To empirically determine the functional penetrance of blue light through brain tissue in terms of neuronal activation, and to test whether we could observe an increase in

vHPC activity after illumination, we used the immediate early gene c-fos as a readout for neural activity. Although we did not observe a change in BLA somata c-fos expression induced by illumination of BLA terminals in the vHPC, c-fos expression was increased in the pyramidal layer of the vHPC extending to ∼1.5 mm below the fiber tip ( Figures S4 and S5). We complemented our c-fos readouts with estimated irradiance levels through brain tissue using an empirically based model and illumination CHIR-99021 supplier during whole-cell patch-clamp recordings (see Supplemental Experimental Procedures). While these data indicate that inhibition of BLA terminals in the vHPC can reduce anxiety-related behaviors, the illumination of ChR2-expressing processes in the vHPC could carry the potential for inducing backpropagating action potentials (Petreanu et al., 2007) or depolarization of axons of passage. To test whether the activation of BLA axon terminals synapsing locally in the vHPC was the underlying mechanism of this light-induced change in anxiety-related behavior, we combined in vivo pharmacological manipulations

with our in vivo optogenetic manipulations during anxiety assays (Figures 3A–3D). To determine whether the robust changes in anxiety-related behaviors that we observed were indeed mediated by monosynaptic, glutamatergic inputs from the BLA to selleck screening library the vHPC, rather than axons of passage or antidromic activation of BLA somata, we performed an additional heptaminol series of experiments (Figure 3). First, we expressed ChR2 in BLA projection

neurons as before and implanted a guide cannula to deliver either saline or glutamate receptor antagonists to the vHPC 30 min prior to testing and illumination on the EPM, OFT, or NSF (Figures 3A and S6). To allow for a within-subject comparison, we tested each animal twice on different days and contexts administering either saline or a glutamate receptor antagonist cocktail, counterbalanced for order. We compared saline trials to trials in which the combination of AMPA and NMDA receptor antagonists, 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione (NBQX) and (2R)-amino-5-phosphono-pentanoate (AP5), respectively, was intracranially administered to the vHPC. In saline trials, mice replicated the light-induced anxiogenic effect on both the EPM (Figure 3B) and the OFT (Figure 3C). However, after vHPC glutamate receptor antagonism, the light-induced changes in open-arm exploration on the EPM, the time spent in the center on the OFT, and the latency to feed on the NSF test were all attenuated (Figures 3B, 3C, and 3D).