Structured tracking of alcohol reinforcement (STAR) for basic and translational alcohol research.

There is inherent tension between methodologies developed to address basic research questions in model species and those intended for preclinical to clinical translation: basic investigations require flexibility of experimental design as hypotheses are rapidly tested and revised, whereas preclinical models emphasize standardized protocols and specific outcome measures. This dichotomy is particularly relevant in alcohol research, which spans a diverse range of basic sciences in addition to intensive efforts towards understanding the pathophysiology of alcohol use disorder (AUD). To advance these goals there is a great need for approaches that facilitate synergy across basic and translational areas of nonhuman alcohol research. In male and female mice, we establish a modular alcohol reinforcement paradigm: Structured Tracking of Alcohol Reinforcement (STAR). STAR provides a robust platform for quantitative assessment of AUD-relevant behavioral domains within a flexible framework that allows direct crosstalk between translational and mechanistically oriented studies. To achieve cross-study integration, despite disparate task parameters, a straightforward multivariate phenotyping analysis is used to classify subjects based on propensity for heightened alcohol consumption and insensitivity to punishment. Combining STAR with extant preclinical alcohol models, we delineate longitudinal phenotype dynamics and reveal putative neuro-biomarkers of heightened alcohol use vulnerability via neurochemical profiling of cortical and brainstem tissues. Together, STAR allows quantification of time-resolved biobehavioral processes essential for basic research questions simultaneous with longitudinal phenotyping of clinically relevant outcomes, thereby providing a framework to facilitate cohesion and translation in alcohol research.

The Actin Bundling Protein Fascin-1 as an ACE2-Accessory Protein.

We have previously shown that angiotensin-converting enzyme 2 (ACE2), an enzyme counterbalancing the deleterious effects of angiotensin type 1 receptor activation by production of vasodilatory peptides Angiotensin (Ang)-(1-9) and Ang-(1-7), is internalized and degraded in lysosomes following chronic Ang-II treatment. However, the molecular mechanisms involved in this effect remain unknown. In an attempt to identify the accessory proteins involved in this effect, we conducted a proteomic analysis in ACE2-transfected HEK293T cells. A single protein, fascin-1, was found to differentially interact with ACE2 after Ang-II treatment for 4 h. The interactions between fascin-1 and ACE2 were confirmed by confocal microscopy and co-immunoprecipitation. Overexpression of fascin-1 attenuates the effects of Ang-II on ACE2 activity. In contrast, downregulation of fascin-1 severely decreased ACE2 enzymatic activity. Interestingly, in brain homogenates from hypertensive mice, we observed a significant reduction of fascin-1, suggesting that the levels of this protein may change in cardiovascular diseases. In conclusion, we identified fascin-1 as an ACE2-accessory protein, interacting with the enzyme in an Ang-II dependent manner and contributing to the regulation of enzyme activity.

Glutamatergic neurons of the paraventricular nucleus are critical contributors to the development of neurogenic hypertension.

KEY POINTS: Recurrent periods of over-excitation in the paraventricular nucleus (PVN) of the hypothalamus could contribute to chronic over-activation of this nucleus and thus enhanced sympathetic drive. Stimulation of the PVN glutamatergic population utilizing channelrhodopsin-2 leads to an immediate frequency-dependent increase in baseline blood pressure. Partial lesions of glutamatergic neurons of the PVN (39.3%) result in an attenuated rise in blood pressure following Deoxycorticosterone acetate (DOCA)-salt treatment and reduced index of sympathetic activity. These data suggest that stimulation of PVN glutamatergic neurons is sufficient to cause autonomic dysfunction and drive the increase in blood pressure during hypertension. ABSTRACT: Neuro-cardiovascular dysregulation leads to increased sympathetic activity and neurogenic hypertension. The paraventricular nucleus (PVN) of the hypothalamus is a key hub for blood pressure (BP) control, producing or relaying the increased sympathetic tone in hypertension. We hypothesize that increased central sympathetic drive is caused by chronic over-excitation of glutamatergic PVN neurons. We tested how stimulation or lesioning of excitatory PVN neurons in conscious mice affects BP, baroreflex and sympathetic activity. Glutamatergic PVN neurons were unilaterally transduced with channelrhodopsin-2 using an adeno-associated virus (CamKII-ChR2-eYFP-AAV2) in wildtype mice (n = 7) to assess the impact of acute stimulation of excitatory PVN neurons selectively on resting BP in conscious mice. Stimulation of the PVN glutamatergic population resulted in an immediate frequency-dependent (2, 10 and 20 Hz) increase in BP from baseline by ∼9 mmHg at 20 Hz stimulation (P < 0.001). Additionally, in vGlut2-cre mice glutamatergic neurons of the PVN were bilaterally lesioned utilizing a cre-dependent caspase (AAV2-flex-taCASP3-TEVp). Resting BP and urinary noradrenaline (norepinephrine) levels were then recorded in conscious mice before and after DOCA-salt hypertension. Partial lesions of glutamatergic neurons of the PVN (39.3%, P < 0.05) resulted in an attenuated rise in BP following DOCA-salt treatment (P < 0.05 at 7 day time point, n = 8). Noradrenaline levels as an index of sympathetic activity between the lesion and wildtype groups showed a significant reduction after DOCA-salt treatment in the lesioned animals (P < 0.05). These experiments suggest that stimulation of PVN glutamatergic neurons is sufficient to cause autonomic dysfunction and drive the increase in BP.

