We observed an EGFP signal in all fibers within the CeA and in axonal terminals in the posterior pituitary (Figure 3C). Similarly, expression of a synaptophysin-EGFP fusion protein revealed synaptic terminals in both structures (Figure 3D). Costaining of synaptophysin-EGFP puncta with antibodies against OT and vesicular glutamate transporter 2 (VGluT2), the mRNA of which was detected in OT neurons (Kawasaki et al., 2005), demonstrated overlap of EGFP, VGluT2, and OT signals (Figure 3E), confirming previous reports on magnocellular neurons enriched

by microvesicles that associate with synaptophysin (Navone et al., 1989) and contain glutamate (Meeker et al., 1991). Higher-resolution electron microscopic analysis revealed the presence of synaptic contacts between immunoreactive axon terminals and local dendrites in the CeL (Figure 3F), Cobimetinib price most likely dendrites of GABAergic interneurons, of which the CeL is predominantly composed (Davis, 2000 and Huber et al., 2005). In the three cases analyzed, we encountered Nutlin-3a concentration synaptic appositions bearing the features of asymmetric (Gray’s type 2) synapses, proposed to be of excitatory nature (Figure 3F). Importantly, we were unable to find synaptic contacts within the CeM (M.E., unpublished data), where fibers are traversing the region without branching and forming varicosities, as was the case in the CeL (Figure S3A). Our

collective findings strongly suggest a presence in the CeL of axonal terminals that originate from OT neurons and form glutamatergic synapses. Based on the anatomical evidence for OT-containing axonal fibers of hypothalamic origin in the CeA, we selectively expressed the blue-light (BL)-sensitive unless ChR2 protein (Nagel et al., 2003) fused to mCherry (Figure 4A) in

all hypothalamic OT neurons via an rAAV. Whole-cell voltage-patch-clamp recordings in vitro in coronal slices of mCherry fluorescent cells (Figures 4A and 4B, top) revealed functional ChR2 expression in PVN, SON, and AN neurons, as evident from the presence of BL induced currents with a characteristic rapidly inactivating peak followed by a stable tail (Boyden et al., 2005; Figures 4C and S4). Because high frequencies of action potentials are thought to be necessary to trigger release of neuropeptides, in contrast to release of classical neurotransmitters (Hökfelt, 1991), we quantified the effect of different BL stimulations on AP frequencies of PVN and SON neurons. Current-clamp recordings from these neurons showed that AP frequencies up to 20 Hz could be reliably induced by stimulation frequencies with short BL pulses of 10 ms applied at 30 Hz, as well as by continuous BL exposure (Figures 4C and S4A). Having shown functional ChR2 expression in the OT cell bodies, we tested whether BL could also release endogenous OT from axonal projections in horizontal slices of the CeA (Figure 4B, bottom).