The actin depolymerizing factor destrin serves as a negative feedback inhibitor of smooth muscle cell differentiation.

We have previously shown that several components of the RhoA signaling pathway control smooth muscle cell (SMC) phenotype by altering serum response factor (SRF)-dependent gene expression. Because our genome-wide analyses of chromatin structure and transcription factor binding suggested that the actin depolymerizing factor, destrin (DSTN), was regulated in a SMC-selective fashion, the goals of the current study were to identify the transcription mechanisms that control DSTN expression in SMC and to test whether it regulates SMC function. Immunohistochemical analyses revealed strong and at least partially SMC-selective expression of DSTN in many mouse tissues, a result consistent with human data from the genotype-tissue expression (GTEx) consortium. We identified several regulatory regions that control DSTN expression including a SMC-selective enhancer that was activated by myocardin-related transcription factor-A (MRTF-A), recombination signal binding protein for immunoglobulin κ-J region (RBPJ), and the SMAD transcription factors. Indeed, enhancer activity and endogenous DSTN expression were upregulated by RhoA and transforming growth factor-ß (TGF-ß) signaling and downregulated by inhibition of Notch cleavage. We also showed that DSTN expression was decreased in vivo by carotid artery injury and in cultured SMC cells by platelet-derived growth factor-BB (PDGF-BB) treatment. siRNA-mediated depletion of DSTN significantly enhanced MRTF-A nuclear localization and SMC differentiation marker gene expression, decreased SMC migration in scratch wound assays, and decreased SMC proliferation, as measured by cell number and cyclin-E expression. Taken together our data indicate that DSTN is a negative feedback inhibitor of RhoA/SRF-dependent gene expression in SMC that coordinately promotes SMC phenotypic modulation. Interventions that target DSTN expression or activity could serve as potential therapies for atherosclerosis and restenosis.NEW & NOTEWORTHY First, DSTN is selectively expressed in SMC in RhoA/SRF-dependent manner. Second, a SMC-selective enhancer just upstream of DSTN TSS harbors functional SRF, SMAD, and Notch/RBPJ binding elements. Third, DSTN depletion increased SRF-dependent SMC marker gene expression while inhibiting SMC migration and proliferation. Taken together, our data suggest that DSTN is a critical negative feedback inhibitor of SMC differentiation.

A murine model of increased coronary sinus pressure induces myocardial edema with cardiac lymphatic dilation and fibrosis.

Myocardial edema is a consequence of many cardiovascular stressors, including myocardial infarction, cardiac bypass surgery, and hypertension. The aim of this study was to establish a murine model of myocardial edema and elucidate the response of cardiac lymphatics and the myocardium. Myocardial edema without infarction was induced in mice by cauterizing the coronary sinus, increasing pressure in the coronary venous system, and inducing myocardial edema. In male mice, there was rapid development of edema 3 h following coronary sinus cauterization (CSC), with associated dilation of cardiac lymphatics. By 24 h, males displayed significant cardiovascular contractile dysfunction. In contrast, female mice exhibited a temporal delay in the formation of myocardial edema, with onset of cardiovascular dysfunction by 24 h. Furthermore, myocardial edema induced a ring of fibrosis around the epicardial surface of the left ventricle in both sexes that included fibroblasts, immune cells, and increased lymphatics. Interestingly, the pattern of fibrosis and the cells that make up the fibrotic epicardial ring differ between sexes. We conclude that a novel surgical model of myocardial edema without infarct was established in mice. Cardiac lymphatics compensated by exhibiting both an acute dilatory and chronic growth response. Transient myocardial edema was sufficient to induce a robust epicardial fibrotic and inflammatory response, with distinct sex differences, which underscores the sex-dependent differences that exist in cardiac vascular physiology.NEW & NOTEWORTHY Myocardial edema is a consequence of many cardiovascular stressors, including myocardial infarction, cardiac bypass surgery, and high blood pressure. Cardiac lymphatics regulate interstitial fluid balance and, in a myocardial infarction model, have been shown to be therapeutically targetable by increasing heart function. Cardiac lymphatics have only rarely been studied in a noninfarct setting in the heart, and so we characterized the first murine model of increased coronary sinus pressure to induce myocardial edema, demonstrating distinct sex differences in the response to myocardial edema. The temporal pattern of myocardial edema induction and resolution is different between males and females, underscoring sex-dependent differences in the response to myocardial edema. This model provides an important platform for future research in cardiovascular and lymphatic fields with the potential to develop therapeutic interventions for many common cardiovascular diseases.

Visual Psychophysics in Head-Fixed Mice.

