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G proteins are major transducers of signals from G-protein coupled receptors (GPCRs). They are made up of α, β, and γ subunits, with 16 Gα, 5 Gβ and 12 Gγ subunits. Though much is known about the specificity of Gα subunits, the specificity of Gβγs activated by a given GPCR and that activate each effector in vivo is not known. Here, we examined the in vivo Gβγ specificity of presynaptic α-adrenergic receptors (αARs) in both adrenergic (auto-αARs) and non-adrenergic neurons (hetero-αARs) for the first time. With a quantitative MRM proteomic analysis of neuronal Gβ and Gγ subunits, and co-immunoprecipitation of tagged αARs from mouse models including transgenic FLAG-αARs and knock-in HA-αARs, we investigated the in vivo specificity of Gβ and Gγ subunits to auto-αARs and hetero-αARs activated with epinephrine to understand the role of Gβγ specificity in diverse physiological functions such as anesthetic sparing, and working memory enhancement. We detected Gβ, Gγ, Gγ, and Gγ with activated auto αARs, whereas we found Gβ and Gγ preferentially interacted with activated hetero-αARs. Further understanding of in vivo Gβγ specificity to various GPCRs offers new insights into the multiplicity of genes for Gβ and Gγ, and the mechanisms underlying GPCR signaling through Gβγ subunits.
Stress contributes to numerous psychiatric disorders. Corticotropin releasing factor (CRF) signaling and CRF neurons in the bed nucleus of the stria terminalis (BNST) drive negative affective behaviors, thus agents that decrease activity of these cells may be of therapeutic interest. Here, we show that acute restraint stress increases cFos expression in CRF neurons in the mouse dorsal BNST, consistent with a role for these neurons in stress-related behaviors. We find that activation of α-adrenergic receptors (ARs) by the agonist guanfacine reduced cFos expression in these neurons both in stressed and unstressed conditions. Further, we find that α- and β-ARs differentially regulate excitatory drive onto these neurons. Pharmacological and channelrhodopsin-assisted mapping experiments suggest that α-ARs specifically reduce excitatory drive from parabrachial nucleus (PBN) afferents onto CRF neurons. Given that the α-AR is a G-linked GPCR, we assessed the impact of activating the G-coupled DREADD hM4Di in the PBN on restraint stress regulation of BNST CRF neurons. CNO activation of PBN hM4Di reduced stress-induced in BNST neurons. Further, using as an additional marker of BNST neuronal identity, we uncovered a female-specific upregulation of the coexpression of in BNST neurons following stress, which was prevented by ovariectomy. These findings show that stress activates BNST CRF neurons, and that α-AR activation suppresses the activity of these cells, at least in part by suppressing excitatory drive from PBN inputs onto CRF neurons. Stress is a major variable contributing to mood disorders. Here, we show that stress increases activation of BNST CRF neurons that drive negative affective behavior. We find that the clinically well tolerated α-AR agonist guanfacine reduces activity of these cells , and reduces excitatory PBN inputs onto these cells Additionally, we uncover a novel sex-dependent coexpression of with in female BNST neurons after stress, an effect abolished by ovariectomy. These results demonstrate input-specific interactions between norepinephrine and CRF, and point to an action by which guanfacine may reduce negative affective responses.
Copyright © 2019 the authors 0270-6474/19/390472-13$15.00/0.
BACKGROUND - Dysregulation of arousal is symptomatic of numerous psychiatric disorders. Previous research has shown that the activity of dopamine (DA) neurons in the ventral periaqueductal gray (vPAG) tracks with arousal state, and lesions of vPAG cells increase sleep. However, the circuitry controlling these wake-promoting DA neurons is unknown.
METHODS - This study combined designer receptors exclusively activated by designer drugs (DREADDs), behavioral pharmacology, electrophysiology, and immunoelectron microscopy in male and female mice to elucidate mechanisms in the vPAG that promote arousal.
RESULTS - Activation of locus coeruleus projections to the vPAG or vPAG neurons induced by DREADDs promoted arousal. Similarly, agonist stimulation of vPAG alpha1-adrenergic receptors (α1ARs) increased latency to fall asleep, whereas α1AR blockade had the opposite effect. α1AR stimulation drove vPAG activity in a glutamate-dependent, action potential-independent manner. Compared with other dopaminergic brain regions, α1ARs were enriched on astrocytes in the vPAG, and mimicking α1AR transmission specifically in vPAG astrocytes via Gq-DREADDS was sufficient to increase arousal. In general, the wake-promoting effects observed were not accompanied by hyperactivity.
CONCLUSIONS - These experiments revealed that vPAG α1ARs increase arousal, promote glutamatergic input onto vPAG neurons, and are abundantly expressed on astrocytes. Activation of locus coeruleus inputs, vPAG astrocytes, or vPAG neurons increase sleep latency but do not produce hyperactivity. Together, these results support an arousal circuit whereby noradrenergic transmission at astrocytic α1ARs activates wake-promoting vPAG neurons via glutamate transmission.
Copyright © 2018 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.
