The Regulatory Mechanism of Central Complement C3a Receptor on LPS-Induced Depressive-Like Behavior

Introduction

With the increasing number of depression patients globally, major depressive disorder (MDD) has become one of the leading causes of disability worldwide. The symptoms, treatment effects, and biological correlations of depression exhibit immense heterogeneity, reflecting the complex pathological process of this disorder. In recent years, mounting evidence has shown that the immune system plays a pivotal role in the pathogenesis of depression. Many immune-related genes are considered risk factors for depression, and patients with depression exhibit immune dysfunction. Moreover, certain anti-inflammatory treatments have shown antidepressant effects, and immune-based therapeutic approaches have been proposed as precision medicine for specific groups of depression patients. However, the mechanism by which immune factors promote depression remains unclear.

The prefrontal cortex (PFC), particularly the medial prefrontal cortex (mPFC) in rodents, has been proven to be one of the reasons for susceptibility to depression due to its functional and structural abnormalities. Although studies have shown dysregulation of immune cell activity in the PFC, such as microglial activation or synaptic pruning, directly regulating PFC neuronal activity through immune pathways has been limited.

The complement system is crucial in innate immunity, and its dysregulation is associated with various neurological and psychiatric disorders, including depression. Studies have shown that the complement C3a receptor (C3AR) regulates monocyte infiltration and microglial activity in chronic stress mouse models. However, how C3AR activity affects PFC neurons in depression models remains unclear.

This study aims to investigate the role of C3AR in the mPFC in systemic lipopolysaccharides (LPS)-induced depressive-like behavior and study its effect on the excitability of mPFC neurons.

Source of the Study

The article was published in the 2024 issue 236 of the journal “Progress in Neurobiology” and was released online on April 17, 2024. The study was a collaboration of researchers from several departments of Zhejiang University, including the Department of Neurology and Psychiatry from the Second Affiliated Hospital and the System Medicine Research Center from the School of Basic Medicine. Corresponding author: Lien Hong.

Study Design and Procedures

Animal Model and Experimental Procedures

All animal experiments followed the guidelines for the care and use of laboratory animals of Zhejiang University and received approval from the Animal Experiment Committee of Zhejiang University. The C3AR knockout (KO) mice used in the study were provided by Dr. Zhou Hong from Nanjing Medical University and were bred with C57BL/6J mice for more than ten generations. Mice were housed in a standard 12-hour light/dark cycle (lights on at 7 AM and off at 7 PM), at a temperature of 22±1℃ and humidity of 55%±5%, with free access to food and water. Behavioral tests were conducted on male mice aged 2-4 months during the light cycle.

Viruses and Cell Culture

The study used a series of AAV viruses to perform extensive gene expression and silencing experiments in specific brain regions and specifically manipulated the expression of C3aR in PFC neurons. Additionally, primary cell culture and electrophysiology experiments played a significant role in the research process. By treating and analyzing primary cultured neurons, astrocytes, and microglial cells, researchers further explored the impact of LPS and C3a on C3aR expression.

Behavioral Testing

Behavioral tests on mice included the Open Field Test (OFT), Elevated Plus Maze Test (EPM), Tail Suspension Test (TST), Forced Swim Test (FST), and Sucrose Preference Test (SPT). These tests helped evaluate the anxiety and depression-like behavior exhibited by mice after LPS treatment.

Data Analysis

Experimental data were analyzed using IBM’s SPSS statistical software or GraphPad Prism 6, and data were presented as mean ± standard deviation. Significance thresholds were set at *p < 0.05, **p < 0.01, ***p < 0.001.

Results

C3AR Knockout Mice Showed Resistance to LPS-Induced Depressive-Like Behavior

Through ELISA and immunoblot analysis, the study showed that LPS-treated WT mice had a significant increase in the expression of C3 and C3aR in the mPFC, whereas C3AR knockout mice exhibited resistance to LPS-induced anxiety and depressive-like behavior. Compared with LPS-treated WT mice, LPS-treated KO mice showed no significant behavioral abnormalities in the OFT, EPM, TST, and FST, indicating that the absence of C3aR effectively suppressed LPS-induced depressive-like behavior.

C3AR in the mPFC Regulates LPS-Induced Depressive-Like Behavior

To further verify the regulatory role of C3AR in the mPFC on LPS-induced behavioral changes, researchers performed experiments by bilaterally implanting cannulae in the mPFC of WT mice and administering a C3aR antagonist (C3ARA) via intracerebral infusion. The results showed that compared with the control group infused with artificial cerebrospinal fluid, LPS-treated WT mice displayed significantly reduced motor impairment in the tail suspension and forced swim tests after C3ARA infusion, indicating that C3AR in the mPFC might play an important role in LPS-induced depressive-like behavior.

C3AR Regulates mPFC Neuronal Excitability and Bidirectionally Modulates LPS-Induced Depressive-Like Behavior

Through electrophysiological recordings of primary cortical neurons and acute brain slices from WT and KO mice, the study found that neurons lacking C3AR had significantly higher action potential numbers with the same depolarizing current injection than WT neurons, indicating higher excitability in KO neurons. Additionally, C3AR activity could bidirectionally regulate neuronal excitability: C3a treatment led to a significant decrease in action potential frequency in WT neurons, whereas LPS treatment increased action potential frequency in KO neurons. This suggests that C3AR deletion not only enhanced baseline excitability but further increased mPFC neuronal excitability after LPS treatment.

C3AR KO Mice Treated with LPS Showed Resistance to Inhibition-Induced Depressive-Like Behavior of mPFC Glutamatergic Neurons

Further exploring the specific functioning cells of C3AR in the mPFC, researchers used chemogenetic tools to specifically inhibit mPFC glutamatergic neurons in C3AR KO mice, finding that LPS-treated KO mice still exhibited normal behavior.

Conclusion

The study revealed key regulatory mechanisms of C3AR in LPS-induced depressive-like behavior and proposed an important pathway for achieving antidepressant effects by regulating the excitability of mPFC glutamatergic neurons. This not only provides a new understanding of the pathogenesis of MDD but also lays the foundation for future development of therapeutic strategies targeting C3AR.

Significance of the Study

By uncovering new mechanisms of the complement system in depression, this study provides a new theoretical basis for developing interventions for MDD in the future. C3AR directly influences depressive-like behavior by regulating the excitability of mPFC neurons, showing potential as a therapeutic target. Future research can further investigate the role of C3AR in different genders and other brain regions and its feasibility in clinical interventions.

This study opens new directions for the etiological research of depression, showing that regulation of the complement system and neuronal activity might bring effective treatment methods, particularly precision treatment for specific patient groups.