DAG-sensitive and Ca(2+) permeable TRPC6 channels are expressed in dentate granule cells and interneurons in the hippocampal formation

Members of the transient receptor potential (TRP) cation channel family play important roles in several neuronal functions. To understand the precise role of these channels in information processing, their presence on neuronal elements must be revealed. In this study, we investigated the localization of TRPC6 channels in the adult hippocampal formation. Immunostainings with a specific antibody, which was validated in Trpc6 knockout mice, showed that in the dentate gyrus, TRPC6 channels are strongly expressed in granule cells. Immunogold staining revealing the subcellular localization of TRPC6 channels clarified that these proteins were predominantly present on the membrane surface of the dendritic shafts of dentate granule cells, and also in their axons, often associated with intracellular membrane cisternae. In addition, TRPC6 channels could be observed in the dendrites of some interneurons. Double immunofluorescent staining showed that TRPC6 channels were present in the dendrites of hilar interneurons and hippocampal interneurons with horizontal dendrites in the stratum oriens expressing mGlu1a receptors, whereas parvalbumin immunoreactivity was revealed in TRPC6-expressing dendrites with radial appearance in the stratum radiatum. Electron microscopy showed that the immunogold particles depicting TRPC6 channels were located on the surface membranes of the interneuron dendrites. Our results suggest that TRPC6 channels are in a key position to alter the information entry into the trisynaptic loop of the hippocampal formation from the entorhinal cortex, and to control the function of both feed-forward and feed-back inhibitory circuits in this brain region

DEFiNE: A Method for Enhancement and Quantification of Fluorescently Labeled Axons

Visualization and quantification of fluorescently labeled axonal fibers are widely employed in studies of neuronal connectivity in the brain. However, accurate analysis of axon density is often confounded by autofluorescence and other fluorescent artifacts. By the time these problems are detected in labeled tissue sections, significant time and resources have been invested, and the tissue may not be easy to replace. In response to these difficulties, we have developed Digital Enhancement of Fibers with Noise Elimination (DEFiNE), a method for eliminating fluorescent artifacts from digital images based on their morphology and fluorescence spectrum, thus permitting enhanced visualization and quantification of axonal fibers. Application of this method is facilitated by a DEFiNE macro, written using ImageJ Macro Language (IJM), which includes an automated and customizable procedure for image processing and a semi-automated quantification method that accounts for any remaining local variation in background intensity. The DEFiNE macro is open-source and used with the widely available FIJI software for maximum accessibility

Allelic mutations of the sodium channel SCN8A reveal multiple cellular and physiological functions

Allelic mutations of Scn8a in the mouse have revealed the range of neurological disorders that can result from alternations of one neuronal sodium channel. Null mutations produce the most severe phenotype, with motor neuron failure leading to paralysis and juvenile lethality. Two less severe mutations cause ataxia, tremor, muscle weakness, and dystonia. The electrophysiological effects have been studied at the cellular level by recording from neurons from the mutant mice. The data demonstrate that Scn8a is required for the complex spiking of cerebellar Purkinje cells and for persistent sodium current in several classes of neurons, including some with pacemaker roles. The mouse mutations of Scn8a have also provided insight into the mode of inheritance of channelopathies, and led to the identification of a modifier gene that affects transcript splicing. These mutations demonstrate the value of mouse models to elucidate the pathophysiology of human disease.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/42795/1/10709_2004_Article_5381441.pd

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Two Subpopulations of Noradrenergic Neurons in the Locus Coeruleus Complex Distinguished by Expression of the Dorsal Neural Tube Marker Pax7

Central noradrenergic neurons, collectively defined by synthesis of the neurotransmitter norepinephrine, are a diverse collection of cells in the hindbrain, differing in their anatomy, physiological and behavioral functions, and susceptibility to disease and environmental insult. To investigate the developmental basis of this heterogeneity, we have used an intersectional genetic fate mapping strategy in mice to study the dorsoventral origins of the En1-derived locus coeruleus (LC) complex which encompasses virtually all of the anatomically defined LC proper, as well as a portion of the A7 and subcoeruleus (SubC) noradrenergic nuclei. We show that the noradrenergic neurons of the LC complex originate in two different territories of the En1 expression domain in the embryonic hindbrain. Consistent with prior studies, we confirm that the majority of the LC proper arises from the alar plate, the dorsal domain of the neural tube, as defined by expression of Pax7Cre. In addition, our analysis shows that a large proportion of the En1-derived A7 and SubC nuclei also originate in the Pax7Cre-defined alar plate. Surprisingly, however, we identify a smaller subpopulation of the LC complex that arises from outside the Pax7Cre expression domain. We characterize the distribution of these neurons within the LC complex, their cell morphology, and their axonal projection pattern. Compared to the broader LC complex, the newly identified Pax7Cre-negative noradrenergic subpopulation has very sparse projections to thalamic nuclei, suggestive of distinct functions. This developmental genetic analysis opens new avenues of investigation into the functional diversity of the LC complex

Recombinase-Dependent Mouse Lines for Chemogenetic Activation of Genetically Defined Cell Types

Chemogenetic technologies, including the mutated human Gq-coupled M3 muscarinic receptor (hM3Dq), have greatly facilitated our ability to directly link changes in cellular activity to altered physiology and behavior. Here, we extend the hM3Dq toolkit with recombinase-responsive mouse lines that permit hM3Dq expression in virtually any cell type. These alleles encode a fusion protein designed to increase effective expression levels by concentrating hM3Dq to the cell body and dendrites. To illustrate their broad utility, we targeted three different genetically defined cell populations: noradrenergic neurons of the compact, bilateral locus coeruleus and two dispersed populations, Camk2a+ neurons and GFAP+ glia. In all three populations, we observed reproducible expression and confirmed that activation of hM3Dq is sufficient to dose-dependently evoke phenotypic changes, without extreme phenotypes associated with hM3Dq overexpression. These alleles offer the ability to non-invasively control activity of diverse cell types to uncover their function and dysfunction at any developmental stage