Introduction

Ketamine, a noncompetitive and nonselective N-methyl-d-aspartate (NMDA) receptor antagonist, has been widely used clinically as an anesthetic (Huang et al. 2016). In addition, a small number of individuals use ketamine as a recreational drug. Ketamine typically induces hallucinogenic effects that alter the sense of sight, sound and touch, which was proven by research in rodents (Nikiforuk et al. 2016; Zhao et al. 2016; Kara et al. 2017). Additionally, at higher doses (>80 mg/kg) in rodent models, the drug can induce DNA damage within 12 h, as confirmed by comet assay (Leffa et al. 2016). Ketamine can also inhibit human sperm function and lead to schizophrenia (He et al. 2016; Favretto et al. 2016).

Schizophrenia is a severe chronic neuropsychiatric disorder characterized by positive symptoms (hallucinations and delusions), negative symptoms (social withdrawal) and cognitive impairments (impaired executive functions and visual memory) (Potasiewicz et al. 2017; Zugno et al. 2016; Bennett 2011). According to a study by Simeone et al. (2015), the 12-month prevalence of schizophrenia is 0.33% (with a range of 0.26–0.51%), and the median estimated lifetime prevalence is 0.48% (with a range of 0.34–0.85%) (Simeone et al. 2015). In 2013, the American Psychiatric Association estimated a lifetime prevalence of schizophrenia of approximately 0.3–0.7%. Due to the lack of biomarkers and mechanisms, while antipsychotic drugs exert beneficial effects on the positive symptoms, they have little effect on negative symptoms or cognitive deficits (Okazaki et al. 2016; Noto et al. 2016; Chen et al. 2016). Since these two types of symptoms negatively affect social functioning throughout the course of the disease, there is an urgent need to develop new effective therapeutic methods to manage them. At this point, a new biomarker for the two types of symptoms would help scientists in monitoring and studying the process.

ErbB4 plays an important role as a cell surface receptor for neuregulins and EGF family members. ErbB4 can regulate the development of the heart (Wadugu and Kuhn 2012), the central nervous system (Huang et al. 2000) and the mammary glands (Muraoka-Cook et al. 2008). ErbB4 and its ligand neuregulin1 (NRG1) have been studied extensively as candidate pathways for schizophrenia (Hahn et al. 2006; Li et al. 2007; Banerjee et al. 2010; Nicodemus et al. 2010). The dysfunction of the NRG1/ErbB4 signaling pathway has been closely associated with the pathogenesis of schizophrenia; cases of schizophrenia and childhood onset of schizophrenia have been shown to be related to mutation of the NRG1/ErbB4 signaling pathway (Dang et al. 2016; Deng et al. 2015; du Bois et al. 2012; Taylor et al. 2011; Yang et al. 2012).

In this study, we aimed to determine the effects of ketamine on ErbB4 in a ketamine-induced rat model for schizophrenia. We found that ErbB4 was expressed only in the CA3 region of the hippocampus and the Cg1 region of the prefrontal cortex under treatment with high-dose ketamine (60 mg/kg), as determined by immunohistochemistry. This work will help scientists better understand the ErbB4-related signaling pathway in schizophrenia.

Materials and Methods

Drugs, Reagents and Apparatus

The reagents/materials were purchased as follows: ketamine hydrochloride (cat. #H35020148, Fujian Gutian Pharmaceutical Co., Ltd.); rabbit monoclonal anti-ErbB4 antibody (cat. #05–1133, MilliporeSigma, Billerica, MA, USA); DAB detection kit (cat. #PV-9000, Beijing Zhong Shan Jin Qiao Biotechnology Co., Ltd.); microscope (Olympus BX53, Japan).

Animals

Three-week-old male Sprague–Dawley (SD) rats, weighing 110–125 g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All the animals were housed in an environment with temperature of 22 ± 1 °C, relative humidity of 50 ± 1% and a light/dark cycle of 12/12 h, and were fed a standard diet (Beijing KeaoXieli Feed Co., Ltd., SPF rat feed); proper water was given. All animal studies (including the rat euthanasia procedure) were performed in compliance with the regulations and guidelines of Kunming Medicine University institutional animal care and conducted according to Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) and institutional animal care and use committee (IACUC) guidelines.

