Probes for INS

LGR5 is expressed in various tumors and has been identified as a putative intestinal stem cell marker. Here we investigated LGR5 expression in colorectal neuroendocrine neoplasms and analyzed the correlation with pathological characteristics. We evaluated the clinicopathological features of 8 neuroendocrine tumor (NET) grade 1 (NET G1), 4 NET Grade 2 (NET G2), and 8 NET Grade 3 (NET G3; also termed neuroendocrine carcinoma, or NEC) cases. We examined LGR5 expression using an RNAscope, a newly developed RNA in situ hybridization technique, with a tissue microarray of the neuroendocrine neoplasm samples. LGR5 staining in individual tumor cells was semi-quantitatively scored using an H-score scale. We also performed a combination of LGR5 RNA in situ hybridization and synaptophysin immunohistochemistry. All cases contained tumor cells with some LGR5-positive dots. For all cases, H-scores showed a positive correlation with nuclear beta-catenin expression. In the NEC group, there was a strong positive correlation between H-score and beta-catenin expression. Our findings suggest that LGR5 may serve as a stem cell marker in NEC, as is the case in colon adenocarcinoma. The positive correlation between H-score and beta-catenin expression suggests that LGR5 expression might be affected by beta-catenin expression in neuroendocrine neoplasms and especially in NEC.

Abstract

Abstract

Stress is a precipitating agent in neuropsychiatric disease and initiates relapse to drug-seeking behavior in addicted patients. Targeting the stress system in protracted abstinence from drugs of abuse with anxiolytics may be an effective treatment modality for substance use disorders. α2A-adrenergic receptors (α2A-ARs) in extended amygdala structures play key roles in dampening stress responses. Contrary to early thinking, α2A-ARs are expressed at non-noradrenergic sites in the brain. These non-noradrenergic α2A-ARs play important roles in stress-responses, but their cellular mechanisms of action are unclear. In humans, the α2A-AR agonist guanfacine reduces overall craving and uncouples craving from stress yet minimally affects relapse, potentially due to competing actions in the brain. Here we show that heteroceptor α2A-ARs postsynaptically enhance dorsal BNST (dBNST) neuronal activity in mice of both sexes. This effect is mediated by hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels, as inhibition of these channels is necessary and sufficient for excitatory actions. Finally, this excitatory action is mimicked by clozapine-N-oxide activation of the Gi-coupled DREADD hM4Di in dBNST neurons, and its activation elicits anxiety-like behavior in the elevated plus maze. Together, this data provides a framework for elucidating cell-specific actions of GPCR signaling and provides a potential mechanism whereby competing anxiogenic and anxiolytic actions of guanfacine may affect its clinical utility in the treatment of addiction.SIGNIFICANCE STATEMENTStress impacts the development of neuropsychiatric disorders including anxiety and addiction. Guanfacine is an α2A-adrenergic receptor (α2A-AR) agonist with actions in the bed nucleus of the stria terminalis (BNST) that produces antidepressant actions and uncouples stress from reward-related behaviors. Here we show that guanfacine increases dBNST neuronal activity through actions at postsynaptic α2A-ARs via a mechanism that involves hyperpolarization-activated cyclic nucleotide gated cation (HCN) channels. This action is mimicked by activation of the designer receptor hM4Di expressed in the BNST, which also induces anxiety-like behaviors. Together, these data suggest 1) that postsynaptic α2A-ARs in BNST have excitatory actions on BNST neurons, and 2) these actions can be phenocopied by the so-called "inhibitory" DREADDs, suggesting care must be taken regarding interpretation of data obtained with these tools.