HD 24355 observed by the Kepler K2 mission: a rapidly oscillating Ap star pulsating in a distorted quadrupole mode

We present an analysis of the first Kepler K2 mission observations of a rapidly oscillating Ap (roAp) star, HD 24355 (V = 9.65). The star was discovered in SuperWASP broad-band photometry with a frequency of 224.31 d−1 (2596.18 μHz; P = 6.4 min) and an amplitude of 1.51 mmag, with later spectroscopic analysis of low-resolution spectra showing HD 24355 to be an A5 Vp SrEu star. The high-precision K2 data allow us to identify 13 rotationally split sidelobes to the main pulsation frequency of HD 24355. This number of sidelobes combined with an unusual rotational phase variation show this star to be the most distorted quadrupole roAp pulsator yet observed. In modelling this star, we are able to reproduce well the amplitude modulation of the pulsation, and find a close match to the unusual phase variations. We show this star to have a pulsation frequency higher than the critical cut-off frequency. This is currently the only roAp star observed with the Kepler spacecraft in short cadence mode that has a photometric amplitude detectable from the ground, thus allowing comparison between the mmag amplitude ground-based targets and the μmag spaced-based discoveries. No further pulsation modes are identified in the K2 data, showing this star to be a single-mode pulsator

Analyzing the mechanisms that facilitate the subtype-specific assembly of γ-aminobutyric acid type A receptors

Impaired inhibitory signaling underlies the pathophysiology of many neuropsychiatric and neurodevelopmental disorders including autism spectrum disorders and epilepsy. Neuronal inhibition is regulated by synaptic and extrasynaptic γ-aminobutyric acid type A receptors (GABAARs), which mediate phasic and tonic inhibition, respectively. These two GABAAR subtypes differ in their function, ligand sensitivity, and physiological properties. Importantly, they contain different α subunit isoforms: synaptic GABAARs contain the α1–3 subunits whereas extrasynaptic GABAARs contain the α4–6 subunits. While the subunit composition is critical for the distinct roles of synaptic and extrasynaptic GABAAR subtypes in inhibition, the molecular mechanism of the subtype-specific assembly has not been elucidated. To address this issue, we purified endogenous α1- and α4-containing GABAARs from adult murine forebrains and examined their subunit composition and interacting proteins using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) and quantitative analysis. We found that the α1 and α4 subunits form separate populations of GABAARs and interact with distinct sets of binding proteins. We also discovered that the β3 subunit, which co-purifies with both the α1 and α4 subunits, has different levels of phosphorylation on serines 408 and 409 (S408/9) between the two receptor subtypes. To understand the role S408/9 plays in the assembly of α1- and α4-containing GABAARs, we examined the effects of S408/9A (alanine) knock-in mutation on the subunit composition of the two receptor subtypes using LC-MS/MS and quantitative analysis. We discovered that the S408/9A mutation results in the formation of novel α1α4-containing GABAARs. Moreover, in S408/9A mutants, the plasma membrane expression of the α4 subunit is increased whereas its retention in the endoplasmic reticulum is reduced. These findings suggest that S408/9 play a critical role in determining the subtype-specific assembly of GABAARs, and thus the efficacy of neuronal inhibition

Locally Reducing KCC2 Activity in the Hippocampus is Sufficient to Induce Temporal Lobe Epilepsy

Mesial temporal lobe epilepsy (mTLE) is the most common form of epilepsy, believed to arise in part from compromised GABAergic inhibition. The neuronal specific K+/Cl− co-transporter 2 (KCC2) is a critical determinant of the efficacy of GABAergic inhibition and deficits in its activity are observed in mTLE patients and animal models of epilepsy. To test if reductions of KCC2 activity directly contribute to the pathophysiology of mTLE, we locally ablated KCC2 expression in a subset of principal neurons within the adult hippocampus. Deletion of KCC2 resulted in compromised GABAergic inhibition and the development of spontaneous, recurrent generalized seizures. Moreover, local ablation of KCC2 activity resulted in hippocampal sclerosis, a key pathological change seen in mTLE. Collectively, our results demonstrate that local deficits in KCC2 activity within the hippocampus are sufficient to precipitate mTLE

