The aim of the present study was to examine central pathways and neurotransmitters through which stressors disrupt GnRH release, particularly via AVP under the influence of oestradiol (E₂). First, in an in vitro perifusion system for hypothalamic slices the effect of E₂ on GABA or noradrenergic control of AVP or GnRH release directly from ewe hypothalamic slices was investigated. Second, a confocal analysis of ewe hypothalamic sections was carried out to establish the presence of close contacts between various neurotransmitters systems involved in stress and reproduction interactions. Third, neuronal responses to in vivo insulin treatment using Fos-mapping of brainstem and hypothalamic nuclei delineated the activation of central mechanisms involved in stress-induced disruption of GnRH release. The validity of the in vitro perifusion system was established by: 1) a robust release of AVP and GnRH in response to a depolarizing dose of KCI (100 mM) at the end of collection period, 2) histological evaluation of the slices which revealed intact neuronal cell bodies near the surface of slice. Exposure to high E₂ (24 pg/ml) increased the release of AVP and GnRH from hypothalamic slices in vitro suggesting direct action of E₂ within the hypothalamus. A marked release of AVP in response to GABA_A or B antagonists (10 mM) revealed an inhibitory GABA control over AVP release that was potentiated through GABA_B receptors in the presence of E₂. However, stimulation of AVP release by both α₁-adrenoreceptor agonist and antagonist (10 mM) indicated the existence of dual stimulatory and inhibitory noradrenergic control over AVP neurones, with lower stimulatory response in the presence of E₂. The release of GnRH in response to GABA receptor antagonists (10 mM) revealed predominant GABA, receptor-mediated inhibitory GABA control that was attenuated in the presence of E₂. However, GnRH release induced by an α₁-adrenoreceptor agonist (10 mM) was potentiated by E₂. With confocal microscopy, close contacts were observed between noradrenergic terminals and CRH or AVP cell bodies in the PVN but not with β-endorphin cell bodies in the ARC. However, GABA terminals closely contact CRH and β-endorphin, but not AVP, cell bodies. No relation was observed between CRH, AVP and β-endorphin terminals and GnRH cell bodies in the mPOA, however, CRH and GnRH terminals were close in the ME. Oestradiol receptor (ERa) colocalisation was not observed in CRH and AVP neurones in the PVN but noradrenergic neurones in the brainstem and β-endorphin neurones in the ARC did colocalise ERα. Following insulin treatment of ewes in vivo, there was robust Fos-immunoreactivity in the noradrenergic neurones in the brainstem and CRH and magnocellular and parvocellular AVP neurones in the PVN; however Fos decreased in the β-endorphin neurones in the ARC. The presence of Fos-positive cells increased in the ARC and the VMN after insulin. The colocalisation ERα and Fos-positive cells decreased in the ARC contrary to the increase in the VMN. In conclusion, E₂-induced stimulation of AVP from the hypothalamus may be involved in E₂-induced augmentation of HPA activity, however, GABA and noradrenergic interactions with AVP neurones in the presence of E₂ do not favor this hypothesis. Oestradiol alone as well as in association with GABA and noradrenergic systems support the potentiation of GnRH release from hypothalamus in vitro. Complex interactions between various neurones in the brainstem and hypothalamus are involved in stress-induced suppression of the GnRH release.