Perinatal Exposure to Western Diet Programs Autonomic Dysfunction in the Male Offspring.

Although the deleterious influence of protein deficiency on fetal programming is well documented, the impact of a Western diet on epigenetic mechanisms is less clear. We hypothesized that high-fat high-sucrose diet (HFHSD) consumption during pregnancy leads to epigenetic modifications within the progeny's compensatory renin-angiotensin system (RAS), affecting autonomic and metabolic functions. Dams were fed HFHSD (45% fat and 30% sucrose) or regular chow (RD) from mating until weaning of the pups (~7 weeks). Offspring from both groups were then maintained on chow and studied in adulthood (3-7 months). Offspring from HFHSD-exposed dams (OH) exhibited no difference in body weight or fasting blood glucose compared to controls (OR). In 3-month-old offspring, DNA methylation was significantly lower for the ACE2 gene (P < 0.05) in the brainstem, kidney and cecum. Moreover, ACE2 activity in the hypothalamus was increased at 7 months (OH: 91 ± 1 vs. OR: 74 ± 4 AFU/mg/min, P < 0.05). Although baseline blood pressure was not different between groups, vagal tone in OH was significantly impaired compared to OR. At the same time, OH offspring had a 1.7-fold increase in AT1a receptor expression and a 1.3-fold increase in ADAM17 mRNA. DOCA-salt treatment further revealed and exacerbated hypertensive response in the OH progeny (OH: 130 ± 6 vs. OR: 108 ± 3 mmHg, P < 0.05). Taken together, our data suggest that perinatal exposure to HFHSD resulted in epigenetic modifications of the compensatory brain RAS, potentially affecting plasticity of neuronal networks leading to autonomic dysfunction in the male offspring.

Systemic kappa opioid receptor antagonism accelerates reinforcement learning via augmentation of novelty processing in male mice.

Selective inhibition of kappa opioid receptors (KORs) is highly anticipated as a pharmacotherapeutic intervention for substance use disorders and depression. The accepted explanation for KOR antagonist-induced amelioration of aberrant behaviors posits that KORs globally function as a negative valence system; antagonism thereby blunts the behavioral influence of negative internal states such as anhedonia and negative affect. While effects of systemic KOR manipulations have been widely reproduced, explicit evaluation of negative valence as an explanatory construct is lacking. Here, we tested a series of falsifiable hypotheses generated a priori based on the negative valence model by pairing reinforcement learning tasks with systemic pharmacological KOR blockade in male C57BL/6J mice. The negative valence model failed to predict multiple experimental outcomes: KOR blockade accelerated contingency learning during both positive and negative reinforcement without altering innate responses to appetitive or aversive stimuli. We next proposed novelty processing, which influences learning independent of valence, as an alternative explanatory construct. Hypotheses based on novelty processing predicted subsequent observations: KOR blockade increased exploration of a novel, but not habituated, environment and augmented the reinforcing efficacy of novel visual stimuli in a sensory reinforcement task. Together, these results revise and extend long-standing theories of KOR system function.

Targeting diacylglycerol lipase reduces alcohol consumption in preclinical models.

Alcohol use disorder (AUD) is associated with substantial morbidity, mortality, and societal cost, and pharmacological treatment options for AUD are limited. The endogenous cannabinoid (eCB) signaling system is critically involved in reward processing and alcohol intake is positively correlated with release of the eCB ligand 2-Arachidonoylglycerol (2-AG) within reward neurocircuitry. Here we show that genetic and pharmacological inhibition of diacylglycerol lipase (DAGL), the rate limiting enzyme in the synthesis of 2-AG, reduces alcohol consumption in a variety of preclinical models ranging from a voluntary free-access model to aversion resistant-drinking and dependence-like drinking induced via chronic intermittent ethanol vapor exposure in mice. DAGL inhibition during either chronic alcohol consumption or protracted withdrawal was devoid of anxiogenic and depressive-like behavioral effects. Lastly, DAGL inhibition also prevented ethanol-induced suppression of GABAergic transmission onto midbrain dopamine neurons, providing mechanistic insight into how DAGL inhibition could affect alcohol reward. These data suggest reducing 2-AG signaling via inhibition of DAGL could represent an effective approach to reduce alcohol consumption across the spectrum of AUD severity.

Next-Generation Tools to Study Autonomic Regulation In Vivo.