A computationally inspired in-vivo approach identifies a link between amygdalar transcriptional heterogeneity, socialization and anxiety

Pharmaceutical breakthroughs for anxiety have been lackluster in the last half-century. Converging behavior and limbic molecular heterogeneity has the potential to revolutionize biomarker-driven interventions. However, current in vivo models too often deploy artificial systems including directed evolution, mutations and fear induction, which poorly mirror clinical manifestations. Here, we explore transcriptional heterogeneity of the amygdala in isogenic mice using an unbiased multidimensional computational approach that segregates intra-cohort reactions to moderate situational adversity and intersects it with high content molecular profiling. We show that while the computational approach stratifies known features of clinical anxiety including nitric oxide, opioid and corticotropin signaling, previously unrecognized druggable biomarkers emerge, such as calpain11 and scand1. Through ingenuity pathway analyses, we further describe a role for neurosteroid estradiol signaling, heat shock proteins, ubiquitin ligases and lipid metabolism. In addition, we report a remarkable behavioral pattern that maps to molecular features of anxiety in mice through counterphobic social attitudes, which manifest as increased, yet spatially distant socialization. These findings provide an unbiased approach for interrogating anxiolytics, and hint toward biomarkers underpinning behavioral and social patterns that merit further exploration

Spectrin-beta 2 facilitates the selective accumulation of GABAA receptors at somatodendritic synapses

Fast synaptic inhibition is dependent on targeting specific GABAAR subtypes to dendritic and axon initial segment (AIS) synapses. Synaptic GABAARs are typically assembled from α1-3, β and γ subunits. Here, we isolate distinct GABAARs from the brain and interrogate their composition using quantitative proteomics. We show that α2-containing receptors co-assemble with α1 subunits, whereas α1 receptors can form GABAARs with α1 as the sole α subunit. We demonstrate that α1 and α2 subunit-containing receptors co-purify with distinct spectrin isoforms; cytoskeletal proteins that link transmembrane proteins to the cytoskeleton. β2-spectrin was preferentially associated with α1-containing GABAARs at dendritic synapses, while β4-spectrin was associated with α2-containing GABAARs at AIS synapses. Ablating β2-spectrin expression reduced dendritic and AIS synapses containing α1 but increased the number of synapses containing α2, which altered phasic inhibition. Thus, we demonstrate a role for spectrins in the synapse-specific targeting of GABAARs, determining the efficacy of fast neuronal inhibition

Isolation and Characterization of Multi-Protein Complexes Enriched in the K-Cl Co-transporter 2 From Brain Plasma Membranes

Kcc2 plays a critical role in determining the efficacy of synaptic inhibition, however, the cellular mechanisms neurons use to regulate its membrane trafficking, stability and activity are ill-defined. To address these issues, we used affinity purification to isolate stable multi-protein complexes of K-Cl Co-transporter 2 (Kcc2) from the plasma membrane of murine forebrain. We resolved these using blue-native polyacrylamide gel electrophoresis (BN-PAGE) coupled to LC-MS/MS and label-free quantification. Data are available via ProteomeXchange with identifier PXD021368. Purified Kcc2 migrated as distinct molecular species of 300, 600, and 800 kDa following BN-PAGE. In excess of 90% coverage of the soluble N- and C-termini of Kcc2 was obtained. In total we identified 246 proteins significantly associated with Kcc2. The 300 kDa species largely contained Kcc2, which is consistent with a dimeric quaternary structure for this transporter. The 600 and 800 kDa species represented stable multi-protein complexes of Kcc2. We identified a set of novel structural, ion transporting, immune related and signaling protein interactors, that are present at both excitatory and inhibitory synapses, consistent with the proposed localization of Kcc2. These included spectrins, C1qa/b/c and the IP3 receptor. We also identified interactors more directly associated with phosphorylation; Akap5, Akap13, and Lmtk3. Finally, we used LC-MS/MS on the same purified endogenous plasma membrane Kcc2 to detect phosphorylation sites. We detected 11 sites with high confidence, including known and novel sites. Collectively our experiments demonstrate that Kcc2 is associated with components of the neuronal cytoskeleton and signaling molecules that may act to regulate transporter membrane trafficking, stability, and activity

