Estrogen has been reported to modify synapse numbers

Estrogen has been reported to modify synapse numbers in several types of neurons. For example, estradiol increases the spine density of hippocampal CA1 pyramidal neurons, neurons in the posterodorsal medial amygdala, and neurons in the ventromedial hypothalamic nucleus (Gould et al., 1990, Calizo and Flanagan-Cato, 2000, de Castilhos et al., 2008). In GnRH neurons, Miconazole synthesis increases the number of synaptic structures at GnRH neurons in OVX rats (Chan et al., 2011). Therefore, estrogen may change the number of PGE2-responsive synapses in GnRH neurons, and this may underlie PGE2’s effect on mEPSC frequency. Further studies are needed to clarify this issue. PGE2 has been reported to alter the efficacy of neurotransmission by activating PGE2 receptor subtypes. For example, in the substantia nigra pars compacta, PGE2 action via EP1 increases the frequency of miniature inhibitory postsynaptic currents (mIPSCs) in neurons (Tanaka et al., 2009). PGE2 also increases the frequency of mEPSCs in neurons cultured from the hippocampus via EP2 presynaptic receptors (Sang et al., 2005). On the other hand, PGE2 decreases the frequency of spontaneous IPSCs in neurons from the supraoptic nucleus and mEPSCs from dorsolateral periaqueductal gray neurons (via EP3) (Shibuya et al., 2002, Lu et al., 2007). These results indicate that EP1 and EP2 promote, whereas EP3 inhibits, the effects of PGE2. Because the effect of PGE2 is an increase in the frequency of mEPSCs, we excluded any analyses of EP3 in the present study. However, we did confirm that the EP4 agonist ONO-AE1-329 is able to mimic the effect of PGE2 on mEPSCs in GnRH neurons. These results indicate that the EP4 receptor subtype mediates the effects of PGE2 in altering mEPSC frequency in GnRH neurons under high estrogen conditions. This hypothesis is further supported by the EP4 receptor antagonist ONO-AE3-208 attenuating PGE2’s effect on mEPSC frequency. Currently, however, the existence of EP4 at presynaptic sites on GnRH neurons remains unclear. Further anatomical studies are needed to answer the question. Notably, it is unlikely that the doses of ONO-DI-004 and butaprost used were too low to have an effect, as these doses were reported previously to alter spontaneous activity, or evoked activity in both GnRH and other neurons (Moriyama et al., 2005, Tanaka et al., 2009, Clasadonte et al., 2011). Although the mechanisms underlying the EP4-mediated increase in the frequency of mEPSCs via PGE2 remain unclear, mechanisms similar to those driven by EP2 may be involved because activation of both these receptor subtypes increases the intracellular concentration of cAMP (Nishigaki et al., 1995, Sugimoto and Narumiya, 2007). The EP2-mediated, PGE2-induced increase in glutamate release from the synaptosomes of cortical neurons has been reported to be accomplished by increasing the concentration of cAMP in the synaptosomes (Lin et al., 2014). Thus, presynaptic EP2 activated by PGE2 could increase the intracellular cAMP concentration, resulting in the increased presynaptic release of vesicles that is reflected by the increase in mEPSC frequency. In support of this hypothesis, mEPSC frequency was increased in pyramidal neurons in the medial prefrontal cortex and in Purkinje cells in the cerebellar cortex by forskolin, which is a drug that elevates the intracellular cAMP concentration by activating adenylate cyclase (Chen and Regehr, 1997, Huang and Hsu, 2006).