In the spinal cord EAAT constitutes of all high affinity

In the spinal cord, EAAT1 constitutes 40% of all high-affinity EAATs, and it is abundantly present in lumbar dorsal horn (Queen et al., 2007). In physiological conditions, EAATs remove glutamate from the synaptic cleft by co-transporting glutamate and sodium into adjacent glial cells. The inefficient function of EAATs can lead to high extracellular glutamate concentration causing neuronal damage (Kanai et al., 2013). In the spinal cord, glutamate transporters have been shown to be downregulated following chronic sciatic nerve injury (Sung et al., 2003, Yan et al., 2009, Ramos et al., 2010). Drugs inhibiting EAATs such as TBOA have shown the pronociceptive effect by increasing synaptic glutamate and hence neuronal excitability (Liaw et al., 2005). Interestingly in pathological chronic pain states, these compounds have shown opposite i.e. antinociceptive effect (Minami et al., 2001, Yang et al., 2015). Tao et al. have proposed various possible causes of this phenomena including sustained higher extracellular glutamate leading to neurotoxicity; glutamate conversion to inhibitory molecule gamma amino butyric silybin (GABA); presynaptic glutamate transporter blockade leading to depletion of presynaptic glutamate and postsynaptic glutamate receptor desensitization (Tao et al., 2005). The exogenous estrogen decreased the EAATs activity in the male spinal cord at 100nM concentration in our study. This concentration of estrogen is higher than the physiological one but was used to allow the penetration into the spinal cord tissue in our ex-vivo experiments. In females, the proestrus phase exhibited less active EAAT state most likely under the effect of high endogenous estrogen. This effect proved to be sustainable and persisted in estrus phase where estrogen usually decreases its circulatory levels. Mitrovic at al. demonstrated that the exogenous progesterone showed no effect on glial glutamate uptake in male rats, alone or in combination with estrogen (Mitrovic et al., 1999). In our study, the glutamate uptake was high in metestrus and diestrus, implying that progesterone does not reduce the uptake in this case. Remarkably, the estrogen receptor ERα gene expression was up-regulated in the estrus phase suggesting an enhanced hormone signaling. Furthermore, studies have shown that selective activation of spinal ERα mimics the pronociceptive effects of 17β-estradiol on visceral sensitivity (Ji et al., 2011). Estrogen generates its effects via the nuclear receptors: estrogen receptors alpha (ERα) and beta (ERβ), which upon their activation instigate transcriptional changes for target genes modulating cellular function (Dechering et al., 2000). Estrogen also has non-genomic effects through cellular membrane estrogen receptors by initiating activation of intracellular signaling pathways depending upon their location e.g. mitogen-activated protein kinase/extracellular signal-regulated kinases (MAPK/ERK) in the spinal cord (Bjornstrom and Sjoberg, 2005). Estrogen potentially exerts its nociceptive effects on visceral pain pathways through ERα leading to activation of ERK pathway (Ji et al., 2011). Our study showed increased expression of spinal ERα mRNA in the estrus phase which may be linked to the decreasing levels of estrogen. We also noted an increase in ERβ in the diestrus phase which is related to decreased estrogen and visceral pain. Moreover, ERβ ligands have an antinociceptive effect in rats during colorectal distension (Cao et al., 2012). Future work is needed to focus on whether this is causally related to the changes in glutamate transporter per se and downstream pain states. This may include the use of selective ERα and ERβ inhibitors while assessing the glutamate uptake. Interestingly, estrogen has been shown to upregulate NMDA receptors (NDMARs), which are heterotetramers composed of two N1 and two N2 or three subunits. NMDA receptor activity is subject to changes in receptor abundance, distribution, phosphorylation status, and subunit composition and may be altered by ischemia, inflammation, aging, or sex hormones (Rajilic-Stojanovic et al., 2015). Estrogen also increases receptor phosphorylation of the NR1 subunit contributing to an increase in NMDA receptor activity in a model of visceral pain (Tang et al., 2008). This may result in a negative feedback mechanism and thus decreased expression of the NR1 subunit mRNA, which is possibly the case in our data as there was a selective reduction of NR1 mRNA in proestrus. This effect was not present in estrus phase of the estrous cycle most likely due to the decreasing concentration of systemic estrogen levels in the estrus phase. We noted no change in the NR2B subunit between the sexes or between cycle stages. This conflicts with other studies possibly due to our use of healthy rats and Ji et al., using a model of inflammatory visceral pain (Ji et al., 2012).