Analysis of neuronal circuit for the gonadotropin-releasing hormone surege generator (GnRH) using an in vitro GnRH surge generator model

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1. We determined morphological changes in an in vitro gonadotropin-releasing hormone (GnRH) surge generator model we developed. GnRH neurons in the POA slice were clearly visible 4 weeks after co-culture with the suprachiasmatic nucleus (SCN). However, we could not stained vasopressin neurons. We tried to identify living GnRH neurons by transfecting a expression vector containing GnRH promoter and Ds-Red as a reporter. Transfection efficiency of this vector is, however, very low even in GT1-7 cell lines.2. We investigated the propagation of electrical stimulus-evoked excitation in the in vitro GnRH surge generator model. We found that the evoked excitation was not propagated to the POA slice when an electrical stimulus was applied in the SCN slice, suggesting that the POA slice could not make synaptic contact with the SCN slice.3. We next measured GnRH releases in tne POA slice obtained from the adult rat on the day of proestrus. Bicuculline, an GABA_A receptor antagonist, significantly increased GnRH releases in the POA slice. Interestingly, when the POA slice was perfused with the SCN slice, we found that GnRH release was significantly fractuated, suggesting that GnRH release was conveyed by the SCN via a humoral information.From these results, we conclude that an acute slice preparation is useful tools to investigate the GnRH surge generator activity.