Estrogen Contributes To Pain-related Aversion

Different with other somatic feelings, pain is a complex experience with a strong emotion which is as the most significant chareacteristics of pain. When experiencing pain and suffering with pain, most people feels that there is always accompanied by strong negative emotions (such as aversion, fear and so on) and the feeling usually associated with actual or potential behavior to avoidance. According to the definition of pain by the International Association for the Study of Pain (IASP), pain is an unpleasant sensory and emotional experience associated with actual or potential tissue damage, which including a sensory-discriminative and an emotional-affective component. Researches on sensory-discriminative component of pain can be traced back to the period of ancient Greece. Along with the advances in technology on animal praxeology, cognitive psychology and human functional brain imaging, studies on pain have got tremendous progress in the entire body, cell, molecular and genetic level. In recent years, the experimental researches on pain-related emotion and affection get more and more attention. However, there are a lot of difficulties to test and quantify emotional-affective component of pain, such as short of well-designed animal models and the relevant detection techniques. Furthermore, clinical observations have increasingly indicated that in chronic pain patients, pain-related negative affect (fear, anxiety, anger, depression and even a suicidal tendency) is more disabling than the pain itself. Therefore, it is important to study and reveal the mechanisms of pain-related emotion and affection. After getting fully understands on pain, we can provide effective treatment to relieve the negative affect in patients who suffer pain.Various regions in brain have different functions and roles in the generation of emotion, memory and pain. The anterior cingulate cortex is an important structure of the limbic system and is implicated in many functions related to emotional processing. Previous studies have demonstrated that the destruction of neurons originating from the rostral ACC (rACC) blocks formalin-induced conditioned place aversion (F-CPA), a pain-related aversive learning that directly reflects the affective component of pain in rats, without reducing acute pain-related behaviors. Positron emission tomography (PET) studies revealed that both noxious stimuli and pain-induced unpleasantness activated the ACC. Surgical ablation of the ACC obviously reduced painrelated unpleasantness/dysphoria without affecting patient’s ability to discriminate the intensity or localization of the noxious stimulus. These data strongly suggested that the ACC might be a pivotal brain area linked to affective processing of pain.Estrogen, a steroid hormone, is involved across an extensive spectrum of neural functions, including learning, memory, nociception and mood disorder.17β-estradiol (E2), a main form of estrogen, acts by binding and activating both intracellular estrogen receptors (ERs) and receptors associated with the plasma membrane (mERs). Despite the circulating gonadal hormones, evidence for the presence of multiple enzymes in the brain that control neurosteroid, including estrogen, biosynthesis has been described in males and females. For brain-derived estrogen with a higher concentration than the circulating hormone, the rapidly modulation of synaptic plasticity and cognitive functions may be their essential functions. Whether and how E2is involved in modulation of affective pain? As a hormone, whether the effect of E2is sex specific? Is E2brain-derived or relied on the modulation of fluctuations in circulating estrogen? Is the action of E2rapid nongenomic or classic genomic? What is the possible mechanism on the effect of E2in rACC? By methods of behavioral testing, whole-cell patch clamp recordings in rACC slices, immunohistochemistry and other techniches, the present study investigated whether and how estrogen in the rACC regulates affective pain and synaptic plasticity. The main findings were as follows:1. Endogenous estrogen contributes to the acquisition of pain-related aversion without sex specificityWhen a unilateral intraplantar (i.pl.) injection of formalin (5%,50μl) was paired with a particular compartment in the place conditioning apparatus, both female and male rats spent less time in this compartment on the post-conditioning day compared with the pre-conditioning day. To investigate the contribution of endogenous estrogen in the induction of pain-related aversion, estrogen receptors (ERs) antagonist ICI182,780(ICI) and an inhibitor of aromatase androstatrienedione (ATD) were bilaterally microinjected into the rACC5min before formalin-paired conditioning. F-CPA acquisition was completely blocked by ICI and ATD in both female and male rats without difference in F-CPA score. F-CPA was reliably induced in OVX rats with significant difference in the time spent in the formalin-paired compartment between the pre-and post-conditioning day, although the F-CPA score in OVX group was less than that in sham OVX group.2. Exogenous estrogen elicits CPA via NMDA receptorsTo test whether estrogen per se is sufficient to produce CPA, E2was directly microinjected into the bilateral rACC without an i.pl. injection of formalin. Rats spent