, mice were water-deprived for 24 h. Then, the two kettles with water or sucrose were weighed 1 h after the formal test began, and sucrose preference was calculated as the consumed sucrose divided by the total consumed volume of both water and sucrose.
Tail suspension test
The mice were suspended by their tails using a plastic clip, 60 cm above the surface. After a 1-min adaptation time, the mice were recorded on video for the next 5 min, and the time they spent motionless was quantified.
The mice were anesthetized with sodium pentobarbital (50 mg/kg) via intraperitoneal injection and placed in a stereotaxic apparatus (RWD, Shenzhen, China). Using a dental drill, the stainless-steel guide cannulas were placed into the lateral ventricle with the following coordinates: 1.0 mm beside the sagittal seam, posterior 0.22 mm and ventral 2.5 mm, according to The Mouse Brain in Stereotaxic Coordinates . The guide cannulas were secured in place with glass ionomer cement; incisions were fixed and covered with glass ionomer cement as well. The behavioral tests were performed 7 days after the surgery. The mice were infused with NRG1 at a flow rate of 1 µL/min for 2 min, and the final concentration of NRG1 was 100 nM.
Mice were anesthetized and perfused through the heart with 0.9% NaCl to clear blood proteins. The mice’s brains were removed to ice-cold 0.9% NaCl solution, where the hippocampus and PFC regions were dissected under a stereoscope. Then, the hippocampus and PFC were homogenized in 1 mL of radioimmunoprecipitation assay lysis buffer and centrifuged at 12 000g for 5 min at 4°C. The supernatants were diluted in loading buffer and boiled at 95°C for 10 min. The proteins were subjected to 10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, Massachusetts, USA) at 120 V within 2 h. The membranes were blocked with 5% BSA in PBS and incubated with the primary antibody for 16–18 h at 4°C. Then, the membranes were washed with PBST (0.05% Tween-20 in PBS) and incubated with the HRP-conjugated secondary antibody for 2 h. Finally, the membranes were washed with PBST and immunodetected with enhanced chemiluminescence reagents (Pierce ECL substrate, Thermo Fisher Scientific, Waltham, MA, USA).
Statistical analysis was performed using the SPSS 18.0 software. The data are shown as the mean ± SEM. Statistical differences were compared using one-way analysis of variance (ANOVA) or two-way ANOVA for multiple groups. P < 0.05 was considered significant.
The chronic social defeat stress model establishment and behavioral test analysis
Among different depression-like behavior models, the CSDS paradigms have been widely used to explore the detailed mechanisms underlying depression-like behaviors [29,30]. In the present study, C57bl/6J male mice were subjected to a 10-day-long social defeat, followed by the social interaction test, sucrose preference test, and tail suspension test used to evaluate the depression-like behaviors. In the social interaction test, the time spent in the interaction zone significantly decreased in the susceptible group compared to the control and resilient groups [Fig. 1b, F (2, 109) = 6.544, P < 0.0021]. However, the time spent in the corner zone was increased in the susceptible group compared to the control and resilient groups [Fig. 1c, F (2, 108) = 14.69, P < 0.0001]. Social interaction and corner-zone ratio showed that mice from the susceptible group spent less time interacting with the CD1 mouse, whereas mice in the control group performed similarly to the resilient group [Fig. 1d, F (2, 55) = 10.81, P = 0.0001; Fig. 1e, F (2, 55) = 15.32, P < 0.0001]. In the sucrose preference test, mice from the susceptible group showed declined sucrose preference compared to the control and resilient groups [Fig. 1F, F (2, 57) = 13.57, P < 0.0001]. In the tail suspension test, the immobility time was increased for mice from the susceptible group [Fig. 1g, F (2, 25) = 1.457, P = 0.2519].
These results indicated that the CSDS model is well-established and clearly shows depression-like behavior.
Reduced Neuregulin1 and ErbB4 expression levels in the medial prefrontal cortex of the chronic social defeat stress mice
A neuroimaging study showed decreased mPFC volume in patients with depression . To investigate the relationship between the NRG1 and ErbB4 expression levels and depression-like behavior, we evaluated the NRG1 and ErbB4 protein expression levels in the mPFC (Fig. 2a). Compared to the control and resilient groups, in mice from the susceptible group NRG1 protein expression level was significantly decreased [Fig. 2b, F (2, 11) = 13.86, P = 0.0010]. In addition, the protein expression level of phosphorylated ErbB4 in the mPFC region of mice from the susceptible group was decreased [Fig. 2c, F (2, 11) = 6.905, P = 0.0114; Fig. 2d, F (2, 13) = 4.807, P = 0.0274; Fig. 2e, F (2, 11) = 0.7599, P = 0.4908]. Compared to the control group, the protein expression level of phosphorylated ErbB4 in the resilient group was decreased as well (Fig. 2c). The results above demonstrated that the NRG1 and ErbB4 expression levels in the CSDS mice mPFC were decreased.