The recent development of tools to decipher the intricacies of neural networks has improved our understanding of brain function. Optogenetics allows one to assess the direct outcome of activating a genetically-distinct population of neurons. Neurons are tagged with light-sensitive channels followed by photo-activation with an appropriate wavelength of light to functionally activate or silence them, resulting in quantifiable changes in the periphery. Capturing and manipulating activated neuron ensembles, is a recently-designed technique to permanently label activated neurons responsible for a physiological function and manipulate them. On the other hand, neurons can be transfected with genetically-encoded Ca2+ indicators to capture the interplay between them that modulates autonomic end-points or somatic behavior. These techniques work with millisecond temporal precision. In addition, neurons can be manipulated chronically to simulate physiological aberrations by transfecting designer G-protein-coupled receptors exclusively activated by designer drugs. In this review, we elaborate on the fundamental concepts and applications of these techniques in research.

ACE2 and ADAM17 Interaction Regulates the Activity of Presympathetic Neurons.

Brain renin angiotensin system within the paraventricular nucleus plays a critical role in balancing excitatory and inhibitory inputs to modulate sympathetic output and blood pressure regulation. We previously identified ACE2 and ADAM17 as a compensatory enzyme and a sheddase, respectively, involved in brain renin angiotensin system regulation. Here, we investigated the opposing contribution of ACE2 and ADAM17 to hypothalamic presympathetic activity and ultimately neurogenic hypertension. New mouse models were generated where ACE2 and ADAM17 were selectively knocked down from all neurons (AC-N) or Sim1 neurons (SAT), respectively. Neuronal ACE2 deletion revealed a reduction of inhibitory inputs to AC-N presympathetic neurons relevant to blood pressure regulation. Primary neuron cultures confirmed ACE2 expression on GABAergic neurons synapsing onto excitatory neurons within the hypothalamus but not on glutamatergic neurons. ADAM17 expression was shown to colocalize with angiotensin-II type 1 receptors on Sim1 neurons, and the pressor relevance of this neuronal population was demonstrated by photoactivation. Selective knockdown of ADAM17 was associated with a reduction of FosB gene expression, increased vagal tone, and prevented the acute pressor response to centrally administered angiotensin-II. Chronically, SAT mice exhibited a blunted blood pressure elevation and preserved ACE2 activity during development of salt-sensitive hypertension. Bicuculline injection in those models confirmed the supporting role of ACE2 on GABAergic tone to the paraventricular nucleus. Together, our study demonstrates the contrasting impact of ACE2 and ADAM17 on neuronal excitability of presympathetic neurons within the paraventricular nucleus and the consequences of this mutual regulation in the context of neurogenic hypertension.

Activation of ADAM17 (A Disintegrin and Metalloprotease 17) on Glutamatergic Neurons Selectively Promotes Sympathoexcitation.

Chronic activation of the brain renin-angiotensin system contributes to the development of hypertension by altering autonomic balance. Beyond the essential role of Ang II (angiotensin II) type 1 receptors, ADAM17 (A disintegrin and metalloprotease 17) is also found to promote brain renin-angiotensin system overactivation. ADAM17 is robustly expressed in various cell types within the central nervous system. The aim of this study was to determine whether ADAM17 modulates presympathetic neuronal activity to promote autonomic dysregulation in salt-sensitive hypertension. To test our hypothesis, ADAM17 was selectively knocked down in glutamatergic neurons using Cre-loxP technology. In mice lacking ADAM17 in glutamatergic neurons, the blood pressure increase induced by deoxycorticosterone acetate-salt treatment was blunted. Deoxycorticosterone acetate-salt significantly elevated cardiac and vascular sympathetic drive in control mice, while such effects were reduced in mice with ADAM17 knockdown. This blunted sympathoexcitation was extended to the spleen, with a lesser activation of the peripheral immune system, translating into a sequestration of circulating T cells within this organ, compared with controls. Within the paraventricular nucleus, Ang II-induced activation of kidney-related presympathetic glutamatergic neurons was reduced in ADAM17 knockdown mice, with the majority of cells no longer responding to Ang II stimulation, confirming the supportive role of ADAM17 in increasing presympathetic neuronal activity. Overall, our data highlight the pivotal role of neuronal ADAM17 in regulating sympathetic activity and demonstrate that activation of ADAM17 in glutamatergic neurons leads to a selective increase of sympathetic output, but not vagal tone, to specific organs, ultimately contributing to dysautonomia and salt-sensitive hypertension.

A Disintegrin and Metalloprotease 17 in the Cardiovascular and Central Nervous Systems.

ADAM17 is a metalloprotease and disintegrin that lodges in the plasmatic membrane of several cell types and is able to cleave a wide variety of cell surface proteins. It is somatically expressed in mammalian organisms and its proteolytic action influences several physiological and pathological processes. This review focuses on the structure of ADAM17, its signaling in the cardiovascular system and its participation in certain disorders involving the heart, blood vessels, and neural regulation of autonomic and cardiovascular modulation.