TREK-1 Mediates The Alleviative Effects of GABAB Receptor Antagonism on Depression-Like Behavior in Chronic Unpredictable Stress Rats

GABA B receptor (GABA B R) antagonists are known to have antidepressant effects. TWIK-related potassium channel-1 (TREK-1) plays a role in GABA B R signaling. However, the role of TREK-1 in the antidepressant actions of GABA B R antagonist is still unclear. This study aimed to investigate whether TREK-1 mediates the antidepressant actions of GABA B R antagonist. To investigate this hypothesis, chronic unpredictable stress rats were treated with a GABA B R antagonist, GABA B R agonist, or TREK-1 blocker. Depression-like behavior was assessed by open eld tests and sucrose preference tests. GABA B R and TREK-1 protein levels were measured by western blotting. The results demonstrated that the GABA B R antagonist alleviated depression-like behavior and reversed the decrease in hippocampal TREK-1 protein expression that characterizes chronic unpredictable stress rats. Conversely, the GABA B R agonist exacerbated depression-like behavior and further decreased hippocampal TREK-1 protein expression. In addition, the TREK-1 blocker alleviated the depression-like behavior of chronic unpredictable stress rats and increased hippocampal TREK-1 protein expression. These results suggest that the alleviative effects of the GABA B receptor antagonist on depression-like behavior in chronic unpredictable stress rats are at least partially mediated through TREK-1. These novel ndings will be helpful for the clinical therapy of depression.

, soluble N-ethyl maleimide-sensitive factor attachment protein receptor (SNARE) signaling pathway (Gassmann et al. 2012), G-protein coupled inwardly rectifying K + channel (GIRK) signaling pathway (Bettler et al. 2004;Misgeld et al. 1995), and extra-cellular signal-regulated protein kinase 1 and 2 (ERK1/2) signaling pathway (Tu et al. 2007 (Deng et al. 2009). TREK-1 and TREK-2 are two-pore-domain potassium (K 2 P) channel proteins, and they share 78% homology in their genomic sequences (Lesage et al. 2000). Furthermore, Sandoz et al. revealed that TREK-1 was an additional target of GABA B R in the hippocampus by using a photoswitchable conditional subunit (Sandoz et al. 2012). Based on the above studies, we speculated that TREK-1 mediates the alleviative effects of GABA B receptor antagonism on depression-like behavior in chronic unpredictable stress rats. To investigate this hypothesis, the GABA B R and TREK-1 proteins were measured by western blotting, and depression-like behavior was assessed by open eld tests (OFT) and sucrose preference tests (SPT).

Grouping and drug administration
Three-month-old adult male Sprague-Dawley (SD) rats (250 ± 50 g, n=59) were obtained from Dalian Medical University. This work was permitted by the Animal Experiments Ethical Committee of Dalian Medical University (AEE19087). The rats were placed in rearing cages (50 cm × 30 cm × 20 cm). The temperature of the rearing environment was 21 ± 1°C, the humidity was 40%-70%, and the rearing environment was dry, clean, and ventilated. The rats were exposed to a 12 h/12 h constant light/dark cycle. The rats were provided normal food and water (n=4 rats/cage). All the rats were adapted to the rearing environment for a week. The experimental rats were divided into nine groups (n=6-8 rats/group). The groups included the control group (C), three drug treatment only groups (C+Baclofen, C+CGP55845, and C+Spadin), the CUS group, and three drug intervention groups (CUS+Baclofen, CUS+CGP55845, CUS+Spadin, and CUS+Citalopram). The chronic unpredictable stress treatments were performed according to a previously described modi ed protocol eld tests and sugar preference tests were used to test the depression-like behavior. The rats without chronic unpredictable stress treatments were tested at the same time. In addition, The drug administration was performed for 4 weeks, during which the chronic stress stimulation was carried out. The route of administration was intraperitoneal injection. The rats without drug treatments were given to the corresponding volume of physiological saline solution (vehicle). The dosages of the intervention drugs were based on the references and pilot study. The dosage of the GABA B R agonist (baclofen, Target Mol Company) was 3 mg/kg/d, the dosage of the GABA B R antagonist (CGP55845, APExBIO Company, USA) was 0.1 mg/kg/d, the dosage of the TREK-1 blocker (spadin, APExBIO Company, USA) was 0.1 mg/kg/d, and the dosage of the positive control drug, a 5-hydroxytryptamine (5-HT) reuptake inhibitor (citalopram, MedChem Express, USA), was 5 mg/kg/d.

