Estrogen Enhances Synaptic Plasticity Of Superficial Dorsal Horn Neurons In Rats:Nongenomic Mechanisms

With heightened social status of female, people increasingly pay more attention to sex difference in pain perception. Estrogen is an important type of female hormone belonged to steroid. Natural estrogen includes17β-estradiol (E2), estriol and estrone. E2is the strongest crude estrogen.Numerous works showed that E2affected pain severity via an action on nervous system. But we cannot yet unequivocally classify estrogens as "pro-nociceptive" or "anti-nociceptive," and the underlying mechanisms have not been identified. In view of many kinds of pain disturbed preferrly female, to seek and find new targets for treatment become more urgent.In the past decades, discussions about pain modulation by E2mostly focused on nER (nucleus estrogen receptor, nER) mediated genomic effects reffered to as chronic action. However, few research gropes for its nongenomic effects or rapid action on pain regulation. Then, whether E2was involved in rapid modulation of pain sensation? If participated, what is the possible mechanism?Previous studies have proved that E2could rapidly regulate excitatory synaptic transmission and LTP in hippocampus. Learnig and memory associated with hippocampus LTP could also be affected. So, whether excitatory synaptic transmission and LTP in pain conduction pathway could be quickly modulated? What is the possible mechanism? By means of whole-cell patch clamp recordings and field potential recordings on spinal cord slices, behavioral test, immunohistochemistry and other techniches, the present study investigated the acute role of E2in pain modulation, the effect of E2on excitatory synaptic transmission and LTP and its possible cellular and molecular mechanisms. The main findings were as follows: 1. Exogenous and endogenous estrogen were involved in rapid pain modulationIntrathecal injection (i.t) of E2rapidly (≤30min) produced a significant mechanical allodynia and thermal hyperalgesia. Both effects could be abrogated by ICI182,780, antagonist of ER, and mimicked by agonist of mER (membrane estrogen receptor, mER). Intrathecal injection of ICI182,780(5nM,20μl) produced antinociceptive effect on formalin-induced late-phase responses. These findings indicate that exogenous and endogenous estrogen were involved in rapid pain modulation and mER was indicated in pro-nociceptive effect.2. Subcellular distribution of estrogen receptorApplication of double immunostaining technique, we fould ERα/β were well colocalized with NeuN, MAP2and glutamate, GPR30were also coexpressed with MAP2and glutamate indicating three types of ERs were all expressed in glutamatergic neurons. Exposure of alive cells to membrane-impermeant estrogens coupled to FITC results in a punctate staining pattern of the plasma membrane suggesting the existence of surface binding sites for estrogen. The punctuate staining were well colocalized with NR1and GluR1.3. NMD A currents and NMDA-eEPSC could be rapidly enhanced by E2in spinal cord dorsal hornConventional whole-cell patch clamp recordings were performed from lamina I and II neurons of spinal cord slices. Short infusion of E2(5-10min) resulted in a rapid enhancement of N-methyl-D-aspartate (NMDA)-induced currents in a dose-dependent manner while α-amino-3-hydroxy-5-methyl-4-isoxazolepropri-anate (AMPA) currents were differently affected in individual cells and remained unaffected in total. The enhancement of NMD A currents by E2could be blocked by intracellular application of GDP-β-S, a G protein inhibitor, suggesting the involvement of GPCR (G protein coupled receptor) in the enhancement.Furthermore, we observed effects of E2on NMDA-eEPSC and AMPA-eEPSC evoked by dorsal root stimulation. Acute infusion of E2(5-10min) could significantly enhance NMDA-eEPSCs/AMPA-eEPSCs ratio which is mainly related to enhanced NMDA-eEPSCs. Morever, NMDAR-eEPSCs were rapidly potentiated by5-10min perfusion of E2and could persist even after E2washout for30min, which was defined as a chemical LTP of NMDA-eEPSCs (chemical LTP, c-LTP). In contrary, AMPAR-mediated currents were slightly reduced during treatment and washout in most cases. C-LTP induced by E2could be replicated by DPN, agonist of ERβ, but not PPT, agonist of ERa, indicating that E2possibly potentiate NMDAR mediated synaptic transmission via activation of ERβ.4. Presynaptic neurotransmitter release probability was increased by E2Paired pulse facilitation ratio (PPR) at35ms and60ms were both reduced after5-10min E2application indicating a potential increase in release probability. Neither frequency nor amplitude of mEPSC or sEPSC was changed by E2administration.5. LTP of spinal cord dorsal horn evoked by primary afferent stimulation could be facilitated by E2via modulation of NR2B of NMDARLTP of LT-fEPSP in superficial dorsal horn could be reliably induced by HFS (high frequency stimutilation, HFS) at LT (Lissauer’s tract, LT).40-60min E2pretreatment could lower the threshold for LTP induction and enhance LTP magnitude. Infusions of ICI-182,780, antagonist of estrogen receptor, prevented the facilitation of LTP by E2. This facilitation of LTP could be mimicked by E2-BSA, agonist of membrane ER, implicating the involvement of mER.Pharmacologically isolated NMDAR mediated LT-eEPSCs were recorded in superficial dorsal horn. NMDA-eEPSCs in40-60min E2pretreatment group showed more potentiation after HFS than control slices indicating the E2-induced increase in NMDAR transmission may underlie the intensified LTP. Morever, in slices pretreated with combined E2and Ro25-6981show no increase in LTP magnitude anymore, suggesting a role of NR2B-containing NMDAR in the hormone effects. Results from primary culture of spinal neurons combined with Western blot indicated expression of pNR2B and NR2B in membrane were increased by10min bath of E2. It implied that functional modulation of NR2B and increased membrane levels of NR2B were both involved in enhanced NMDA transmission and LTP.6. PKA/ERK signaling pathway may be involved in facilitated spinal LTP by E2pPKA expression was upregulated by10min treatment in cultured spinal cord neurons. Results from combined immunostaining technique with acute drug infusion in spinal cord slices indicated the number of pERK-labeled neurons peaked at10min and activated pERK were well colocalized with pPKA. Intrathecal E2could significantly upregulate pERK expression in spinal dorsal horn which was peaked at10min and then declined to normal levels at30min. The effects could be mimicked by E2-BSA, agonist of mER. Electrophysiological results showed that both H89(1μM) and PD98059(500.M), antagonist of PKA and ERK respectively, not only blocked enhanced LTP by E2, but also agrobated LTP induction indicating an indispensable role of PKA and ERK in the induction of spinal LTP. In sum, it suggested the potential role of PKA and ERK pathway in the facilitated LTP.In conclusion, the present work demonstrated that exogenous and endogenous estrogen could rapidly modulate pain sensitivity. Moreover, E2could acutely enhance NMDAR mediated excitatory synaptic transmission and LTP of spinal dorsal horn and pNR2B/pPKA/pERK signaling pathway was possibly involved in the process. It may contributed greatly to mechanisms underlying E2-induced behavioral sensitization.