br Experimental procedures br Results Fluorescence attributa

Experimental procedures
Results Fluorescence attributable to GnRH-eGFP neurons was observed through the diagonal band of Broca, to the POA of the hypothalamus in GnRH-eGFP transgenic rats for all groups as described previously (Kato et al., 2003, Tada et al., 2013).
Discussion The mEPSCs were recorded at GnRH-eGFP neurons as reported previously using GnRH-eGFP rats (Kato et al., 2003, Tada et al., 2013) and indicate spontaneous vesicle discharge from excitatory synapses, independent of action potentials. According to the quantal theory of neurotransmitter release, a change in the amplitude of the mEPSC represents a change in the sensitivity of the postsynaptic membrane, whereas a change in the frequency of mEPSCs reflects a change in the total presynaptic output (including the probability of vesicle release from the presynaptic terminal and the number of synapses) (Kerchner and Nicoll, 2008). We show that bath application of PGE2 increases the frequency but not the amplitude of mEPSCs in GnRH neurons in ProE but not D1 rats, suggesting that PGE2 alters the presynaptic excitatory input in GnRH neurons in a manner independent of estrous cycle stage. A higher basal frequency of mEPSCs in GnRH neurons in ProE relative to D1 rats was previously reported (Tada et al., 2013), and is inconsistent with our data. The reasons for this difference are unknown, but could be related to differences in the time of Mdivi 1 receptor slice preparation and/or ages of the rats. The observation that PGE2 modulates neurotransmission in GnRH neurons and the high estrogen levels present on the day of proestrus are suggestive of a role for estrogen (Butcher et al., 1974). Our data support this, and show that PGE2 treatment increases the frequency of mEPSCs in GnRH neurons in OVX+E2, but not OVX+C rats. As the dose of estradiol used in the present study could induce a surge of luteinizing hormone (Nishihara et al., 1994, Tada et al., 2013), our results suggest that the estrogen's effect could be related to the positive feedback effect of estrogen on luteinizing hormone release. Although the baseline amplitudes of OVX+E2 rat mEPSCs were slightly lower than those seen in OVX+C rats, E2 treatment itself did not seem to affect the baseline amplitudes of mEPSCs in GnRH neurons, since there was no significant difference between OVX+C and OVX+E2 rats in baseline amplitude in either the agonist or antagonist treatment studies. The reason for the difference observed the in OVX+E2 rats used in the PGE2 treatment study is unclear. A possible mechanism for PGE2’s effect on the frequency of mEPSCs in GnRH neurons is that estrogen modulates the presynaptic expression of PGE2 receptors. Estradiol and/or progesterone increase the levels of prostaglandin receptor proteins, including EP4, in the uterus of OVX rats, suggesting that estrogen modulates PGE2 receptor expression (Blesson et al., 2012). Thus, increased presynaptic PGE2 receptor expression (induced by estrogen) could further amplify responses to PGE2, increasing the frequency of mEPSCs recorded at GnRH neurons. Another plausible mechanism is that estrogen may change the synapse number and/or population in GnRH neurons. 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, estrogen 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).