Activation of Lateral Parabrachial Nucleus (LPBn) PACAP-Expressing Projection Neurons to the Bed Nucleus of the Stria Terminalis (BNST) Enhances Anxiety-like Behavior

Abstract

Anxiety disorders are among the most common psychiatric disorders, and understanding the underlying neurocircuitry of anxiety- and stress-related behaviors may be important for treatment. The bed nucleus of the stria terminalis (BNST) has been studied for its role in many stress-related pathologies, such as anxiety, pain, depression, and addiction. Our prior work has demonstrated that pituitary adenylate cyclase-activating polypeptide (PACAP) receptor activation in the BNST mediates many of the behavioral consequences of chronic stress. While the BNST contains local PACAP-expressing neurons, a major source of afferent PACAP is the lateral parabrachial nucleus (LPBn), and excitotoxic lesions of the LPBn substantially decreasess PACAP immunostaining in the BNST. Here, we first assessed Cre-dependent reporter expression by injecting AAV2-hSyn-DIO-mCherry into the LPBn of PACAP-IRES-Cre mice for circuit mapping studies and identified PACAP projections to the BNST, lateral capsular central nucleus of the amygdala (CeLC), and ventromedial hypothalamus (VMH). In a second study, we assessed the effects of chemogenetically activating LPBn PACAP afferents in the BNST by injecting AAV2-hSyn-DIO-hM3D(Gq)-mCherry into the LPBn of PACAP-IRES-Cre mice for Cre-dependent expression of excitatory designer receptors exclusively activated by designer drugs (DREADDs). Before behavioral testing, clozapine-N-oxide (CNO), the selective agonist of our DREADD, was infused directly into the BNST. We found that after specific activation of LPBn PACAP afferents in the BNST, mice had increased anxiety-like behavior compared with controls, while total locomotor activity was unaffected. These results indicate that activation of PACAPergic LPBn projections to the BNST may play an important role in producing anxiety-like behavior.

Keywords: Anxiety; DREADD; Extended amygdala; PAC1 receptor; Post-traumatic stress disorder; Stress.

Figure 1

LPBn PACAP neuron reporter expression in soma and axonal projections. A AAV2-hSyn-DIO-mCherry vector infusion in to LPBn of PACAP-IRES-Cre mice resulted in reporter expression in a large population of multipolar neurons (B, high magnification). C Inspection of anterior forebrain tissue sections showed that the mCherry LPBn neurons have axonal projections to several distal target site such as the CeLC; D high magnification of the CeLC revealed high density of reporter axonal terminal puncta. Scale bar, high magnification 20 μm

Figure 2

LPBn PACAP neurons project to multiple distal emotional/behavioral target sites. Right panels: AAV2-hSyn-DIO-mCherry vector was infused into the LPBn of PACAP-IRES-Cre mice (right panel D), and reporter terminal expression was observed in the BNST, CeLC, and VMH (right panels A–C, respectively). Left and middle panels, schematic illustration of mouse brain with corresponding coronal sections of LPBn (reporter infusion site, coronal section D), and axonal terminal projections to the BNST, CeLC, and VMH (coronal sections A–C, respectively). Micrographs of mCherry reporter expression correspond to locations denoted by the colored dots on the coronal sections. ac, anterior commissure; 3v, third ventricle; scp, superior cerebellar peduncle

Figure 3

Activation of LPBn PACAP neuronal terminals in BNST enhances anxiety-like responses. Following injections of AAV2-hSyn-DIO-hM3D(Gq)-mCherry or control virus into the LPBn of PACAP-IRES-Cre mice, CNO was infused directly into the BNST prior to behavioral testing on the elevated plus maze. A Schematic diagram illustrating LPBn virus injections and BNST CNO infusions. B DREADD or control virus injections were localized to the LPBn. C After CNO activation of LPBn PACAP afferents in the BNST, mice exhibited increased anxiety-like behavior compared with controls, exploring the open arms of the elevated-plus maze significantly less (top panel). Total locomotor activity on the elevated plus maze was unaffected (bottom panel). scp, superior cerebellar peduncle shown in dotted line