Open eld test (OFT)
A SuperMaze V2.0 animal behavior video analysis system (Shanghai Xinruan Technology Co., Ltd.) was used to evaluate the behavior of the rats in the open eld test. The autonomous behavior and exploratory behavior of the rats in the novel environment were recorded and analyzed. The rat was placed in the center of the box (50 cm × 50 cm × 40 cm), and video was recorded for 5 min. The inner wall of the box was black, and the bottom surface was divided into a lattice containing 25 squares (10 cm × 10 cm per square) for analysis. The light was approximately 40 lx. The background noise of the laboratory was below 65 dB. The total time spent and the total distance moved at the central region of the open eld were analyzed for each rat. To avoid the in uence of odor, 75% alcohol was used to clean the cages after each test was performed.

Sucrose preference test (SPT)
The sucrose preference test is the most commonly used assessment method for the symptoms of

Western blotting
Hippocampal samples were isolated according to The Brain Atlas by using a brain mold. The hippocampal protein was extracted according to the instructions of the Ketyl Total Protein Extraction Kit (Nanjing Kaiji Biotechnology Development Co., Ltd). The target protein concentration was evaluated by using the BCA Protein Content Detection Kit (Cat: KGPBCA, Nanjing Kaiji Biotechnology Development Co., Ltd.). Denatured protein (30 μg/well) was loaded into a 7.5% sodium dodecyl sulfate gel and measured by polyacrylamide gel electrophoresis. Afterwards, the target proteins were transferred onto polyvinyl di uoride (PVDF) membranes. The PVDF membranes were incubated with 5% skim milk for 2 h at 37°C. After that, the PVDF membranes were immunoblotted with the following antibodies: GABA B R-Ab (primary antibody, 1:5000, Abcam Co., Ltd.), TREK-1-Ab (primary antibody, 1:1000, BioVision), and GAPDH-Ab (primary antibody, 1:1000, ImmunoWay Company). Tris-buffered saline containing Tween 20 (TBST) was used to wash the membranes (10 min × 3); afterward, the membranes were placed in an incubation box with horseradish peroxidase (HRP)-labeled secondary antibody (anti-goat IgG/HRP, 1:5000, ZSJQ-BIO Company, Beijing, China; anti-mouse, IgG/HR, 1:5000, ZSJQ-BIO Company, Beijing, China) at room temperature for 2 h. Subsequently, the membranes were washed in TBST (10 min × 3), and Bio-Rad analysis software (Hercules, CA, USA) was used to measure the signal intensities of the protein bands. TREK-1 and GABA B R protein expression was normalized to GAPDH (n=5/group).

Statistics
In the present study, SPSS 23.0 (Aramonk, NY, USA) was used. All data are presented as the means ± standard deviation. One-way ANOVA was used for comparison of samples among multiple groups.
Differences between two groups were analysed using Student's t-tests. To evaluate the possible interaction effect of chronic unpredictable stress and drug treatments on depressive-like behavior and GABA B R and TREK-1 protein expression among rats, two-way ANOVA was used. p < 0.05 was regarded as statistically signi cant. The double-blind method was used in this study.