significantly less time in the E2-paired context on the postconditioning day compared with the preconditioning day. The CPA scores of E2-induced CPA (E-CPA) is also not sex specific. A membrane-impermeable estrogen conjugate E2-BSA that has been observed to act only on membrane ERs can mimic E2-induced CPA, which strongly indicates that E2-induced aversive is predominantly mediated by membrane ERs (mERs). We microinjected selective NMDA receptor (NMDAR) antagonist APV into the rACC during E2conditioning, and found that E-CPA was completely blocked, which indicates that the NMDAR may be downstream of the ERs-mediated action.3. Estrogen phosphorylates ERK-CREB and NMDA receptors subunit NR2BBath application of E2for10min produced a robust increase in pERK and pCREB in rACC slices. E2-induced pERK and pCREB were significantly suppressed by APV. Also, intra-rACC microinjection of E2produced a robust increase in pNR2B expression. NR1and NR2B expression levels were not influenced in the rACC. These data suggest that E2may exert non-genonic effect via activating rapid signalings.4. Subcellular distribution of estrogen receptorImmunochemical data revealed a wide distribution of ERα-, ERβ-and GPR30-positive cells in the rACC. ERα-and ERβ-immunoreactive signals were detected in the nucleus, cytoplasm, membrane and dendrites, and GPR30was mainly expressed in the cytoplasm and membrane. Both ERα and ERβ colocalized with cytoskeleton marker microtubule-associated protein2(MAP-2) and βⅢ-tubulin. Real-time PCR assays showed that the mRNA of all the three ERs was expressed in the rACC without significant difference between sexes.5. Estrogen enhances the excitatory postsynaptic currents by increasing the ratio of NMDA to AMPA transmission and presynaptic glutamate releaseWhole-cell recordings were performed on visually identified pyramidal neurons in layer Ⅱ/Ⅲ of rACC slices from15-to21-day-old rats of both sexes whose E2concentrations had no detectable difference in perfusate of rACC slices. Bath application of E2(10nM,100nM and1μM) rapidly potentiated eEPSC amplitude within minutes in most of the recorded neurons and the increased frequency of EPSC potentiation with increasing concentrations of E2. We calculated a ratio by dividing the average maximum NMDA-EPSCs by the average maximum AMPA-EPSCs. E2significantly increased the NMDA/AMPA ratio dose dependently, which could be occluded by ICI, indicating that estrogen modulated rACC neuron excitatory synaptic transmission under a distinct manner of NMDAR and AMPAR. To determine presynaptic effects of E2, we computed paired-pulse ratio (PPR). E2significantly decreased the PPR within minutes. ICI completely blocked E2-induced attenuation, as represented by the PPR. This result suggests that the E2-induced potentiation of EPSC amplitude is mediated, at least in part, by increasing glutamate release probability.6. Estrogen is sufficient to induce NMDA receptor-mediated LTP via the GPCR-associated PKA or NR2B pathwayWe examined the long-term effects of E2on NMDA-EPSCs. As expected, upon a brief application of100nM E2for6min, the onset of NMDA-EPSCs was rapid, and long-lasting potentiation was seen for more than40min (termed NMDA-LTP). A bath application of membrane-impermeable estrogen conjugate E2-BSA can also directly elicit NMDA-LTP, suggesting an effect of mERs. Different from NMDA-EPSCs, AMPA-EPSCs were obviously depressed for20min by a6min application of E2, after which, they gradually recovered to baseline. G-protein inhibitor GDP-(3-S in the intracellular solution completely prevented E2-induced NMDA-LTP. E2-induced NMDA-LTP was also blocked by bathing a specific PKA inhibitor H89andNMDAR subunit NR2B antagonist Ifenprodil. We used theta-burst stimulation (TBS) to induce LTP. This paradigm is thought to be physiological. TBS produced a significant, long-lasting potentiation of synaptic responses in the rACC pyramidal neurons. The amplitude of LTP was significantly suppressed by ERs antagonist ICI. 7. Estrogen rapidly induces formation of dendritic spinesTo investigate whether estrogen could rapidly increase the connectivity of neurons, we examined assessed the dendritic spine morphology in pyramidal neurons following treatment by of100nM E2for a maximum of120min. A significant increase in both dendritic spine and dendritic filopodia density occurred at5min and lasted103min after E2. The average spine length and number were significantly increased at all time points observed.Taken together, the present study has shown that E2plays a critical role in the induction of affective pain. Formalin nociceptive stimulation induced a rapid increase in extracellular concentration of E2in the rACC and CPA acquisition. Blocking ERs by an administration of ICI182,780or inhibiting E2local synthesis by androstatrienedione completely blocked F-CPA. Furthermore, we demonstrated that E2acutely and persistently potentiated excitatory synaptic transmission in the rACC through mERs-initated G-protein-dependent rapid signalings, by increasing the ratio of NMDA-EPSCs to AMPA-EPSCs and regulating dendritic spine morphogenesis. To the best of our knowledge, this is the first example demonstrating E2plays a critical role in the induction of affective pain and nociceptor-driven brain-derived estrogen is necessary for pain-related negative affect without sex specificity.