We describe a set of protocols for doing visual psychophysical experiments in head-fixed mice. The goal of this approach was to conduct in mice the same type of precise and well-controlled tests of visual perception and decision making as is commonly done in primates. For example, these experimental protocols were the basis for our demonstration that mice are capable of visual selective attention in paradigms adapted from classic attention cueing paradigms in primates. Basic Protocol 1 describes how to construct the experimental apparatus, including the removable wheel assembly on which the mice run during the visual tasks, the lick spout used to deliver rewards and detect licks, and the behavioral box that places these components together with the visual displays. We also describe the functions of the computerized control system and the design of the customized head fixture. Basic Protocol 2 describes the preparation of mice for the experiments, including the detailed surgical steps. Basic Protocol 3 describes the transition to a food schedule for the mice and how to operate the experimental apparatus. Basic Protocol 4 outlines the logic of the task design and the steps necessary for training the mice. Finally, Basic Protocol 5 describes how to obtain and analyze the psychometric data. Our methods include several distinctive features, including a custom quick-release method for holding the head and specific strategies for training mice over multiple weeks. Published 2020. U.S. Government. Basic Protocol 1: Experimental apparatus Basic Protocol 2: Head fixture surgery Basic Protocol 3: General operation of the experimental apparatus Basic Protocol 4: Behavioral task design and training Basic Protocol 5: Psychometric data collection and analysis.

Activation of Striatal Neurons Causes a Perceptual Decision Bias during Visual Change Detection in Mice.

The basal ganglia are implicated in perceptual decision-making, although their specific contributions remain unclear. Here, we tested the causal role of the basal ganglia by manipulating neuronal activity in the dorsal striatum of mice performing a visual orientation-change detection (yes/no) task. Brief unilateral optogenetic stimulation caused large changes in task performance, shifting psychometric curves upward by increasing the probability of "yes" responses with only minor changes in sensitivity. For the direct pathway, these effects were significantly larger when the visual event was expected in the contralateral visual field, demonstrating a lateralized bias in responding to sensory inputs rather than a generalized increase in action initiation. For both direct and indirect pathways, the effects were specific to task epochs in which choice-relevant visual stimuli were present. These results indicate that the causal link between striatal activity and decision-making includes an additive perceptual bias in favor of expected or valued visual events.

The functional micro-organization of grid cells revealed by cellular-resolution imaging.

Establishing how grid cells are anatomically arranged, on a microscopic scale, in relation to their firing patterns in the environment would facilitate a greater microcircuit-level understanding of the brain's representation of space. However, all previous grid cell recordings used electrode techniques that provide limited descriptions of fine-scale organization. We therefore developed a technique for cellular-resolution functional imaging of medial entorhinal cortex (MEC) neurons in mice navigating a virtual linear track, enabling a new experimental approach to study MEC. Using these methods, we show that grid cells are physically clustered in MEC compared to nongrid cells. Additionally, we demonstrate that grid cells are functionally micro-organized: the similarity between the environment firing locations of grid cell pairs varies as a function of the distance between them according to a "Mexican hat"-shaped profile. This suggests that, on average, nearby grid cells have more similar spatial firing phases than those further apart.

Overexpression of neurotrophin-3 stimulates a second wave of dopaminergic amacrine cell genesis after birth in the mouse retina.

Dopaminergic amacrine (DA) cells play multiple and important roles in retinal function. Neurotrophins are known to modulate the number and morphology of DA cells, but the underlying regulatory mechanisms are unclear. Here, we investigate how neurotrophin-3 (NT-3) regulates DA cell density in the mouse retina. We demonstrate that overexpression of NT-3 upregulates DA cell number and leads to a consequent increase in the density of DA cell dendrites. To examine the mechanisms of DA cell density increase, we further investigate the effect of NT-3 overexpression on retinal apoptosis and mitosis during development. We find that NT-3 does not affect the well known wave of retinal cell apoptosis that normally occurs during the first 2 weeks after birth. Instead, overexpression of NT-3 promotes additional mitosis of DA cells at postnatal day 4, but does not affect cell mitosis before birth, the peak period of amacrine cell genesis in wild-type retinas. We next show that retinal explants cultured from birth to day 7 without extra NT-3 produced by lens exhibit similar number of DA cells as in wild type, further supporting the notion that postnatal overexpression of lens-derived NT-3 affects DA cell number. Moreover, the additional mitosis after birth in NT-3-overexpressing mice does not occur in calretinin-positive amacrine cells or PKC-positive rod ON bipolar cells. Thus, the NT-3-triggered wave of cell mitosis after birth is specific for the retinal DA cells.

Direction-specific disruption of subcortical visual behavior and receptive fields in mice lacking the beta2 subunit of nicotinic acetylcholine receptor.

Retinotopic mapping is a basic feature of visual system organization, but its role in processing visual information is unknown. Mutant mice lacking the beta2 subunit of nicotinic acetylcholine receptor have imprecise maps in both visual cortex (V1) and the superior colliculus (SC) due to the disruption of spontaneous retinal activity during development. Here, we use behavioral and physiological approaches to study their visual functions. We find that beta2-/- mice fail to track visual stimuli moving along the nasotemporal axis in a subcortical optomotor behavior, but track normally along the dorsoventral axis. In contrast, these mice display normal acuity along both axes in the visual water task, a behavioral test of cortical functions. Consistent with the behavioral results, we find that V1 neurons in beta2-/- mice have normal response properties, while SC neurons have disrupted receptive fields, including enlarged structure and decreased direction and orientation selectivity along the nasotemporal axis. The subcortical-specific deficits indicate that retinotopic map disruption has different impacts on the development of functional properties in V1 and the SC.