No abstract

Anxiety disorders are prevalent across the United States and result in a large personal and societal burden. Currently, numerous therapeutic and pharmaceutical treatment options exist. However, drugs to classical receptor targets have shown limited efficacy and often come with unpleasant side effects, highlighting the need to identify novel targets involved in the etiology and treatment of anxiety disorders. GPR83, a recently deorphanized receptor activated by the abundant neuropeptide PEN, has also been identified as a glucocorticoid regulated receptor (and named GIR) suggesting that this receptor may be involved in stress-responses that underlie anxiety. Consistent with this, GPR83 null mice have been found to be resistant to stress-induced anxiety. However, studies examining the role of GPR83 within specific brain regions or potential sex differences have been lacking. In this study, we investigate anxiety-related behaviors in male and female mice with global knockout and following local GPR83 knockdown in female mice. We find that a global knockdown of GPR83 has minimal impact on anxiety-like behaviors in female mice and a decrease in anxiety-related behaviors in male mice. In contrast, a local GPR83 knockdown in the basolateral amygdala leads to more anxiety-related behaviors in female mice. Local GPR83 knockdown in the central amygdala or nucleus accumbens (NAc) showed no significant effect on anxiety-related behaviors. Finally, dexamethasone administration leads to a significant decrease in receptor expression in the amygdala and NAc of female mice. Together, our studies uncover a significant, but divergent role for GPR83 in different brain regions in the regulation of anxiety-related behaviors, which is furthermore dependent on sex.

Antibodies represent powerful tools to examine signal transduction pathways. Here, we present a strategy integrating multiple state-of-the-art methods to produce, validate, and utilize antibodies. Focusing on understudied synaptic proteins, we generated 137 recombinant antibodies. We used yeast display antibody libraries from the B cells of immunized rabbits, followed by FACS sorting under stringent conditions to identify high affinity antibodies. The antibodies were validated by high-throughput functional screening, and genome editing. Next, we explored the temporal dynamics of signaling in single cells. A subset of antibodies targeting opioid receptors were used to examine the effect of treatment with opiates that have played central roles in the worsening of the ‘opioid epidemic.’ We show that morphine and fentanyl exhibit differential temporal dynamics of receptor phosphorylation. In summary, high-throughput approaches can lead to the identification of antibody-based tools required for an in-depth understanding of the temporal dynamics of opioid signaling.