The bed nucleus of the stria terminalis (BNST) is a brain region important for regulating anxiety-related behavior in both humans and rodents. Here we used a chemogenetic strategy to investigate how engagement of G protein-coupled receptors (GPCR) signaling cascades in genetically defined GABAergic BNST neurons modulates anxiety-related behavior and downstream circuit function. We saw that stimulation of vesicular γ-Aminobutyric acid (GABA) transporter (VGAT)-expressing BNST neurons using hM3Dq, but neither hM4Di nor rM3Ds Designer Receptors Exclusively Activated by a Designer Drug (DREADDs), promotes anxiety-like behavior. Further, we identified that activation of hM3Dq receptors in BNST VGAT neurons can induce a long-term depression (LTD)-like state of glutamatergic synaptic transmission, indicating DREADD-induced changes in synaptic plasticity. Further, we used DREADD-assisted metabolic mapping (DREAMM) to profile brain-wide network activity following activation of Gq-mediated signaling in BNST VGAT neurons and saw increased activity within ventral midbrain structures, including the ventral tegmental area (VTA), and hindbrain structures such as the locus coeruleus (LC) and parabrachial nucleus (PB). These results highlight that Gq-mediated signaling in BNST VGAT neurons can drive downstream network activity that correlates with anxiety-like behavior, and points to the importance of identifying endogenous GPCRs within genetically defined cell populations. We next used a microfluidics approach to profile the receptorome of single BNST VGAT neurons. This approach yielded multiple Gq-coupled receptors that are associated with anxiety-like behavior and several potential novel candidates for regulation of anxiety-like behavior. From this, we identified that stimulation of the Gq-coupled receptor 5-HT2CR in the BNST is sufficient to elevate anxiety-like behavior in an acoustic startle task. Together, these results provide a novel profile of receptors within genetically defined BNST VGAT neurons that may serve as therapeutic targets for regulating anxiety states and provide a blueprint for examining how G-protein mediated signaling in a genetically defined cell type can be used to assess behavior and brain-wide circuit function.

Highlights d Pnoc CeA neurons are activated following palatable food consumption d Pnoc CeA neurons promote palatable food consumption specifically d Pnoc CeA neuronal projections in the CeA, vBNST, PBN, and NTS promote reward

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Serotonin (5-hydroxytryptamine; 5-HT) coordinates behavioral responses to stress through a variety of presynaptic and postsynaptic receptors distributed across functionally diverse neuronal networks in the central nervous system. Efferent 5-HT projections from the dorsal raphe nucleus (DRN) to the bed nucleus of the stria terminalis (BNST) are generally thought to enhance anxiety and aversive learning by activating 5-HT 2C receptor (5-HT 2C R) signaling in the BNST, although an opposing role for postsynaptic 5-HT 1A receptors has recently been suggested. In the present study, we sought to delineate a role for postsynaptic 5-HT 1A receptors in the BNST in aversive behaviors using a conditional knockdown of the 5-HT 1A receptor. Both males and females were tested to dissect out sex-specific effects. We found that male mice have significantly reduced fear memory recall relative to female mice and inactivation of 5-HT 1A receptor in the BNST increases contextual fear conditioning in male mice so that they resemble the females. This coincided with an increase in neuronal excitability in males, suggesting that 5-HT 1A receptor deletion may enhance contextual fear recall by disinhibiting fear memory circuits in the BNST. Interestingly, 5-HT 1A receptor knockdown did not significantly alter anxiety-like behavior in male or female mice, which is in agreement with previous findings that anxiety and fear are modulated by dissociable circuits in the BNST. Overall, these results suggest that BNST 5-HT 1A receptors do not

Cell adhesion and migration play important roles in physiological and pathological states, including embryonic development and cancer invasion and metastasis. The type I transmembrane protein with epidermal growth factor and two follistatin motifs 2 (TMEFF2) is expressed mainly in brain and prostate and its expression is deregulated in prostate cancer. We have previously shown that TMEFF2 can function as a tumor suppressor by inhibiting cell migration and invasion of prostate cells. However, the molecular mechanisms involved in this inhibition are not clear. In this study we demonstrate that TMEFF2 affects cell adhesion and migration of prostate cancer cells and that this effect correlates with changes in integrin expression and RhoA activation. Deletion of a 13 basic-rich amino acid region in the cytoplasmic domain of TMEFF2 prevented these effects. Overexpression of TMEFF2 reduced cell attachment and migration on vitronectin and caused a concomitant decrease in RhoA activation, stress fiber formation and expression of αv, β1 and β3 integrin subunits. Conversely, TMEFF2 interference in 22Rv1 prostate cancer cells resulted in increased integrin expression. Results obtained with a double TRAMP/TMEFF2 transgenic mouse also indicated that TMEFF2 expression reduced integrin expression in the mouse prostate. In summary, the data presented here indicate an important role of TMEFF2 in regulating cell adhesion and migration that involves integrin signaling and is mediated by its cytoplasmic domain.