Both the GABA B R antagonist and the TREK-1 blocker alleviate the depression-like behavior of chronic unpredictable stress (CUS) rats
In the open eld test (Fig.1 A), the CUS rats showed decreased exploratory behavior. Compared with the control rats, the CUS rats exhibited decreased time spent in the center area. In addition, the GABA B R antagonist, TREK-1 blocker, and positive control (citalopram) treatments alleviated the decreased exploratory behavior of the CUS rats. The above results demonstrated that the autonomous behavior and exploratory behavior of the rats were weakened in a new environment under unpredictable chronic stress. The TREK-1 blocker, GABA B R antagonist, and 5-hydroxytryptamine reuptake inhibitor (positive control) treatments alleviated the above abnormal behaviors, while treatment with the GABA B R agonist exacerbated the above abnormal behaviors.
In the sucrose preference test (Fig.1 B), compared with that in the control group, the ratio of sucrose solution consumption was signi cantly decreased in the CUS group, and there were no signi cant differences between the control group and the drug treatment only groups. Compared with that in the CUS group, the ratio of sucrose solution consumption was signi cantly increased in the CUS+CGP55845 group, CUS+Spadin group, and CUS+Citalopram group, and the ratio of sucrose solution consumption decreased signi cantly in the CUS+Baclofen group. These results indicated that the demand for sucrose solution was signi cantly decreased in the CUS rats. The TREK-1 blocker, GABA B R antagonist, and positive control treatments increased the demand for sucrose solution in the CUS rats, while treatment with the GABA B R agonist reduced the consumption of sucrose solution in the CUS rats.
3.2 Both the GABA B R antagonist and the TREK-1 blocker alleviate the low TREK-1 protein expression in the hippocampus of chronic unpredictable stress (CUS) rats In the western blotting, hippocampal TREK-1 protein expression decreased signi cantly in the CUS group compared with the control group. The spadin, CGP55845, and citalopram treatments all alleviated the above decrease in hippocampal TREK-1 protein expression in the CUS rats. However, baclofen exacerbated the decreased TREK-1 protein expression in the CUS rats. In addition, hippocampal GABA B R protein expression also decreased signi cantly in the CUS group compared with the control group. Only CGP55845 alleviated the above decrease in hippocampal GABA B R protein expression in the CUS rats,   (Evenseth et al. 2020). It has been found that the antidepressant effect of CGP56433A can be eliminated via depletion of serotonin by the tryptophan hydroxylase inhibitor chlorophenylalanine (PCPA). It has been suggested that GABA B R antagonists may play an antidepressant role by interacting with the serotonin system (Slattery et al. 2005). Consistent with the above studies, the results of this study demonstrate that the GABA B R antagonist alleviated depression-like behaviors in the CUS rats, whereas the GABA B R agonist exacerbated depression-like behaviors in the CUS rats. The ndings of this study and the above published reports provide evidence to support that GABA B Rs are key regulatory molecular targets for depression-like behavior. The ndings of this study and the above published reports provide evidence indicating that GABA B Rs should be considered the key molecular targets for regulating depression-like behavior.

TREK-1and depression
TWIK-related potassium channel-1 (TREK-1) is a potential therapeutic target for depression (Gordon and Hen 2006;Heurteaux et al. 2006;Honoré 2007). Blocking TREK-1 and inhibiting the function of TREK-1 can have antidepressant effects (Djillani et al. 2019;Ye et al. 2015). TREK-1 knockout mice display a depression-resistant phenotype and show an increase in serotonin (5-HT) neurotransmission in the dorsal raphe nucleus (DRN) (Heurteaux et al. 2006). In agreement with the published ndings, the results of this study demonstrate that the TREK-1 blocker alleviated depression-like behaviors in the CUS rats. The ndings of this study and the above published reports con rm that TREK-1 is a molecular target for antidepressant action. ; conversely, long-term use of receptor agonists will reduce the number of receptors, resulting in downregulation of receptors (Böhm et al. 1997). Moreover, the present results show that TREK-1 blockers can increase the expression of TREK-1 because long-term application of channel blockers will increase the number of channels and, through a negative feedback mechanism, increase the expression of channel proteins.
More importantly, the present results show that the GABA B R antagonist and the TREK-1 blocker had the same antidepressant effect. In CUS rats, the GABA B R antagonist increased TREK-1 expression, the GABA B R agonist decreased TREK-1 expression, and the TREK-1 blocker had no effect on GABA B R expression.
These results suggest that the GABA B R may affect the expression of TREK-1 through downstream signaling molecules, but TREK-1 cannot impact GABA B R protein expression (Fig. 3). These results suggest that the antidepressant effect of the GABA B R is mainly mediated by TREK-1. TREK-1 affects the resting membrane potential by producing background leak-type potassium currents, thus improving the generation of bioelectricity in hippocampal neurons after stress stimulation and restoring their functions. Maintaining appropriate TREK-1 expression in the hippocampus may be the key cause of the antidepressant activity of GABA B R antagonists. Therefore, this study proves not only that TREK-1 is involved in antidepressant activity as a downstream target of GABA B R but also that GABA B R antagonists could be applied to TREK-1 channel-related diseases.
The limitation is that the study does not provide evidence that the GABA B R antagonist is ineffective in alleviating depression-like behavior in the absence of TREK-1 function or that the impact of the GABA B R agonist on depression-like behavior can be suppressed by decreased TREK-1 function. In addition, other highly relevant brain regions, including the amygdala and frontal cortex, also need to be studied. We will continue to study the unclear mechanisms described above.

Conclusion
The novel ndings in this study demonstrate that TREK-1 mediates the alleviative effects of GABA B receptor antagonists on depression-like behavior in chronic unpredictable stress rats.