Development of a generic activities model of command and control

This paper reports on five different models of command and control. Four different models are reviewed: a process model, a contextual control model, a decision ladder model and a functional model. Further to this, command and control activities are analysed in three distinct domains: armed forces, emergency services and civilian services. From this analysis, taxonomies of command and control activities are developed that give rise to an activities model of command and control. This model will be used to guide further research into technological support of command and control activities

The ASTRA Spectrophotometer: A July 2004 Progress Report

A cross-dispersed spectrophotometer with CCD detector and its automated 0.5-m telescope at the Fairborn Observatory, now under construction, should begin observations in 6 to 9 months. The Citadel ASTRA Telescope will be able to observe Vega the primary standard, make rapid measurements of the naked-eye stars, use 10 minutes per hour to obtain photometric measurements of the nightly extinction, and obtain high quality observations of V=10.5 mag. stars in an hour. The approximate wavelength range is 3300-9000A with a resolution of 14A in first and 7A in second order. Filter photometric magnitudes and indices will be calibrated in part for use as quality checks. Science observations for major projects such as comparisons with model atmospheres codes and for exploratory investigations should also begin in the first year. The ASTRA team realizes to deal with this potential data flood that they will need help to make the best scientific uses of the data. Thus they are interested in discussing possible collaborations. In less than a year of normal observing, all isolated stars in the Bright Star Catalog that can be observed can have their fluxes well measured. Some A Star related applications are discussed

KCC2 is required for the survival of mature neurons but not for their development

The K+/Cl- co-transporter KCC2 (SLC12A5) allows mature neurons in the CNS to maintain low intracellular Cl- levels that are critical in mediating fast hyperpolarizing synaptic inhibition via type A γ-aminobutyric acid receptors (GABAARs). In accordance with this, compromised KCC2 activity results in seizures, but whether such deficits directly contribute to the subsequent changes in neuronal structure and viability that lead to epileptogenesis, remains to be assessed. Canonical hyperpolarizing GABAAR currents develop postnatally which reflect a progressive increase in KCC2 expression levels and activity. To investigate the role that KCC2 plays in regulating neuronal viability and architecture we have conditionally ablated KCC2 expression in developing and mature neurons. Decreasing KCC2 expression in mature neurons resulted in the rapid activation of the extrinsic apoptotic pathway. Intriguingly, direct pharmacological inhibition of KCC2 in mature neurons was sufficient to rapidly induce apoptosis, an effect that was not abrogated via blockade of neuronal depolarization using Tetrodotoxin (TTX). In contrast, ablating KCC2 expression in immature neurons had no discernable effects on their subsequent development, arborization or dendritic structure. However, removing KCC2 in immature neurons was sufficient to ablate the subsequent postnatal development of hyperpolarizing GABAAR currents. Collectively, our results demonstrate that KCC2 plays a critical role in neuronal survival by limiting apoptosis, and mature neurons are highly sensitive to the loss of KCC2 function. In contrast, KCC2 appears to play a minimal role in mediating neuronal development or architecture

Identification of a Core Amino Acid Motif within the α Subunit of GABAᴀRs that Promotes Inhibitory Synaptogenesis and Resilience to Seizures

The fidelity of inhibitory neurotransmission is dependent on the accumulation of γ-aminobutyric acid type A receptors (GABAARs) at the appropriate synaptic sites. Synaptic GABAARs are constructed from α(1-3), β(1-3), and γ2 subunits, and neurons can target these subtypes to specific synapses. Here, we identify a 15-amino acid inhibitory synapse targeting motif (ISTM) within the α2 subunit that promotes the association between GABAARs and the inhibitory scaffold proteins collybistin and gephyrin. Using mice in which the ISTM has been introduced into the α1 subunit (Gabra1-2 mice), we show that the ISTM is critical for axo-axonic synapse formation, the efficacy of GABAergic neurotransmission, and seizure sensitivity. The Gabra1-2 mutation rescues seizure-induced lethality in Gabra2-1 mice, which lack axo-axonic synapses due to the deletion of the ISTM from the α2 subunit. Taken together, our data demonstrate that the ISTM plays a critical role in promoting inhibitory synapse formation, both in the axonic and somatodendritic compartments