Decreased Neuregulin1 and ErbB4 protein expression levels in the chronic social defeat stress mice hippocampus
A rodent study showed that chronic stress affects hippocampus neurons, decreasing their size, length, and number and significantly inhibiting neurogenesis . We speculated that neurotrophic factor NRG1 and its receptor ErbB4 in the hippocampus might play an important role in developing MDD via decreased neurogenesis. We compared the NRG1 and ErbB4 protein expression levels in the hippocampus between mice from the control, resilient, and susceptible groups (Fig. 3a). Similar to the mPFC, in the hippocampus region, the NRG1 protein expression level was significantly lower in mice from the susceptible group [Fig. 3b, F (2, 11) = 6.24, P = 0.0154]. However, no significant difference between the NRG1 expression levels in control and resilient groups was observed (Fig. 3b). Furthermore, the phosphorylated ErbB4 protein expression levels between the three groups were not significantly different [Fig. 3c, F (2, 11) = 0.7473, P = 0.4962; Fig. 3d, F (2, 13) = 0.3804, P = 0.6910; Fig. 3e, F (2, 11) = 1.009, P = 0.3673].
Increased Neuregulin1 levels in the brain can recover depression behavior
To determine the exact relationship between the NRG1-ErbB4 pathway and depression, we performed intraventricular ACSF or NRG1 injections for the control, resilient, and susceptible groups. Before the injections, the mice were evaluated in the social interaction test. Compared to the control group, the social interaction ratio was significantly decreased in mice from the resilient and susceptible groups, while the corner ratio was increased in the susceptible group only [Fig. 4b, F (2, 60) = 36.6, P < 0.0001; Fig. 4d, F (2, 60) = 14.17, P < 0.0001]. However, 1 h after the NRG1 injection, the social interaction ratio of mice from the susceptible group was notably increased, and the corner ratio was significantly decreased compared to the ACSF treatment [Fig. 4c, F (2, 60) = 5.131, P = 0.0088; Fig. 4e, F (2, 60) = 4.257, P = 0.0187]. These results indicated that NRG1 intraventricular injection plays a role in ameliorating depression behavior, but this effect may be time-dependent.
A recent genomic study showed that NRG1-ErbB4 signaling may play an important role in schizophrenia, MDD, and bipolar disorder . Our previous study revealed that NRG1-ErbB4 expression is significantly decreased in the brains of symptomatic epilepsy patients . Moreover, the ErbB4 knockout mice showed decreased activity-dependent GABAergic FS-PN transmission, which may underlie the etiology of schizophrenia disorders . The relationship between epilepsy and MDD is complex and bidirectional. Some studies showed that epilepsy patients are at greater risk of developing depression, and depression patients have a higher risk of developing epilepsy [35,36]. Thus, we hypothesized that the NRG1-ErbB4 pathway may play an important role in depression. In our present study, we demonstrated that the NRG1 and Erb4 protein expression levels are significantly reduced in the mPFC and hippocampus of the CSDS mice. Moreover, increasing the brain NRG1 protein level could rescue the CSDS mice depression behavior. These results suggest that the expression levels of NRG1 and ErbB4 play an essential role in MDD.
Several previous studies have shown that volume alterations in the PFC and hippocampus brain regions are highly related to MDD [37–39]. The atrophy of the PFC and hippocampus structures may be due to the decreased expression levels of neurotrophic factors, and the neurotrophic role of antidepressants could reverse neuron loss and atrophy . Most studies focus on the BDNF, demonstrating that acute or chronic stress model could decrease its expression level in the PFC and hippocampus and decrease the peripheral serum BDNF level [15,41].
The NRG1 and ErbB4 expression levels were decreased in the serum of depressed mice . Likewise, the present results clearly show that the NRG1 protein expression level is significantly decreased in the mPFC and hippocampus regions of CSDS mice. Thus, we hypothesized that the ErbB4 receptor stimulation by its natural ligand NRG1 may play an important role in depression.
Our previous studies have demonstrated that intracerebral infusion of NRG1 can rescue epilepsy-related symptoms. In the CSDS model, NRG1 intracerebral treatment can significantly rescue depression-related behavior. However, the NRG1 and ErbB4 expression profiles were unknown, and the effect of the hippocampus and mPFC NRG1-ErbB4 signaling on depression was unclear as well. It was demonstrated on hippocampal slices that the NRG1 treatment can significantly disrupt 2-arachidonoylglycerol signaling and affect long-term depression, while the aberrant NRG1 level can alter endocannabinoid signaling in brain stress circuitry . Whether in the present CSDS model the aberrant NRG1-ErbB4 signaling affects endocannabinoid signaling or synaptic plasticity in the hippocampus remains unclear and requires detailed investigation.
In the CSDS model, the NRG1 and ErbB4 expression levels are significantly decreased in both mPFC and hippocampus regions. Meanwhile, NRG1 intracerebral infusion treatment of CSDS mice can significantly rescue depression-related behaviors. Our study demonstrates for the first time the protective role of the NRG1-ErbB4 signaling in a depression model and provides a new perspective on the treatment of depression.
This work was supported by the Zhejiang Provincial Natural Science Foundation (grant number: LY17C090004) and the Natural Science Foundation of Bengbu Medical College (grant number: BYKF1819).
W.W.J. and L.H. conceived and designed the study. W.W.J., Q.Y., L.H., L.C.Y. and Q.H.Y. performed the experiments. W.W.J., Z.L., M.L.L. and L.C.Y. wrote the paper. W.W.J., L.H., Q.Y., Z.L., M.L.L., L.C.Y. and Q.H.Y. reviewed and edited the manuscript. All authors read and approved the manuscript.
The animal experiments were approved by the Institutional Animal Care and Use Committee of Zhejiang University and conducted in accordance with the UK Animals (Scientific Procedures) Act, 1986.
Conflicts of interest
There are no conflicts of interest.
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