Sustained hyperactivity of parasubthalamic nucleus by PACAP signaling mediates stress-induced anxiety

Stress represents a major risk factor for anxiety disorders, yet the underlying circuit and molecular substrates through which stress causes anxiety remain elusive. We employed an established stress paradigm to induce anxiety-like behaviors in mice and found that stress causes robust and sustained activation of glutamatergic neurons in the parasubthalamic nucleus (PSTh). Moreover, inhibition of those neurons significantly reduced anxiety-like behavior in stressed animals. At circuit level, inhibition of excitatory inputs from the lateral parabrachial nucleus (LPB) decreased PSTh activation during stress and alleviated anxiety-like behaviors following stress. RNA sequencing revealed that the pituitary adenylate cyclase-activating polypeptide type 1 receptor (PAC1R, encoded by Adcyap1r1) is enriched in the PSTh. Ex vivo patch-clamp recordings showed that PACAP, an endogenous agonist of PAC1R, increases the excitability of PSTh neurons. Interestingly, pharmacological blockade of PAC1R within PSTh was sufficient to prevent anxiety-like behaviors induced by stress. These findings suggest that stress induces sustained activation of PSTh neurons through the LPB-PSTh excitatory circuitry and PACAP-PAC1R signaling, ultimately leading to anxiety-like state.