The Role And Mecahnism Of Estrogen Receptor GPR30 In Anxiety

BackgroundAnxiety is the most prevalent mental diorsder, which brings great burden to individuals and society. According to large population-based surveys from Europe, up to 33.7% of the population are affected by anxiety disorders during their lifetime; and the lifetime prevalence of anxiety disorders in the United States is about 30%.The pathogenesis of anxiety disorder is complicated and still not fully understood. The brain limbic system play a crucial role in mood regulation, and the abnormality of neuronal networks is closely related to anxiety disorders. Glutamate and GABA are two main neurotransmitters in central nervous system(CNS), which mediate excitatory and inhibitory neurotransmission and creat the opposite forces. They account for vast majority of all neurotransmission in the CNS. The banlance of excitatory/inhibitory(E/I) transmission is crucial to maintain the neuronal homeostasis, and the imbanlance of E/I is critically involved in anxiety disorders.Estrogen, the most important sex hormone of the female, also plays key roles in the CNS in addition to maintaining the female reproductive function. The prevalence of mood disorders in women increase significantly after menopause, which is related with decreased estrogen levels. Clinically, estrogen replacement therapy(ERT) is used to improve the mood and cognition of the menopausal women. The physiological functions of estrogen are mainly mediated by its classical nuclear receptor α(ERα) and β(ERβ), which activate the genomic pathways. ERα is mainly associated with the repoductive function and ERβ is mainly associated with the non-reproductive function of estrogen. In the CNS, ERβ is critically involved in estrogen-mediated neural protection, synaptic plasticity, neurogenesis and mood modualtion.The G-protein coupled receptor 30(GPR30), also called G-protein coupled estrogen recptor(GPER), is a new estrogen receptor discovered in recent years. GPR30 is localized in the membrane sturcture of the cell and mediates the fast non-genomic effects of estrogen, which is independent of the nuclear transcription and causes cell response within a very short time through activating intracellular kinases. GPR30 is distributed widely in the reproductive system, cardiovascular system, immune system and neuronal system. But the role of GPR30 in mood regulation is still not clear. In the CNS, GPR30 is expressed in multiple brain regions related to mood regualtion, including hippocampus, amygdala and cerebral cortex. The tissue distribution suggests that GPR30 may be involved in the regulation of emotion. AimsTo investigate the effects of GPR30 in basolateral amygdala(BLA) on anxiety-like behaviors induced by stress or chronic pain and to reveal the possible mechanisms. To evaluate the possibility of GPR30 as target for anti-anxiety therapy and provide theoretical basis for the development of anxiolytic drugs. Methods 1. The detection of GPR30 expression in amygdalaThe mice were perfused by 4% paraformaldehyde through the heart under anesthesia, the whole brain was removed and postfixed in 4% paraformaldehyde for 6 h at 4 oC and then transferred to sucrose for dehydration. Coronal brain slices(25μm) containing the BLA were cut on a cryostat and immediately mounted on the slides. Slices were washed with PBS and blocked with 10% goat serum, then incubated overnight with primary rabbit anti- GPR30 at 4 oC. Slice were washed and incubated with Cy3-conjuated secondary antibody for 2 h at room temperature. Slides were then coverslipped with glycerine, photographed and analyzed under confocal microscope. 2. The surgery of ovariectomy(OVX) in miceThe mice were anesthetized by pentobarbital sodium and fixed on the operating platform with face down. The incision was made at the suitable site of its back, the bilateral ovary was exposed and then ablated. The wound was sterilized and sutured. 3. The surgery of amygdala cannulation in OVX miceThe mice were anesthetized and fixed on the platform of stereotaxic frame. The skull of mice was exposed and the localization of basal amygdala was decided according to its relative position with Bregma. Two stainless steel cannulas(guide cannula, length 8 mm, internal diameter 0.32 mm, external diameter 0.48 mm) were bilaterally aimed at 0.5 mm above intended sites of BLA. The guide cannulas were affixed to the skull with machine screws and dental cement, and stylets were inserted into the cannula to keep them patent. 4. The mouse model of stress-induced anxiety1) Restraint stress: OVX mice were restrained in well-ventilated perspex restraining tubes for 2 h without physical compression or pain. The mice were exposed to a single 2 h restraint or a 2 h restraint once daily for two consecutive days. 2) Forced swimming(FS) stress: the mice of the FS stress group were forced to swim in a tank filled with water with suitable depth and temperature. The mice were exposed to a single 30 min swimming or a 30 min swimming once daily for two consecutive days. 5. The mouse model of chronic pain-induced anxiety1) Chronic inflammatory pain-induced anxiety model: the mice were injected with a single dose of complete Freund’s adjuvant(CFA)(10 μL) into plantar of right hindpaw. Pain threshold was detected and other experiments were performed two weeks later.2) Chronic neuropathic pain-induced anxiety model: chronic constriction injury of sciatic nerve(CCI) was induced by four ligatures(chromic gut) tied around the sciatic nerve. The related experiments were performed three weeks later. 