, 2005) and higher levels of Venus expression in lactating rats, we found many more fine Venus-positive axons in all major forebrain regions than by direct staining for OT. Moreover, classical immunohistochemistry does not

reveal the sources of these fibers, which may originate from the PVN, SON, or AN. According to our results, the PVN and AN neurons project extensively to forebrain structures, whereas the SON contributes less to forebrain innervation. But even from the SON, which features only magnocellular neurons, a moderate number of fibers were observed in five forebrain regions (the horizontal limb of the diagonal band of Broca, Acb, CeA, lateral septum, and CA1 of the ventral Cisplatin purchase hippocampus). Additional evidence that magnocellular neurons project to higher brain regions was obtained with PS-Rab delivered into the CeA or the Acb. After injection of EGFP-expressing PS-Rab into these structures, we observed EGFP-positive back-labeled OT neurons residing in magnocellular nuclei, as well as their axonal terminals in the posterior pituitary. Importantly, only magnocellular hypothalamic

neurons, but no other neuronal cell types, project to the posterior pituitary lobe (Brownstein et al., 1980, Sofroniew, 1983, Swanson and Sawchenko, 1983 and Burbach et al., 2001). In support of our observations, injection of the retrograde marker fluorogold learn more into the Acb of voles led to the appearance of back-labeled OT neurons in the PVN and SON, with fluorogold-containing terminals in the posterior pituitary (Ross et al., 2009). In contrast, injection of PS-Rab into the NTS (Figure S6B) resulted in back-labeling of PVN parvocellular OT neurons, which all do not project to the posterior pituitary (Sawchenko and Swanson, 1983 and Swanson and Sawchenko, 1983). Collectively, the PS-Rab data in conjunction with light and, in particular, electron microscopic results provide compelling evidence that the

fibers in the CeA and Acb are axonal collaterals of magnocellular OT neurons. Our finding that magnocellular OT neurons simultaneously project to forebrain structures and the posterior pituitary is consistent with results demonstrating that the induced central and peripheral OT releases can be associated, for instance, in a situation of stress (Neumann, 2007). More specifically, it was previously demonstrated that an ethologically relevant stressor (such as forced swim in rats) induces an increase in OT plasma levels (Wotjak et al., 1998), as well as OT release within the CeA. Our anatomical results provide the basis for OT action within the CeA in both virgin and lactating rats. Although the density of OT fibers is lower in virgin than in lactating animals, the profile of axonal innervation of the CeA was similar in animals of both groups. In the CeM, we detected smooth nonbranching fibers which exceed the axons in the CeL in length. This type of fiber appears to represent transitory axons, traversing the CeM with no synaptic contacts.