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BACKGROUND - Relapse is a critical barrier to effective long-term treatment of alcoholism, and stress is often cited as a key trigger to relapse. Numerous studies suggest that stress-induced reinstatement to drug-seeking behaviors is mediated by norepinephrine (NE) and corticotropin-releasing factor (CRF) signaling interactions in the bed nucleus of the stria terminalis (BNST), a brain region critical to many behavioral and physiologic responses to stressors. Here, we sought to directly examine the effects of NE on BNST CRF neuron activity and determine whether these effects may be modulated by chronic intermittent EtOH (CIE) exposure or a single restraint stress.
METHODS - Adult male CRF-tomato reporter mice were treatment-naïve, or either exposed to CIE for 2 weeks or to a single 1-hour restraint stress. Effects of application of exogenous NE on BNST CRF neuron activity were assessed via whole-cell patch-clamp electrophysiological techniques.
RESULTS - We found that NE depolarized BNST CRF neurons in naïve mice in a β-adrenergic receptor (AR)-dependent mechanism. CRF neurons from CIE- or stress-exposed mice had significantly elevated basal resting membrane potential compared to naïve mice. Furthermore, CIE and stress individually disrupted the ability of NE to depolarize CRF neurons, suggesting that both stress and CIE utilize β-AR signaling to modulate BNST CRF neurons. Neither stress nor CIE altered the ability of exogenous NE to inhibit evoked glutamatergic transmission onto BNST CRF neurons as shown in naïve mice, a mechanism previously shown to be α-AR-dependent.
CONCLUSIONS - Altogether, these findings suggest that stress and CIE interact with β-AR signaling to modulate BNST CRF neuron activity, potentially disrupting the α/β-AR balance of BNST CRF neuronal excitability. Restoration of α/β-AR balance may lead to novel therapies for the alleviation of many stress-related disorders.
© 2019 by the Research Society on Alcoholism.
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.
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.
The cellular mechanism(s) linking macrophages to norepinephrine (NE)-mediated regulation of thermogenesis have been a topic of debate. Here we identify sympathetic neuron-associated macrophages (SAMs) as a population of cells that mediate clearance of NE via expression of solute carrier family 6 member 2 (SLC6A2), an NE transporter, and monoamine oxidase A (MAOA), a degradation enzyme. Optogenetic activation of the sympathetic nervous system (SNS) upregulates NE uptake by SAMs and shifts the SAM profile to a more proinflammatory state. NE uptake by SAMs is prevented by genetic deletion of Slc6a2 or inhibition of the encoded transporter. We also observed an increased proportion of SAMs in the SNS of two mouse models of obesity. Genetic ablation of Slc6a2 in SAMs increases brown adipose tissue (BAT) content, causes browning of white fat, increases thermogenesis, and leads to substantial and sustained weight loss in obese mice. We further show that this pathway is conserved, as human sympathetic ganglia also contain SAMs expressing the analogous molecular machinery for NE clearance, which thus constitutes a potential target for obesity treatment.
The contribution of hormone-independent counterregulatory signals in defense of insulin-induced hypoglycemia was determined in adrenalectomized, overnight-fasted conscious dogs receiving hepatic portal vein insulin infusions at a rate 20-fold basal. Either euglycemia was maintained () or hypoglycemia (≈45 mg/dl) was allowed to occur. There were three hypoglycemic groups: one in which hepatic autoregulation against hypoglycemia occurred in the absence of sympathetic nervous system input (), one in which autoregulation occurred in the presence of norepinephrine (NE) signaling to fat and muscle (), and one in which autoregulation occurred in the presence of NE signaling to fat, muscle, and liver (). Average net hepatic glucose balance (NHGB) during the last hour for was -0.7 ± 0.1, 0.3 ± 0.1 ( < 0.01 vs. ), 0.7 ± 0.1 ( = 0.01 vs. ), and 0.8 ± 0.1 ( = 0.7 vs. ) mg·kg·min, respectively. Hypoglycemia per se () increased NHGB by causing an inhibition of net hepatic glycogen synthesis. NE signaling to fat and muscle () increased NHGB further by mobilizing gluconeogenic precursors resulting in a rise in gluconeogenesis. Lowering glucose per se decreased nonhepatic glucose uptake by 8.9 mg·kg·min, and the addition of increased neural efferent signaling to muscle and fat blocked glucose uptake further by 3.2 mg·kg·min The addition of increased neural efferent input to liver did not affect NHGB or nonhepatic glucose uptake significantly. In conclusion, even in the absence of increases in counterregulatory hormones, the body can defend itself against hypoglycemia using glucose autoregulation and increased neural efferent signaling, both of which stimulate hepatic glucose production and limit glucose utilization.