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. α-adrenergic receptors (α-ARs) in extended amygdala structures play key roles in dampening stress responses. Contrary to early thinking, α-ARs are expressed at non-noradrenergic sites in the brain. These non-noradrenergic α-ARs play important roles in stress responses, but their cellular mechanisms of action are unclear. In humans, the α-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 α-ARs postsynaptically enhance dorsal bed nucleus of the stria terminalis (dBNST) neuronal activity in mice of both sexes. This effect is mediated by hyperpolarization-activated cyclic nucleotide-gated cation channels because inhibition of these channels is necessary and sufficient for excitatory actions. Finally, this excitatory action is mimicked by clozapine--oxide activation of the G-coupled DREADD hM4Di in dBNST neurons and its activation elicits anxiety-like behavior in the elevated plus maze. Together, these data provide a framework for elucidating cell-specific actions of GPCR signaling and provide a potential mechanism whereby competing anxiogenic and anxiolytic actions of guanfacine may affect its clinical utility in the treatment of addiction. Stress affects 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 dorsal BNST neuronal activity through actions at postsynaptic α2A-ARs via a mechanism that involves hyperpolarization-activated cyclic nucleotide gated cation 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 that postsynaptic α2A-ARs in BNST have excitatory actions on BNST neurons and that these actions can be phenocopied by the so-called "inhibitory" DREADDs, suggesting that care must be taken regarding interpretation of data obtained with these tools.
Copyright © 2018 the authors 0270-6474/18/388923-21$15.00/0.
The poor norepinephrine innervation and high density of Gi/o-coupled α- and α-adrenoceptors in the striatum and the dense striatal dopamine innervation have prompted the possibility that dopamine could be an effective adrenoceptor ligand. Nevertheless, the reported adrenoceptor agonistic properties of dopamine are still inconclusive. In this study, we analyzed the binding of norepinephrine, dopamine, and several compounds reported as selective dopamine D-like receptor ligands, such as the D receptor agonist 7-OH-PIPAT and the D receptor agonist RO-105824, to α-adrenoceptors in cortical and striatal tissue, which express α-adrenoceptors and both α- and α-adrenoceptors, respectively. The affinity of dopamine for α-adrenoceptors was found to be similar to that for D-like and D-like receptors. Moreover, the exogenous dopamine receptor ligands also showed high affinity for α- and α-adrenoceptors. Their ability to activate Gi/o proteins through α- and α-adrenoceptors was also analyzed in transfected cells with bioluminescent resonance energy transfer techniques. The relative ligand potencies and efficacies were dependent on the Gi/o protein subtype. Furthermore, dopamine binding to α-adrenoceptors was functional, inducing changes in dynamic mass redistribution, adenylyl cyclase activity, and ERK1/2 phosphorylation. Binding events were further studied with computer modeling of ligand docking. Docking of dopamine at α- and α-adrenoceptors was nearly identical to its binding to the crystallized D receptor. Therefore, we provide conclusive evidence that α- and α-adrenoceptors are functional receptors for norepinephrine, dopamine, and other previously assumed selective D-like receptor ligands, which calls for revisiting previous studies with those ligands.
Late-onset Alzheimer disease (AD) has a complex genetic etiology, involving locus heterogeneity, polygenic inheritance, and gene-gene interactions; however, the investigation of interactions in recent genome-wide association studies has been limited. We used a biological knowledge-driven approach to evaluate gene-gene interactions for consistency across 13 data sets from the Alzheimer Disease Genetics Consortium. Fifteen single nucleotide polymorphism (SNP)-SNP pairs within 3 gene-gene combinations were identified: SIRT1 × ABCB1, PSAP × PEBP4, and GRIN2B × ADRA1A. In addition, we extend a previously identified interaction from an endophenotype analysis between RYR3 × CACNA1C. Finally, post hoc gene expression analyses of the implicated SNPs further implicate SIRT1 and ABCB1, and implicate CDH23 which was most recently identified as an AD risk locus in an epigenetic analysis of AD. The observed interactions in this article highlight ways in which genotypic variation related to disease may depend on the genetic context in which it occurs. Further, our results highlight the utility of evaluating genetic interactions to explain additional variance in AD risk and identify novel molecular mechanisms of AD pathogenesis.
Copyright © 2016 Elsevier Inc. All rights reserved.
Heart rate recovery (HRR) after exercise is an independent predictor of adverse cardiovascular outcomes. HRR is mediated by both parasympathetic reactivation and sympathetic withdrawal and is highly heritable. We examined whether common genetic variants in adrenergic and cholinergic receptors and transporters affect HRR. In our study 126 healthy subjects (66 Caucasians, 56 African Americans) performed an 8 min step-wise bicycle exercise test with continuous computerized ECG recordings. We fitted an exponential curve to the postexercise R-R intervals for each subject to calculate the recovery constant (kr) as primary outcome. Secondary outcome was the root mean square residuals averaged over 1 min (RMS1min), a marker of parasympathetic tone. We used multiple linear regressions to determine the effect of functional candidate genetic variants in autonomic pathways (6 ADRA2A, 1 ADRA2B, 4 ADRA2C, 2 ADRB1, 3 ADRB2, 2 NET, 2 CHT, and 1 GRK5) on the outcomes before and after adjustment for potential confounders. Recovery constant was lower (indicating slower HRR) in ADRA2B 301-303 deletion carriers (n = 54, P = 0.01), explaining 3.6% of the interindividual variability in HRR. ADRA2A Asn251Lys, ADRA2C rs13118771, and ADRB1 Ser49Gly genotypes were associated with RMS1min. Genetic variability in adrenergic receptors may be associated with HRR after exercise. However, most of the interindividual variability in HRR remained unexplained by the variants examined. Noncandidate gene-driven approaches to study genetic contributions to HRR in larger cohorts will be of interest.
