The Role Of Periaqueductal Gray Astrocytes In Acute Stress-induced Anxiety-like Behavior

With the rapid development of social economy and the continuous increase of competitive pressure,anxiety has aroused widespread concern in the society.As an unpleasant physiological reaction and emotional experience,moderate anxiety has a positive effect on the body,whereas long-term excessive anxiety may damage healthy,even producing anxiety disorder.Several brain areas are involved in anxiety regulation including prefrontal lobe,amygdala,hippocampus,locus coeruleus,bed nucleus of stria terminalis,hypothalamus,ventral tegmental area of midbrain,parabrachial nucleus,raphe nucleus,periaqueductal gray of midbrain,etc.Among them,periaqueductal gray plays an important role in the regulation of anxiety.In the aspect of anxiety regulation,it not only has direct pathway connections with amygdala,prefrontal lobe,hypothalamus and raphe nucleus,but also has indirect pathway connections with bed nucleus of stria terminalis,locus coeruleus,parabrachial nucleus,ventral tegmental area of midbrain and other brain areas to regulate anxiety.The role of periaqueductal gray in the regulation of anxiety has been reported widely.For example,in animal models,electrical or chemical stimulation of the ventrolateral area of periaqueductal gray(VLPAG)can cause inhibition of fight escape response and low reactive immobility;injection of galanin into the dorsal medial dorsal lateral area of periaqueductal gray can inhibit the acquisition of anxiety-like behavior and destroy acquired anxiety;injection of galanin into the ventrolateral area of periaqueductal gray can inhibit the acquisition of anxiety-like behavior and destroy acquired anxiety.VLPAG injection of angiotensin can induce anxiety-like behavior in rats.These evidences suggest that the periaqueductal gray plays an important role in the regulation of anxiety.5-HT system is closely related to stress reaction,anxiety and depression.A large number of experimental animal studies have shown that a variety of psychological or physical stress stimuli(such as exposure to natural enemies,foot shock,restraint stress,conditioned fear,forced swimming,etc.)can induce anxiety-like behavior and increase the level or turnover of 5-HT and its metabolite 5-HIAA in brain regions related to emotion regulation,while activating or blocking the corresponding 5-HT receptor has the anxiogenic or anxiolytic effect.PAG is one of the main origins of 5-HTergic neurons in the brain.Studies have shown that there is a close relationship between the 5-HT system in PAG and anxiety.For example,injecting 5-HT into PAG or blocking the function of presynaptic 5-HT receptors to feedback inhibit the activity of 5-HTergic neurons can alleviate the anxiety-like behavior produced by stimulating dorsal PAG;on the contrary,enhancing the activity of PAG 5-HTergic neurons can induce anxiety.However,the way and mechanism of regulating anxiety behavior are still unclear.In addition,the occurrence of anxiety-like behavior may be related to the functional changes of astrocytes in PAG.As one of the main cell components of the central nervous system,astrocytes not only have the function of support and nourish neurons,but also have complex and diverse regulatory effects on brain functions such as emotional response,learning and memory by releasing a variety of glial cytokines.On the one hand,astrocytes can form "triple synapses" with neurons to participate in synaptic transmission;on the other hand,astrocytes can regulate the excitability of neurons by releasing glial factors.These characteristics of astrocytes provide a structural and functional basis for astrocytes to induce anxiety-like behavior by regulating the activity of 5-HTergic neurons in PAG.Although there is evidence that astrocytes are involved in the development of chronic stress-induced mental disorders through the release of inflammatory factors,whether astrocytes play a rapid regulatory role in anxiety like-behavior induced by acute stress is still lack of experimental evidence.Based on this,we propose that acute stress can activate PAG astrocytes,and activated astrocytes can enhance the activity of 5-HT neurons in VLPAG by regulating their synaptic input and excitability,therefore resulting in anxiety-like behavior.In order to verify the hypothesis,we used acute stress stimulation of foot shock to induce anxiety-like behavior in mice.Whole cell patch clamp recording was performed in isolated brain slices to analyze the changes of synaptic input and excitability of VLPAG 5-HT neurons during anxiety attack.The effects of chemical genetic method on anxietylike behavior and VLPAG astrocyte activity were observed.The results are as follows:1.Acute stress can induce anxiety-like behavior and increase the excitability of 5HTergic neurons in VLPAGAcute stress could significantly reduce the residence time in the center of OFT,the residence time in the LDT,the times of entries in open arms and the residence time in the open arm of EPM.Acute stress significantly increased the frequency and amplitude of mEPSC,the total area of SIC,and the firing numbers stimulated by step injecting current of 5-HTergic neurons in VLPAG.These results suggest that acute stress could induce anxiety-like behaviors and enhance the excitability of 5-HTergic neurons in VLPAG.2.Inhibition of VLPAG astrocytes can rescue anxiety-like behavior induced by acute stress in mice,and reverse the changes of synaptic transmission and excitability of 5-HT neurons in VLPAG after acute stressInhibition of VLPAG astrocytes significantly inhibited the decrease of the residence time in the white box,the decrease of entries in the open arm caused by acute stress.Inhibition of astrocytes could significantly reduce the increase of the mEPSC frequency,the total area of SIC production and the firing numbers of 5-HT neurons caused by acute stress.The results imply that inhibition of astrocytes in VLPAG could inhibit the anxietylike behavior of mice and inhibit the increase of excitability of 5-HT neurons in VLPAG induced by acute stress.3.Activation of astrocytes in VLPAG can induce anxiety-like behavior and increase the excitability of 5-HT neurons in VLPAGActivation of VLPAG astrocytes significantly reduced the residence time in the central area of OFT,the residence time in the white box of LDT,and the numbers of entries in the open arm of EPM.Activation of astrocytes significantly increased the frequency of mEPSC,the total area of spontaneous SICs,and the firing numbers stimulated by step injecting current of 5-HT neurons in VLPAG.The results show that activation of astrocytes in VLPAG could induce anxiety-like behavior and increase the excitability of 5-HT neurons in VLPAG.Taken together,the following conclusions are drawn:The generation of anxiety-like behavior induced by acute stress in mice is related to the activation of astrocytes in VLPAG.The pathway is to enhance the excitatory synaptic input and excitatory level of 5-HTergic neurons,so as to enhance the activity of 5-HTergic neurons.Different from the previous studies on the chronic regulation of astrocytes,this study preliminarily revealed the pathway of astrocytes influencing behavior by rapidly regulating the activities of related neurons,which provides a new idea for the study of the biological mechanism of anxiety.