Evidence for a nuclear compartment of transcription and splicing located at chromosome domain boundaries

The nuclear topography of splicing snRNPs, mRNA transcripts and chromosome domains in various mammalian cell types are described. The visualization of splicing snRNPs, defined by the Sm antigen, and coiled bodies, revealed distinctly different distribution patterns in these cell types. Heat shock experiments confirmed that the distribution patterns also depend on physiological parameters. Using a combination of fluorescencein situ hybridization and immunodetection protocols, individual chromosome domains were visualized simultaneously with the Sm antigen or the transcript of an integrated human papilloma virus genome. Three-dimensional analysis of fluorescence-stained target regions was performed by confocal laser scanning microscopy. RNA transcripts and components of the splicing machinery were found to be generally excluded from the interior of the territories occupied by the individual chromosomes. Based on these findings we present a model for the functional compartmentalization of the cell nucleus. According to this model the space between chromosome domains, including the surface areas of these domains, defines a three-dimensional network-like compartment, termed the interchromosome domain (ICD) compartment, in which transcription and splicing of mRNA occurs

A Digital Repository and Execution Platform for Interactive Scholarly Publications in Neuroscience

The CARMEN Virtual Laboratory (VL) is a cloud-based platform which allows neuroscientists to store, share, develop, execute, reproduce and publicise their work. This paper describes new functionality in the CARMEN VL: an interactive publications repository. This new facility allows users to link data and software to publications. This enables other users to examine data and software associated with the publication and execute the associated software within the VL using the same data as the authors used in the publication. The cloud-based architecture and SaaS (Software as a Service) framework allows vast data sets to be uploaded and analysed using software services. Thus, this new interactive publications facility allows others to build on research results through reuse. This aligns with recent developments by funding agencies, institutions, and publishers with a move to open access research. Open access provides reproducibility and verification of research resources and results. Publications and their associated data and software will be assured of long-term preservation and curation in the repository. Further, analysing research data and the evaluations described in publications frequently requires a number of execution stages many of which are iterative. The VL provides a scientific workflow environment to combine software services into a processing tree. These workflows can also be associated with publications and executed by users. The VL also provides a secure environment where users can decide the access rights for each resource to ensure copyright and privacy restrictions are met

Hyperexcitability of the local cortical circuit in mouse models of tuberous sclerosis complex

Tuberous sclerosis complex (TSC) is a neurogenetic disorder associated with epilepsy, intellectual disabilities, and autistic behaviors. These neurological symptoms result from synaptic dysregulations, which shift a balance between excitation and inhibition. To decipher the synaptic substrate of hyperexcitability, we examined pan-neuronal Tsc1 knockout mouse and found a reduction in surface expression of a GABA receptor (GABAR) subunit but not AMPA receptor (AMPAR) subunit. Using electrophysiological recordings, we found a significant reduction in the frequency of GABAR-mediated miniature inhibitory postsynaptic currents (GABAR-mIPSCs) but not AMPAR-mediated miniature excitatory postsynaptic currents (AMPAR-mEPSCs) in layer 2/3 pyramidal neurons. To determine a subpopulation of interneurons that are especially vulnerable to the absence of TSC1 function, we also analyzed two strains of conditional knockout mice targeting two of the prominent interneuron subtypes that express parvalbumin (PV) or somatostatin (SST). Unlike pan-neuronal knockout mice, both interneuron-specific Tsc-1 knockout mice did not develop spontaneous seizures and grew into adults. Further, the properties of AMPAR-mEPSCs and GABAR-mIPSCs were normal in both Pv-Cre and Sst-Cre x Tsc1fl/fl knockout mice. These results indicate that removal of TSC1 from all neurons in a local cortical circuit results in hyperexcitability while connections between pyramidal neurons and interneurons expressing PV and SST are preserved in the layer 2/3 visual cortex. Our study suggests that another inhibitory cell type or a combination of multiple subtypes may be accountable for hyperexcitability in TSC. Keywords: Tuberous sclerosis complex; E/I balance; AMPA receptor; GABA receptor; Autism; Epilepsy; mTOR pathwa

Optimization of interneuron function by direct coupling of cell migration and axonal targeting

Neural circuit assembly relies on the precise synchronization of developmental processes, such as cell migration and axon targeting, but the cell-autonomous mechanisms coordinating these events remain largely unknown. Here we found that different classes of interneurons use distinct routes of migration to reach the embryonic cerebral cortex. Somatostatin-expressing interneurons that migrate through the marginal zone develop into Martinotti cells, one of the most distinctive classes of cortical interneurons. For these cells, migration through the marginal zone is linked to the development of their characteristic layer 1 axonal arborization. Altering the normal migratory route of Martinotti cells by conditional deletion of Mafb—a gene that is preferentially expressed by these cells—cell-autonomously disrupts axonal development and impairs the function of these cells in vivo. Our results suggest that migration and axon targeting programs are coupled to optimize the assembly of inhibitory circuits in the cerebral cortex