Copyright © 2015 the American Physiological Society.
Dopaminergic innervation of the extended amygdala regulates anxiety-like behavior and stress responsivity. A portion of this dopamine input arises from dopamine neurons located in the ventral lateral periaqueductal gray (vlPAG) and rostral (RLi) and caudal linear nuclei of the raphe (CLi). These neurons receive substantial norepinephrine input, which may prime them for involvement in stress responses. Using a mouse line that expresses eGFP under control of the tyrosine hydroxylase promoter, we explored the physiology and responsiveness to norepinephrine of these neurons. We find that RLi dopamine neurons differ from VTA dopamine neurons with respect to membrane resistance, capacitance and the hyperpolarization-activated current, Ih. Further, we found that norepinephrine increased the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) on RLi dopamine neurons. This effect was mediated through the α1 adrenergic receptor (AR), as the actions of norepinephrine were mimicked by the α1-AR agonist methoxamine and blocked by the α1-AR antagonist prazosin. This action of norepinephrine on sEPSCs was transient, as it did not persist in the presence of prazosin. Methoxamine also increased the frequency of miniature EPSCs, indicating that the α1-AR action on glutamatergic transmission likely has a presynaptic mechanism. There was also a modest decrease in sEPSC frequency with the application of the α2-AR agonist UK-14,304. These studies illustrate a potential mechanism through which norepinephrine could recruit the activity of this population of dopaminergic neurons.
Copyright © 2014 Elsevier Ltd. All rights reserved.
α2-adrenergic receptors (AR) within the bed nucleus of the stria terminalis (BNST) reduce stress-reward interactions in rodent models. In addition to their roles as autoreceptors, BNST α(2A)-ARs suppress glutamatergic transmission. One prominent glutamatergic input to the BNST originates from the parabrachial nucleus (PBN) and consists of asymmetric axosomatic synapses containing calcitonin gene-related peptide (CGRP) and vGluT2. Here we provide immunoelectron microscopic data showing that many asymmetric axosomatic synapses in the BNST contain α(2A)-ARs. Further, we examined optically evoked glutamate release ex vivo in BNST from mice with virally delivered channelrhodopsin2 (ChR2) expression in PBN. In BNST from these animals, ChR2 partially colocalized with CGRP, and activation generated EPSCs in dorsal anterolateral BNST neurons that elicited two cell-type-specific outcomes: (1) feedforward inhibition or (2) an EPSP that elicited firing. We found that the α(2A)-AR agonist guanfacine selectively inhibited this PBN input to the BNST, preferentially reducing the excitatory response in ex vivo mouse brain slices. To begin to assess the overall impact of α(2A)-AR control of this PBN input on BNST excitatory transmission, we used a Thy1-COP4 mouse line with little postsynaptic ChR2 expression nor colocalization of ChR2 with CGRP in the BNST. In slices from these mice, we found that guanfacine enhanced, rather than suppressed, optogenetically initiated excitatory drive in BNST. Thus, our study reveals distinct actions of PBN afferents within the BNST and suggests that α(2A)-AR agonists may filter excitatory transmission in the BNST by inhibiting a component of the PBN input while enhancing the actions of other inputs.
Copyright © 2014 the authors 0270-6474/14/349319-13$15.00/0.
The worldwide prevalence of obesity is steadily increasing, nearly doubling between 1980 and 2008. Obesity is often associated with insulin resistance, a major risk factor for type 2 diabetes mellitus (T2DM): a costly chronic disease and serious public health problem. The underlying cause of T2DM is a failure of the beta cells of the pancreas to continue to produce enough insulin to counteract insulin resistance. Most current T2DM therapeutics do not prevent continued loss of insulin secretion capacity, and those that do have the potential to preserve beta cell mass and function are not effective in all patients. Therefore, developing new methods for preventing and treating obesity and T2DM is very timely and of great significance. There is now considerable literature demonstrating a link between inhibitory guanine nucleotide-binding protein (G protein) and G protein-coupled receptor (GPCR) signaling in insulin-responsive tissues and the pathogenesis of obesity and T2DM. These studies are suggesting new and emerging therapeutic targets for these conditions. In this review, we will discuss inhibitory G proteins and GPCRs that have primary actions in the beta cell and other peripheral sites as therapeutic targets for obesity and T2DM, improving satiety, insulin resistance and/or beta cell biology.