br Experimental procedures br Results Fluorescence

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 Miconazole 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.