Wide-Field Multi-Parameter FLIM: Long-Term Minimal Invasive Observation of Proteins in Living Cells.

Time-domain Fluorescence Lifetime Imaging Microscopy (FLIM) is a remarkable tool to monitor the dynamics of fluorophore-tagged protein domains inside living cells. We propose a Wide-Field Multi-Parameter FLIM method (WFMP-FLIM) aimed to monitor continuously living cells under minimum light intensity at a given illumination energy dose. A powerful data analysis technique applied to the WFMP-FLIM data sets allows to optimize the estimation accuracy of physical parameters at very low fluorescence signal levels approaching the lower bound theoretical limit. We demonstrate the efficiency of WFMP-FLIM by presenting two independent and relevant long-term experiments in cell biology: 1) FRET analysis of simultaneously recorded donor and acceptor fluorescence in living HeLa cells and 2) tracking of mitochondrial transport combined with fluorescence lifetime analysis in neuronal processes

T Cell Activation Results in Conformational Changes in the Src Family Kinase Lck to Induce Its Activation

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Astrocyte-to-neuron communication through integrin-engaged Thy-1/CBP/Csk/Src complex triggers neurite retraction via the RhoA/ROCK pathway.

Two key proteins for cellular communication between astrocytes and neurons are αvβ3 integrin and the receptor Thy-1. Binding of these molecules in the same (cis) or on adjacent (trans) cellular membranes induces Thy-1 clustering, triggering actin cytoskeleton remodeling. Molecular events that could explain how the Thy-1-αvβ3 integrin interaction signals have only been studied separately in different cell types, and the detailed transcellular communication and signal transduction pathways involved in neuronal cytoskeleton remodeling remain unresolved. Using biochemical and genetic approaches, single-molecule tracking, and high-resolution nanoscopy, we provide evidence that upon binding to αvβ3 integrin, Thy-1 mobility decreased while Thy-1 nanocluster size increased. This occurred concomitantly with inactivation and exclusion of the non-receptor tyrosine kinase Src from the Thy-1/C-terminal Src kinase (Csk)-binding protein (CBP)/Csk complex. The Src inactivation decreased the p190Rho GTPase activating protein phosphorylation, promoting RhoA activation, cofilin, and myosin light chain II phosphorylation and, consequently, neurite shortening. Finally, silencing the adaptor CBP demonstrated that this protein was a key transducer in the Thy-1 signaling cascade. In conclusion, these data support the hypothesis that the Thy-1-CBP-Csk-Src-RhoA-ROCK axis transmitted signals from astrocytic integrin-engaged Thy-1 (trans) to the neuronal actin cytoskeleton. Importantly, the β3 integrin in neurons (cis) was not found to be crucial for neurite shortening. This is the first study to detail the signaling pathway triggered by αvβ3, the endogenous Thy-1 ligand, highlighting the role of membrane-bound integrins as trans acting ligands in astrocyte-neuron communication

3D-Image Browser: Funktions- und Strukturmerkmale am Nagerhirn Sachlicher Abschlussbericht

SIGLEAvailable from TIB Hannover: DtF QN1(97,52) / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekBundesministerium fuer Bildung und Forschung (BMBF), Bonn (Germany)DEGerman

Mega Scan Kamera (2K x 2K Pixel) fuer die Elektronenmikroskopie Schlussbericht

Due to the progress in CCD-camera and computer technology digital image acquisition systems have actually reached a quality standard, which is equal to exposed film negatives by traditional photography. The goal of the present project was to install a high resolution slow scan camera system at a transmission electronmicroscope with special requirements for the use in neurobiology. Moreover, a software was generated for the electron microscope that allows the automatic acquisition and montage of multiple individual images (meanderscan). High resolution digital image acquistion in combination with a digital operated electronmicroscope is a prerequisite for efficient ultrastructural investigations of connected areas in the brain following learning processes or neurodegenerative diseases. (orig.)Available from TIB Hannover: DtF QN1(55,10) / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekSIGLEBundesministerium fuer Bildung, Wissenschaft, Forschung und Technologie, Bonn (Germany)DEGerman

Enhanced synaptic excitation-inhibition ratio in hippocampal interneurons of rats with temporal lobe epilepsy

A common feature of all epileptic syndromes is the repetitive occurrence of pathological patterns of synchronous neuronal activity, usually combined with increased neuronal discharge rates. Inhibitory interneurons of the hippocampal formation control both neuronal synchronization as well as the global level of activity and are therefore of crucial importance for epilepsy. Recent evidence suggests that changes in synaptic inhibition during temporal lobe epilepsy are rather specific, resulting from selective death or alteration of interneurons in specific hippocampal layers. Hence, epilepsy-induced changes have to be analysed separately for different types of interneurons. Here, we focused on GABAergic neurons located at the border between stratum radiatum and stratum lacunosum-moleculare of hippocampal area CA1 (SRL interneurons), which are included in feedforward inhibitory circuits. In chronically epileptic rats at 6-8 months after pilocarpine-induced status epilepticus, frequencies of spontaneous and miniature inhibitory postsynaptic currents were reduced, yielding an almost three-fold increase in excitation-inhibition ratio. Consistently, action potential frequency of SRL interneurons was about two-fold enhanced. Morphological alterations of the interneurons indicate that these functional changes were accompanied by remodelling of the local network, probably resulting in a loss of functional inhibitory synapses without conceivable cell death. Our data indicate a strong increase in activity of interneurons in dendritic layers of the chronically epileptic CA1 region. This alteration may enhance feedforward inhibition and rhythmogenesis and--together with specific changes in other interneurons--contribute to seizure susceptibility and pathological synchronization

Excitatory and inhibitory neurons express c-Fos in barrel-related columns after exploration of a novel environment

Recent work has shown that behaviorally meaningful sensory information processing is accompanied by the induction of several transcription factors in the barrel cortex of rodents. It is now generally accepted that stimulus-transcription coupling is an important step in the sequence of events leading to long-term plastic changes in neuronal structure and function. Nevertheless, so far few data are available as to what types of neurons are involved in such a genomic response. Here, we determined the morphological and neurochemical identity of neurons in rat barrel cortex showing a c-Fos-immunoreactive nucleus after exploration of an enriched environment. Double stainings of c-Fos and glial fibrillary acidic protein excluded astrocytes as a possible cell type expressing this transcription factor. By morphological phenotyping with intracellular Lucifer Yellow injections, it was found that a large majority were probably excitatory pyramidal cells, but inhibitory interneurons were also found to contain c-Fos-immunoreactive nuclei. By neurochemical phenotyping of GABAergic interneurons with specific antibodies, a significant induction was found, in a layer-dependent manner, for the populations of glutamic acid decarboxylase, parvalbumin-, calbindin- and vasoactive intestinal polypeptide-immunoreactive neurons but not for calretinin-immunoreactive cells in experimental compared to control columns.From these data we conclude that thalamic afferents effectively drive cortical excitatory as well as inhibitory intra-cortical circuits. Thus, the adaptations of receptive field properties of cortical neurons after different manipulations of the sensory periphery arc likely to be caused by plastic changes in excitatory and inhibitory networks. (C) 2002 IBRO. Published by Elsevier Science Ltd. All rights reserved