Drug Administration

A total of 24 SD rats were acclimatized to the environment for 1 week. Subsequently, rats were randomly divided into four experimental groups (n = 6/group): control, low-dose, medium-dose and high-dose groups. The low-dose, medium-dose and high-dose groups were intraperitoneally administered 15 mg/kg, 30 mg/kg and 60 mg/kg ketamine, respectively, twice each day (9:00 a.m. and 9:00 p.m); the control group was administered normal saline. The ketamine was diluted with normal saline (Bian et al. 2009). The treatment lasted for 7 days.

Tissue Preparation

Rats were euthanized 13 h after the last injection and perfused with normal saline until the liver became white and the effluent was clarified. A blade was used to remove the unwanted parts of the brain, rostral and caudal to the region of interest. In the case of the hippocampus, the brain was placed ventral side down, the superior colliculus was located and a transverse cut was made, and the caudal part was discarded (see details in supplemental figure). Brain tissues from the hippocampus and prefrontal cortex regions were collected, fixed in 4% paraformaldehyde (PFA) and subsequently embedded in paraffin.

Immunohistochemistry

The paraffin-embedded tissues from the two regions were cut coronally into 5-μm sections. Slices containing coronal sections were incubated at 70 °C for 3 h, deparaffinized and subsequently rehydrated using a graded series of ethanol to PBS. Slices were then treated with 1% (w/w) citrate buffer (pH 6.0) and endogenous peroxidase inhibitors. The measured areas were delineated with an immunohistochemical pen. The samples were sequentially incubated with rabbit monoclonal anti-ErbB4 antibody (1:400) and goat anti-mouse/rabbit IgG polymer-marked horseradish peroxidase. Lastly, the sections were stained with DAB stain agent for 5 min, and then rendered transparent with dimethylbenzene (Liu et al. 2013; Ho et al. 2014; Li et al. 2014). Stained sections were visualized under a microscope (Olympus BX53, Japan).

Quantitative Methods

All images and data were acquired and analyzed using Image-Pro Plus 6.0 software (Media Cybernetics, Rockville, MD, USA). The gray value of ErbB4 protein was measured by the Image-Pro Plus 6.0 analysis system, and then converted into an optical density (OD) value. The ratio of optical density to the whole image, referred to as OD/area, was calculated, and the expression of ErbB4 protein was quantified by the value of the OD/area ratio (Liu et al. 2014; Koh et al. 2016; Onaka et al. 2016; Farahmandfar et al. 2016; Zhang et al. 1631).

Statistical Methods

All data ae presented as mean ± standard error of the mean (SEM). Statistical analysis and generation of graphs were carried out using GraphPad Prism 5 software (San Diego, CA, USA). Statistical analysis was performed using one-way analysis of variance (ANOVA), followed by the Newman–Keuls post hoc test (*p < 0.05; **p < 0.01; ***p < 0.001).

Results

Expression and Localization of ErbB4 in the Hippocampus Region

To investigate the effect of ketamine on the expression and localization of ErbB4 in the hippocampus, we performed an immunohistochemistry (IHC) staining experiment for ErbB4. As shown in Fig. 1a, ErbB4-positive cells were localized in the CA3 region of the hippocampus. Our data indicate that treatment with ketamine decreased the expression of ErbB4 in a dose-dependent manner (Fig. 1a and b). The expression of ErbB4 was significantly decreased in the medium-dose and high-dose groups (p < 0.01), while the low-dose group showed no significant reduction (Newman–Keuls post hoc test) (Fig. 1a and b). Together, the results showed that ketamine decreased the ErbB4 protein level in the CA3 region of the hippocampus.

Fig. 1
figure 1

The expression of ErbB4 in tissues of the CA3 region of the hippocampus. a Data shown are representative images of staining for ErbB4 protein in tissues of the CA3 region of the hippocampus. Scale bar, 20 μm. Four-week-old male Sprague–Dawley (SD) rats were randomly assigned to one of four groups (n = 6 per group): the control group (treated with equal volume of normal saline), low-dose group (treated with ketamine at a dose of 15 mg /kg body weight), medium-dose group (treated with ketamine at a dose of 30 mg/kg body weight), and high-dose group (treated with ketamine at a dose of 60 mg/kg body weight). Rats were intraperitoneally administered ketamine or normal saline twice daily (9 a.m. and 9 p.m.) for 7 consecutive days. Rats were euthanized 13 h after the last injection of ketamine or saline, and the hippocampal tissues were subjected to immunohistochemistry staining. b Quantitation of the ErbB4 protein expression levels in the CA3 region of the hippocampus tissues from rats treated with normal saline or different doses of ketamine. The relative ErbB4 expression levels in tissues from the CA3 region of the hippocampus from the four groups were quantitated by OD/area. n = 6 for each group; **p < 0.01, compared with the control group.