6. Amygdala microinjectionThe mice were anesthetized with isoflurane in oxygen with a delivery rate of 0.5 L/min. Drugs were bilaterally delivered into BLA via a syringe driven by an infusion pump and the velocity of the pump was set at 0.5μL/min. Drugs were injected over a 1 min period with the injector left in place for another 1 min to permit sufficient diffusion. 7. Determining the microinjection sites with crystal violet stainingCoronal brain slices containing the cannula trace were collected on a cryostat and immediately mounted on the slides. Slices were washed with PBS and stained with 0.1% crystal violet for 30 min at room temperature. Slides were then coverslipped with glycerine, photographed under a light microscope equipped with a CCD camera. 8. The test of pain threshold in mice with chronic painIn chronic pain-induced anxiety model, thermal hyperalgesia and mechanical allodynia were measured to examine the effect of the drugs on pain threshold. Thermal hyperalgesia was assessed by measuring the latency of paw withdrawal in response to a radiant heat source. Mechanical allodynia was assessed with a set of von frey filaments using the up-down paradigm. 9. Anxiety-like behavior tests1) Elevated plus maze(EPM): The apparatus comprised two open arms and two closed arms that extended from a common central platform. The mouse was placed in the central area and faced to the open arms, it was allowed to explore the maze for 5 min freely, which was videoed with a camera fixed above the maze. The time spent in open arms and the entries into open arms were analyzed.2) Open field test(OF): The open field was a square area with clear Plexiglas walls and floor. The mouse were placed in the central area and allowed to explore freely for 15 min, which was videoed with an above camera. The total traveled distance and the time spent in central area were analyzed. 10. Examination of glucocorticoid(GC) levelsIn the stress-induced anxiety model, GC levels in the plasma and total homogenate of the brain were detected using the ELISA kit following the instruction. 11. The related proteins expression was detected by western-blot in both stress and chronic pain-induced anxiety models, the effects of the drugs on their expression were also examined. 12. The excitatory and inhibitory synaptic transmission were investigated by whole cell patch in both stress and chronic pain-induced anxiety models, the effects of the drugs were also examined. Results 1. GPR30 was highly expressed in basolateral amygdala, and it was expressed in both glutamatergic pyramidal neurons and the GABAergic interneurons of the BLA. Both restraint and FS stress can induce evident anxiety-like behaviors in OVX mice with concomitant up-regulation of GPR30 expression in BLA. 2. Local microinjection of GPR30 receptor agonists into BLA evidently increased the time spent in open arms and central area in EPM and OF test respectively. GPR30 receptor specific antagonist G15 blocked the effects of agonist. In unstressed OXV mice, the local infusion of GPR30 receptor specific agonist G1 had no evident effects. However, the infusion of G15 notably decreased the time spent in the open arms or in the central area in the EPM and OF tests. 3. To exclude the acute influence of the neuroendocrine system on behaviors, the levels of glucocorticoid(GC) in brain and plasma were tested immediately after the behavioral tests. The GC levels were similar to the basal levels in the brain and the plasma among the different groups. 4. Both chronic inflammatory pain(CFA) and chronic neuropathic pain(CCI) model can cause evident anxiety-like behaviors in OVX mice. Either systematic or BLA local infusion of G1 could attenuate pain-induced anxiety. G15 could block the effect of G1, and the anxiolytic effect of G1 could be blocked by BLA local infusion of GPR30 shRNA beforehand. 5. Both CFA injection and CCI surgery caused pain hypersensitivity in OVX mice. The agonist or antagonist of GPR30 had no obvious effects on the thermal and mechanical pain threshold of mice. 6. In the BLA of anxiety mice induced by stress or chronic pain, the expression of GABAA-α2 or(and) GABAA-γ2 decreased significantly and the expression of glutamate receptors(exclude NR2B) increased evidently. Activation of GPR30 reversed the above tendency, G15 or GPR30 shRNA blocked the effects of G1. 7. Brain slices recording showed decreased frequency and amplitude of spontaneous inhibitory postsynaptic currents(sIPSCs) in the anxiety mice induced by stress. Perfusion of G1 or BSA-E2 increased s IPSCs amplitude and frequency, which was inhibited by G15. Spontaneous excitatory postsynaptic currents(sEPSCs) did not change evidently among different groups. 8. The frequency and amplitude of sIPSCs decreased and the frequency and amplitude of sEPSCs increased markedly in the BLA of anxiety mice induced by chronic inflammatory pain. However, treating the anxiety mice with G1 for five days notably inhibited the changes of s IPSCs and sEPSCs. Simultaneous G15 treatment abolished the effects of G1. ConclusionOur study demonstrated that both stress and chronic pain could induce evident anxiety-like behaviors in OVX mice, which was accompanied by increased GPR30 expression in BLA. Activation of GPR30 had obvious anxiolytic effects in both models, which may be attributed to up-regulation of GABAA receptor and down-regulation of glutamate receptors, enhancement of inhibitory neurotransmission and attenuation of excitatory neurotransmission.In present study, we first clarified the role of GPR30 in the BLA as an anxiolytic target, which provided a new strategy and theoretical basis for the treatment of anxiety disorders.