In this study, we found a decrease in the number of PNNs and percentage of PNN+ PV+ cells among PV+ cells in the PrL and IL of the mPFC in four weeks of CUMS-exposed rats. CUMS also caused decreased levels of PNNs components aggrecan, brevican, GAD67, GLuA1, and PSD95 in the PrL and IL of the mPFC. Electroacupuncture and fluoxetine reversed the depression-like behavior that was induced by CUMS and the decrease in the number of PNNs and percentage of PNN+ PV+ cells among PV+ cells and normalized the levels of aggrecan, brevican, GAD67, GLuA1, and PSD95 in the PrL and IL of the mPFC. In summary, these results suggest that PNNs in the mPFC are important contributors of depression etiology and the antidepressant-like effects of EA.
EA has been widely applied to treat depression for decades, usually using acupuncture points on the head [30, 31]. Traditional Chinese medicine considers two acupoints, Baihui (GV20) and Yintang (GV29), to govern the meridian and correlate directly with the brain via multiple collaterals and channels [28, 32]. Acupuncture on Baihui and Yintang can clear the mind, lift the spirits, regulate the Du meridian, and calm the mind to promote good mood and peaceful sleep [33, 34]. And research shows that electroacupuncture treatment every other day for three weeks can improve depression symptoms[17]. Therefore, we chose these two acupoints for stimulation with EA instruments in this study. On the other hand, fluoxetine (FLX) is a selective serotonin reuptake inhibitor (SSRI) used widely for depression treatment, and also can alleviate depressive behaviors in animal studies[35, 36]. We found that electroacupuncture and fluoxetine could reverse the depression- and anxiety-like behaviors induced by CUMS. We also found no significant difference between the EA and FLX groups, suggesting that electrotherapy had similar effects to fluoxetine.
The mPFC is involved in the pathogenesis of various mental illnesses, and its volume reduction is one of the most documented abnormalities in major depression [37, 38]. The mPFC is the main area responsible for executive function in the cortex. The dysfunction of the mPFC is related to the cognitive and emotional defects caused by stress, and the neuronal atrophy and synaptic structural and functional changes of the mPFC are closely associated to depression pathogenesis [39, 40]. In rodents, it was observed that mPFC-injured rats were more susceptible to stress than normal rats and developed depression- and anxiety-like behaviors, which increased the activation of brain regions involved in neuroendocrine and autonomic response [41]. PNNs mainly surround γ-aminobutyric acid (GABA) interneurons with PV-positive neurons [42]. Van De Werd found that the expression level of parvalbumin (PV) in the mPFC was not significantly different between the PrL and IL [43], but the distribution difference of PNNs in these two sub-regions of the brain has not been studied and compared.
According to the current relevant research, ECM level changes in the brain are related to the occurrence of depression [44]. As a special component of the ECM, PNNs are widely present in the central nervous system and important participants of the signal transmission between neurons and glial cells. The functions of PNNs are abnormal in many neuropsychiatric diseases, such as epilepsy, schizophrenia, and bipolar disorder, and have become the focus of depression research in recent years [45, 46]. These behavioral abnormalities caused by psychiatric diseases may be improved when the changes in the number of PNNs are reversed [47–49]. Generally speaking, PNNs include chondroitin sulfate proteoglycans (CSPGs), hyaluronan, tenascin-R, and link proteins [50]. Among them, CSPGs are mainly composed of aggrecan, brevican, versican, and neurocan [51, 52]. Human autopsy studies have shown that in schizophrenia and bipolar disorder, the neurons expressed by aggrecan and PV are significantly abnormal [53]. Studies have shown that chronic stress in young age or adulthood can change PNN density in the mPFC brain area and affect the plasticity and structure of inhibitory neurons, especially PV-expressing interneurons [54, 55]. Besides, long-term antidepressant therapeutic agents, such as venlafaxine and fluoxetine, could also change the PV and PNN densities in adult cerebral cortex and hippocampus [56, 57]. Some showed that FLX can decrease PNNs formation during critical period which is the time of onset and close of PNNs formation[58]. There are also studies which found that PNNs are required for the antidepressant effect of ketamine, which is a rapid antidepressant drug[59, 60]. In this experiment, we found that electroacupuncture and fluoxetine treatments could reverse the drop in the number of PNNs and the percentage of PNN+ PV+/PV+ the mPFC after CUMS. Additionally, electroacupuncture treatment could reverse the decrease in the protein expression of PNNs components aggrecan and brevican in the mPFC caused by stress. Previous studies indicated that fluoxetine administration would make the inhibitory neurons in the adult cerebral cortex undergo structural changes, which probably occurs by alternating the plasticity-related molecules in neurons or adjacent ECM, and these molecules present in interneurons are crucial for the development of plasticity in juvenile brains [61]. The results in EA and FLX groups in our study were similar, indicating that both treatments had a similar outcome.
The PNN is a complex of extracellular matrix molecules that mostly surrounds GABAergic neurons in various brain regions, participates in synaptic plasticity, and protects and buffers against external oxidative stress [62, 63]. Reduced expression of GABA synthetase has been observed in the brain tissue of suicidal patients in previous studies; the main GABA synthetase, glutamic acid decarboxylase 67 (GAD67), is specifically expressed in GABAergic neurons [64]. Decreased expression levels of GABA and GAD67 can be observed in the brains of depression patients and animals under long-term stress [38]. The decrease in PNN levels may make GABA neurons more sensitive to stress and other stimuli, resulting in abnormal neuronal function. Reduced levels of PNNs and disruption of GABAergic circuits have also been found in psychiatric disorders such as schizophrenia [65]. Our experimental results show that the reduction of PNNs in rats with depression induced by CUMS may lead to the functional damage of GABAergic neurons. Electroacupuncture at Baihui and Yintang acupoints in the depression rats increased the expression level of PNNs, which could protect GABA neurons from external stress stimulation.
During brain development, PNNs appear when the critical period for developmental plasticity ends and affect the onset and closure of that period. Recent work has revealed the crucial role of PNNs in controlling CNS plasticity [63, 66]. Abnormalities in the structure and composition of PNNs may lead to changes in synaptic plasticity and neuronal function [67]. Destroying PNNs or degrading ECM can reduce the glutamatergic input of GABAergic neurons and reduce the excitability of neurons [68, 69]. GLuA1 is a subunit of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor, which exists in the postsynaptic membrane of excitatory synapses and participates in the regulation of synaptic plasticity. Its abnormal expression is a common mechanism of mental illnesses such as depression, schizophrenia, and chronic drug addiction [70]. PSD95 is the main scaffold protein located in the excitatory postsynaptic synapse density [71] and an important participant of synaptic plasticity, glutamatergic transmission, and dendritic spine morphogenesis during neural development [72]. The protein expression levels of GLuA1 and PSD95 in the PrL and IL were increased after electroacupuncture treatment. Thus, electroacupuncture at Baihui and Yintang can exert an antidepressant effect by affecting the expression level of excitatory synaptic proteins on neurons.
In summary, the present study shows that the mechanism of electroacupuncture’s anti-depression effect may work through reversing the stress-induced decline in PNN expression, the functional impairment of GABA neurons, and the regulation of excitatory synaptic expression. The results of this study provide a new theoretical basis for the study of the antidepressant mechanism of acupuncture.