BACKGROUND Previous results from our lab indicate a tumor suppressor role for the transmembrane protein with epidermal growth factor and two follistatin motifs 2 (TMEFF2) in prostate cancer (PCa). Here, we further characterize this role and uncover new functions for TMEFF2 in cancer and adult prostate regeneration. METHODS The role of TMEFF2 was examined in PCa cells using Matrigel™ cultures and allograft models of PCa cells. In addition, we developed a transgenic mouse model that expresses TMEFF2 from a prostate specific promoter. Anatomical, histological and metabolic characterizations of the transgenic mouse prostate were conducted. The effect of TMEFF2 in prostate regeneration was studied by analyzing branching morphogenesis in the TMEFF2-expressing mouse lobes and alterations in branching morphogenesis were correlated with the metabolomic profiles of the mouse lobes. The role of TMEFF2 in prostate tumorigenesis in whole animals was investigated by crossing the TMEFF2 transgenic mice with the TRAMP mouse model of PCa and analyzing the histopathological changes in the progeny. RESULTS Ectopic expression of TMEFF2 impairs growth of PCa cells in Matrigel or allograft models. Surprisingly, while TMEFF2 expression in the TRAMP mouse did not have a significant effect on the glandular prostate epithelial lesions, the double TRAMP/TMEFF2 transgenic mice displayed an increased incidence of neuroendocrine type tumors. In addition, TMEFF2 promoted increased branching specifically in the dorsal lobe of the prostate suggesting a potential role in developmental processes. These results correlated with data indicating an alteration in the metabolic profile of the dorsal lobe of the transgenic TMEFF2 mice. CONCLUSIONS Collectively, our results confirm the tumor suppressor role of TMEFF2 and suggest that ectopic expression of TMEFF2 in mouse prostate leads to additional lobe-specific effects in prostate regeneration and tumorigenesis. This points to a complex and multifunctional role for TMEFF2 during PCa progression.

Injection of a DIO-mCherry to the BNST of a VGAT-Cre mouse to label projection fibers. (b) mCherry fluorescence observed in the BNST injection site (left) and fluorescent fibers in the VTA (middle) and PBN/LC (right). Scale bars: 1 mm. (c) μPET imaging timeline. Mice were injected with vehicle or CNO and 5 min later injected with FDG. Twenty-five minutes later, mice were anesthetized with isoflurane and placed on the scanning bed for a 20 min scanning session. (d) Representative image of a DIO-hM3Dq-mCherry BNST injection site. Scale bar: 500 μm. (e) Increased FDG uptake in areas corresponding to the BNST (left), VTA (middle) and PBN/LC (right) following activation of hM3Dq in BNST VGAT neurons. (f) Representative injection of a DIO-hM3Dq-mCherry to a Cre-negative control mouse. (g) No change in FDG uptake in the BNST (left), VTA (middle), or PBN/LC (right) from pooled adjacent controls. a.c., anterior commissure; BNST, bed nucleus of the stria terminalis; LC, locus coeruleus; N, four mice per group; PBN, parabrachial nucleus; VTA, ventral tegmental area. For more information on this topic, please refer to the article by Mazzone et al. on pages 143-153.

Ex vivo lung perfusion (EVLP) is a novel lung preservation strategy that facilitates the use of marginal allografts; however, it is more expensive than static cold storage (SCS). To understand how preservation method might affect postoperative costs, we compared outcomes and index hospitalization costs among matched EVLP and SCS preserved lung transplant (LTx) recipients at a single, high‐volume institution. A total of 22 EVLP and 66 matched SCS LTx recipients were included; SCS grafts were further stratified as either standard‐criteria (SCD) or extended‐criteria donors (ECD). Median total preservation time was 857, 409, and 438 min for EVLP, SCD, and ECD lungs, respectively (p < .0001). EVLP patients had similar perioperative outcomes and posttransplant survival compared to SCS SCD and ECD recipients. Excluding device‐specific costs, total direct variable costs were similar among EVLP, SCD, and ECD recipients (median $200,404, vs. $154,709 vs. $168,334, p =  .11). The median direct contribution margin was positive for EVLP recipients, and similar to that for SCD and ECD graft recipients (all p > .99). These findings demonstrate that the use of EVLP was profitable at an institutional level; however, further investigation is needed to better understand the financial implications of EVLP in facilitating donor pool expansion in an era of broader lung sharing.