Stress contributes to numerous psychiatric disorders. Corticotropin releasing factor (CRF) signaling and CRF neurons in the bed nucleus of the stria terminalis (BNST) drive negative affective behaviors, thus agents that decrease activity of these cells may be of therapeutic interest. Here, we show that acute restraint stress increases cFos expression in CRF neurons in the mouse dorsal BNST, consistent with a role for these neurons in stress-related behaviors. We find that activation of ␣ 2A-adrenergic receptors (ARs) by the agonist guanfacine reduced cFos expression in these neurons both in stressed and unstressed conditions. Further, we find that ␣and ␤-ARs differentially regulate excitatory drive onto these neurons. Pharmacological and channelrhodopsin-assisted mapping experiments suggest that ␣ 2A-ARs specifically reduce excitatory drive from parabrachial nucleus (PBN) afferents onto CRF neurons. Given that the ␣ 2A-AR is a G i-linked GPCR, we assessed the impact of activating the G i-coupled DREADD hM4Di in the PBN on restraint stress regulation of BNST CRF neurons. CNO activation of PBN hM4Di reduced stress-induced Fos in BNST Crh neurons. Further, using Prkcd as an additional marker of BNST neuronal identity, we uncovered a female-specific upregulation of the coexpression of Prkcd/Crh in BNST neurons following stress, which was prevented by ovariectomy. These findings show that stress activates BNST CRF neurons, and that ␣ 2A-AR activation suppresses the in vivo activity of these cells, at least in part by suppressing excitatory drive from PBN inputs onto CRF neurons.

The dorsal region of the bed nucleus of the stria terminalis (dBNST) receives substantial dopaminergic input which overlaps with norepinephrine input implicated in stress responses. Using ex vivo fast scan cyclic voltammetry in male C57BL6 mouse brain slices, we demonstrate that electrically stimulated dBNST catecholamine signals are of substantially lower magnitude and have slower uptake rates compared with caudate signals. Dopamine terminal autoreceptor activation inhibited roughly half of the catecholamine transient, and noradrenergic autoreceptor activation produced an ;30% inhibition. Dopamine transporter blockade with either cocaine or GBR12909 significantly augmented catecholamine signal duration. We optogenetically targeted dopamine terminals in the dBNST of transgenic (TH:Cre) mice of either sex and, using ex vivo whole-cell electrophysiology, we demonstrate that optically stimulated dopamine release induces slow outward membrane currents and an associated hyperpolarization response in a subset of dBNST neurons. These cellular responses had a similar temporal profile to dopamine release, were significantly reduced by the D2/D3 receptor antagonist raclopride, and were potentiated by cocaine. Using in vivo fiber photometry in male C57BL/6 mice during training sessions for cocaine conditioned place preference, we show that acute cocaine administration results in a significant inhibition of calcium transient activity in dBNST neurons compared with saline administration. These data provide evidence for a mechanism of dopamine-mediated cellular inhibition in the dBNST and demonstrate that cocaine augments this inhibition while also decreasing net activity in the dBNST in a drug reinforcement paradigm.

Animals must learn to predict constantly varying threats in the environment to survive by enacting defensive behaviors. Dopamine is involved in the prediction of rewards, encoding a reward prediction error in a similar manner to temporal difference learning algorithm. However, the corresponding molecular and computational form of threat prediction errors is not as well-characterized, although norepinephrine and other neuromodulators and neuropeptides participate in fear learning. Here, we utilized fluorescent norepinephrine recordings over the course of fear learning in concert with reinforcement learning modeling to identify its role in the prediction of threat. By varying timing and sensory uncertainty in the formation of threat associations, we were able to define a precise computational role for norepinephrine in this process. Norepinephrine release approximates the strength of fear associations, and its temporal dynamics are compatible with a prediction error signal. Intriguingly, the release of norepinephrine is influenced by time and sensory feedback, serving as an antithesis of the classical reward prediction error role of dopamine. Thus, these results directly demonstrate a combined cognitive and affective role of norepinephrine in the prediction of threat, with implications for neuropsychiatric disorders such as anxiety and PTSD.