Copyright © 2017 the American Physiological Society.
Context - Dopamine β-hydroxylase (DBH) deficiency is a rare genetic disorder characterized by failure to convert dopamine to norepinephrine. DBH-deficient patients lack sympathetic adrenergic function and are therefore predisposed to orthostatic hypotension. DBH-deficient mice exhibit hyperinsulinemia, lower plasma glucose levels, and insulin resistance due to loss of tonic sympathetic inhibition of insulin secretion. The impact of DBH deficiency on glucose homeostasis in humans is unknown.
Case Description - We describe the metabolic profile of an adolescent female DBH-deficient patient. The patient underwent genetic testing, cardiovascular autonomic function testing, and evaluation of insulin secretion and sensitivity with hyperglycemic clamp under treatment-naive conditions. All procedures were repeated after 1 year of treatment with the norepinephrine prodrug droxidopa (300 mg, 3 times a day). Genetic testing showed a homozygous mutation in the DBH gene (rs74853476). Under treatment-naive conditions, she had undetectable plasma epinephrine and norepinephrine levels, resulting in sympathetic noradrenergic failure and orthostatic hypotension (-32 mm Hg supine to seated). She had high adiposity (41%) and fasting plasma insulin levels (25 μU/mL), with normal glucose (91 mg/dL). Hyperglycemic clamp revealed increased glucose-stimulated insulin secretion and insulin resistance. Droxidopa restored plasma norepinephrine and improved orthostatic tolerance, with modest effects on glucose homeostasis.
Conclusions - We provide evidence for impairment in cardiovascular autonomic regulation, hyperinsulinemia, enhanced glucose-stimulated insulin secretion, and insulin resistance in a DBH-deficient patient. These metabolic derangements were not corrected by chronic droxidopa treatment. These findings provide insight into the pathophysiology and treatment of DBH deficiency and into the importance of catecholaminergic mechanisms to resting metabolism.
Copyright © 2017 by the Endocrine Society
Modulation of neurotransmission by the catecholamine dopamine (DA) is conserved across phylogeny. In the nematode Caenorhabditis elegans, excess DA signaling triggers Swimming-Induced Paralysis (Swip), a phenotype first described in animals with loss of function mutations in the presynaptic DA transporter (dat-1). Swip has proven to be a phenotype suitable for the identification of novel dat-1 mutations as well as the identification of novel genes that impact DA signaling. Pharmacological manipulations can also induce Swip, though the reagents employed to date lack specificity and potency, limiting their use in evaluation of dat-1 expression and function. Our lab previously established the mammalian norepinephrine transporter (NET) inhibitor nisoxetine to be a potent antagonist of DA uptake conferred by DAT-1 following heterologous expression. Here we demonstrate the ability of low (μM) concentrations of nisoxetine to trigger Swip within minutes of incubation, with paralysis dependent on DA release and signaling, and non-additive with Swip triggered by dat-1 deletion. Using nisoxetine in combination with genetic mutations that impact DA release, we further demonstrate the utility of the drug for demonstrating contributions of presynaptic DA receptors and ion channels to Swip. Together, these findings reveal nisoxetine as a powerful reagent for monitoring multiple dimensions of DA signaling in vivo, thus providing a new resource that can be used to evaluate contributions of dat-1 and other genes linked to DA signaling without the potential for compensations that attend constitutive genetic mutations.
Copyright © 2016 Elsevier Ltd. All rights reserved.
The serotonergic raphe nuclei of the midbrain are principal centers from which serotonin neurons project to innervate cortical and sub-cortical structures. The dorsal raphe nuclei receive light input from the circadian visual system and indirect input from the biological clock nuclei. Dysregulation of serotonin neurotransmission is implicated in neurobehavioral disorders, such as depression and anxiety, and alterations in the serotonergic phenotype of raphe neurons have dramatic effects on affective behaviors in rodents. Here, we demonstrate that day length (photoperiod) during development induces enduring changes in mouse dorsal raphe serotonin neurons—programming their firing rate, responsiveness to noradrenergic stimulation, intrinsic electrical properties, serotonin and norepinephrine content in the midbrain, and depression/anxiety-related behavior in a melatonin receptor 1 (MT1)-dependent manner. Our results establish mechanisms by which seasonal photoperiods may dramatically and persistently alter the function of serotonin neurons.
Copyright © 2015 Elsevier Ltd. All rights reserved.