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Expression and Localization of ErbB4 in the Prefrontal Cortex

The same approach as that described above was used to examine the expression and localization of ErbB4 in the prefrontal cortex of rats. As shown in Fig. 2a, ErbB4-positive cells were localized in the Cg1 region of the prefrontal cortex. Moreover, the quantitative results indicated that ketamine reduced the protein level of ErbB4 in a dose-depended manner. The expression of ErbB4 was significantly lower in medium-dose and high-dose groups than in the control group (p < 0.01), and the protein level in the low-dose group did not show a significant reduction (Newman–Keuls post hoc test) (Fig. 2a and b). Together, the results showed that ketamine could decrease the ErbB4 protein level in the Cg1 region of the prefrontal cortex.

Fig. 2
figure 2

The expression of ErbB4 in the Cg1 region of the prefrontal cortex. a. Data shown are representative images of staining for ErbB4 protein in the Cg1 region of the prefrontal cortex. Scale bar, 20 μm. Experimental procedures were as described in Fig. 1a, except that the ErbB4 protein expression was examined in the postmortem tissue from rat prefrontal cortex. b. Quantitation of the ErbB4 protein expression levels in the Cg1 region of the prefrontal cortex from rats treated with normal saline or different doses of ketamine. The relative expression levels of ErbB4 protein in the Cg1 region of the prefrontal cortex from the four groups were quantitated by OD/area. n = 6 for each group; **p < 0.01, compared with the control group.

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Discussion

Similar to methylamphetamine-induced animal models, ketamine can induce both positive and negative symptoms of schizophrenia (Frohlich and Van Horn 2014). In this study, a ketamine-induced model was used to investigate the effect of the drug on the expression of ErbB4 in the hippocampus and prefrontal cortex of rats.

The experimental results showed that ketamine treatment indeed influenced the expression of ErbB4 specifically in both the CA3 region of the hippocampus and the Cg1 region of the prefrontal cortex. In addition, the effects of ketamine acted in a strictly dose-dependent manner. The study also demonstrated that a dose of 60 mg/kg (ketamine) was the best dosage for establishing a rat model of schizophrenia. Although ketamine doses of 30 mg/kg and 60 mg/kg caused similar effects, the higher-dose ketamine (60 mg/kg) led to more obvious symptoms of schizophrenia. Therefore, based on these results, the higher dose (60 mg/kg) is recommended in order to ensure a reliable, stable and secure model.

The high-dose group (60 mg/kg) showed significant positive symptoms of schizophrenia, including apparent aggressive behavior starting from the third day of ketamine treatment. On the other hand, the medium-dose and low-dose groups (15 mg/kg and 30 mg/kg) exhibited less significant symptoms of schizophrenia than the high-dose group, even after all scheduled treatments. A dose higher than 60 mg/kg led to the death of the rats in our study. This suggests that high-dose ketamine is a requirement in a chronic ketamine exposure-induced rat model of schizophrenia.

Importantly, there are no relevant articles arguing that changes in ErbB4 expression in different encephalon regions are associated with ketamine. Our work thus reveals the relationship between ketamine and protein reduction (ErbB4) in different brain tissues, which could improve our knowledge regarding schizophrenia, and all the results here also provide a potential biomarker to aid in drug discovery. However, schizophrenia is a complex disorder involving multiple disease pathways, and therefore a comprehensive therapeutic methodology is needed. This work may provide a potential target and open a new path to the final objective.

Conclusions

In this study, we showed that in vivo administration of ketamine in rats could reduce the expression of the ErbB4 protein in specific regions of the hippocampus (CA3 region) and the prefrontal cortex (Cg1 region), and that the reduction occurred in a dose-dependent manner. Moreover, ketamine at 60 mg/kg was found to be effective for establishing an optimal rat model of schizophrenia. In summary, this study investigated the protein distribution and expression of ErbB4 in the encephalon regions of a schizophrenia-induced rat model chronically treated with high-dose ketamine, and provides novel insights that will deepen our understanding of the pathogenic mechanisms of schizophrenia and aid in drug discovery. Further studies are still needed to elucidate the exact molecular mechanisms involved in the pathogenesis of schizophrenia.