Post hoc immunostaining of GABAergic neuronal subtypes following in vivo two-photon calcium imaging in mouse neocortex

GABAergic neurons in the neocortex are diverse with regard to morphology, physiology, and axonal targeting pattern, indicating functional specializations within the cortical microcircuitry. Little information is available, however, about functional properties of distinct subtypes of GABAergic neurons in the intact brain. Here, we combined in vivo two-photon calcium imaging in supragranular layers of the mouse neocortex with post hoc immunohistochemistry against the three calcium-binding proteins parvalbumin, calretinin, and calbindin in order to assign subtype marker profiles to neuronal activity. Following coronal sectioning of fixed brains, we matched cells in corresponding volumes of image stacks acquired in vivo and in fixed brain slices. In GAD67-GFP mice, more than 95% of the GABAergic cells could be unambiguously matched, even in large volumes comprising more than a thousand interneurons. Triple immunostaining revealed a depth-dependent distribution of interneuron subtypes with increasing abundance of PV-positive neurons with depth. Most importantly, the triple-labeling approach was compatible with previous in vivo calcium imaging following bulk loading of Oregon Green 488 BAPTA-1, which allowed us to classify spontaneous calcium transients recorded in vivo according to the neurochemically defined GABAergic subtypes. Moreover, we demonstrate that post hoc immunostaining can also be applied to wild-type mice expressing the genetically encoded calcium indicator Yellow Cameleon 3.60 in cortical neurons. Our approach is a general and flexible method to distinguish GABAergic subtypes in cell populations previously imaged in the living animal. It should thus facilitate dissecting the functional roles of these subtypes in neural circuitry

Whole-body tissue stabilization and selective extractions via tissue-hydrogel hybrids for high-resolution intact circuit mapping and phenotyping

To facilitate fine-scale phenotyping of whole specimens, we describe here a set of tissue fixation-embedding, detergent-clearing and staining protocols that can be used to transform excised organs and whole organisms into optically transparent samples within 1–2 weeks without compromising their cellular architecture or endogenous fluorescence. PACT (passive CLARITY technique) and PARS (perfusion-assisted agent release in situ) use tissue-hydrogel hybrids to stabilize tissue biomolecules during selective lipid extraction, resulting in enhanced clearing efficiency and sample integrity. Furthermore, the macromolecule permeability of PACT- and PARS-processed tissue hybrids supports the diffusion of immunolabels throughout intact tissue, whereas RIMS (refractive index matching solution) grants high-resolution imaging at depth by further reducing light scattering in cleared and uncleared samples alike. These methods are adaptable to difficult-to-image tissues, such as bone (PACT-deCAL), and to magnified single-cell visualization (ePACT). Together, these protocols and solutions enable phenotyping of subcellular components and tracing cellular connectivity in intact biological networks

Identifying associations between diabetes and acute respiratory distress syndrome in patients with acute hypoxemic respiratory failure: an analysis of the LUNG SAFE database

Background: Diabetes mellitus is a common co-existing disease in the critically ill. Diabetes mellitus may reduce the risk of acute respiratory distress syndrome (ARDS), but data from previous studies are conflicting. The objective of this study was to evaluate associations between pre-existing diabetes mellitus and ARDS in critically ill patients with acute hypoxemic respiratory failure (AHRF). Methods: An ancillary analysis of a global, multi-centre prospective observational study (LUNG SAFE) was undertaken. LUNG SAFE evaluated all patients admitted to an intensive care unit (ICU) over a 4-week period, that required mechanical ventilation and met AHRF criteria. Patients who had their AHRF fully explained by cardiac failure were excluded. Important clinical characteristics were included in a stepwise selection approach (forward and backward selection combined with a significance level of 0.05) to identify a set of independent variables associated with having ARDS at any time, developing ARDS (defined as ARDS occurring after day 2 from meeting AHRF criteria) and with hospital mortality. Furthermore, propensity score analysis was undertaken to account for the differences in baseline characteristics between patients with and without diabetes mellitus, and the association between diabetes mellitus and outcomes of interest was assessed on matched samples. Results: Of the 4107 patients with AHRF included in this study, 3022 (73.6%) patients fulfilled ARDS criteria at admission or developed ARDS during their ICU stay. Diabetes mellitus was a pre-existing co-morbidity in 913 patients (22.2% of patients with AHRF). In multivariable analysis, there was no association between diabetes mellitus and having ARDS (OR 0.93 (0.78-1.11); p = 0.39), developing ARDS late (OR 0.79 (0.54-1.15); p = 0.22), or hospital mortality in patients with ARDS (1.15 (0.93-1.42); p = 0.19). In a matched sample of patients, there was no association between diabetes mellitus and outcomes of interest. Conclusions: In a large, global observational study of patients with AHRF, no association was found between diabetes mellitus and having ARDS, developing ARDS, or outcomes from ARDS. Trial registration: NCT02010073. Registered on 12 December 2013