Project description:Profiling of transcriptional changes in rat astrocytes when co-cultured with neurons: comparison of astrocytes cultured alone with astrocytes co-cultured with mouse hippocampal neurons. Co-cultured astrocytes are isolated using cold jet, a novel tool for these neuron-glia cultures. Over the last decade, the importance of astrocyte-neuron communication in neuronal development and synaptic plasticity has become increasingly clear. Since neuron-astrocyte interactions represent highly dynamic and reciprocal processes, we hypothesized that at least part of the involved astrocyte genes may be regulated as a consequence of their interactions with maturing neurons. In order to identify such neuron-induced astrocyte genes in vitro, we tested the effectiveness of the ‘cold jet’, a new method for separation of neurons from co-cultured astrocytes. The cold jet method is performed under ice-cold conditions and avoids protease-mediated isolation of astrocytes or time-consuming centrifugation, yielding intact astrocyte mRNA with approximately 90% of neuronal RNA removed. Using this method, we executed genome-wide profiling in which RNA derived from astrocyte-only cultures was compared with astrocyte RNA derived from differentiating neuron-astrocyte co-cultures. Data analysis revealed changes in expression of a large number of mRNAs and biological processes, including novel findings. Thus, cold jet is an efficient method to separate astrocytes from neurons in co-culture, and in this study reveals that neurons induce robust gene-expression changes in co-cultured astrocytes.
Project description:Comparison of expression data of rat forebrain astrocytes from P1, P7 acutely isolated by immunopanning or cultured with astrocytes prepared by McCarthy and de Vellis' (1980) method. Elucidating the genes induced by serum in immunopannedrat astrocytes. Three biological replicates for each sample were done. MD-astrocytes were prepared as described in McCarthy and de Vellis 1980 and harvested for mRNA after 7DIV. IP-astrocytes were isolated from P1 or P7 Sprague Dawley rats and processed for RNA immediately (IP-astrocytes P1/P7), or cultured for 7 days in HBEGF before harvesting (Cult. IP-astrocytes P1/P7). For the serum studies, we plated IP-astrocytes P7 in MD-astrocyte media containing 10% fetal calf serum immediately after isolation and cultured them for 7 days. After 7 days, the cultures were either processed for total RNA or washed 3x with dPBS and astrocyte base media with HBEGF was added. The cells were cultured for an additional 7 days and then processed for RNA. We isolated total RNA with the QIAshredder and Qiagen RNeasy Mini Kit. We used the 3’IVT Express kit for preparation of the RNA and the Rat Genome 230 2.0 Array chip (Affymetrix, Santa Clara). RT-PCR was used to elucidate the level of contamination in each cell sample.
Project description:We have grown C6 glioma cells and rat astrocytes, as well as astrocyte cells co-cultured together with C6 glioma. We performed proteome-wide LC-MS analysis of this experimental groups. The data including LC-MS/MS raw files and exported MaxQuant report. For our co-cultivated in vitro model we used astrocytes and C6 glioma cells. Astrocytes cell lines isolated from rat brain tissue. We analyzed astrocytes in two conditions: beforeand after co-cultivation. Proteins were assessed in an untargeted label-free bottom-up proteomic experiment using IDA approach (i.e. InformationDependent Acquisition) on AB Sciex TripleTOF 6600 Q-TOF mass-spectrometer coupled with LFQ (label-free quantification) approach by MaxQuant software. Dataset covers 165 samples (11 biological rand 5 technical replicates)
Project description:Neurons induce a dramatic transformation in developing astrocytes, causing them to develop a complex stellate morphology resembling their appearance in vivo. However, the transcriptional changes that accompany this transformation are not known, nor are the signalling mechanisms responsible. Similarly, whether synaptic activity controls astrocytic gene expression and whether this leads to altered astrocytic function is unclear. This experiment seeks to investigate this non-cell-autonomously regulated gene expression by co-culturing astrocytes and neurons derived from closely related species (mouse and rat), and separating RNA-seq reads derived from each cell type in silico, thus shedding light on the signalling mechanisms underlying neuron-to-astrocyte communication and the functional consequences for astrocytes.
Project description:Primary cultures of astrocytes from rat optic nerve heads were treated with EGFR ligand, EGF. Two cell lines from two different rat donors were used. The sister cell cultures were set as control and EGF treated groups. Experiment Overall Design: experiment #1: compared control astrocyte cultures to sister cultures treated with EGF for 4 hours. Experiment Overall Design: experiment #2: compared control astrocyte cultures to sister cultures treated with EGF for 12 hours
Project description:We and other groups doumented that astrocytes modulate migration, maturation and myelin sythesis of oligodendrocytes through release of neurotransmitters, cytokins and other signaling molecules. However, much less is known about on how the oligodendrocytes affects the astrocytes. We compared the transcriptome of cortical astrocytes when cultured alone and co-cultured with non-touching immortalized precursor oligodendrocytes (Oli-neu) in insert systems. Experimental data indicate that the oligodendrocyte-conditioning medium has a substantial effect on the the gene expression in astrocytes. Moreover, oligodendendrocyte proximity remodels major astrocyte functional pathways.
Project description:Aubert2005 - Interaction between astrocytes
and neurons on energy metabolism
Enocded non-curated model. Issues:
- Confusing equations A.41 and A.42
- Missing values for parameters Vv,0 and dHb,0
This model is described in the article:
Interaction between
astrocytes and neurons studied using a mathematical model of
compartmentalized energy metabolism.
Aubert A, Costalat R.
J. Cereb. Blood Flow Metab. 2005 Nov;
25(11): 1476-1490
Abstract:
Understanding cerebral energy metabolism in neurons and
astrocytes is necessary for the interpretation of functional
brain imaging data. It has been suggested that astrocytes can
provide lactate as an energy fuel to neurons, a process
referred to as astrocyte-neuron lactate shuttle (ANLS). Some
authors challenged this hypothesis, defending the classical
view that glucose is the major energy substrate of neurons, at
rest as well as in response to a stimulation. To test the ANLS
hypothesis from a theoretical point of view, we developed a
mathematical model of compartmentalized energy metabolism
between neurons and astrocytes, adopting hypotheses highly
unfavorable to ANLS. Simulation results can be divided between
two groups, depending on the relative neuron versus astrocyte
stimulation. If this ratio is low, ANLS is observed during all
the stimulus and poststimulus periods (continuous ANLS), but a
high ratio induces ANLS only at the beginning of the stimulus
and during the poststimulus period (triphasic behavior).
Finally, our results show that current experimental data on
lactate kinetics are compatible with the ANLS hypothesis, and
that it is essential to assess the neuronal and astrocytic
NADH/NAD+ ratio changes to test the ANLS hypothesis.
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Project description:Astrocytes, the most abundant cells in the central nervous system, promote synapse formation and help refine neural connectivity. Although they are allocated to spatially distinct regional domains during development, it is unknown whether region-restricted astrocytes are functionally heterogeneous. Here we show that postnatal spinal cord astrocytes express several region-specific genes, and that ventral astrocyte-encoded Semaphorin3a (Sema3a) is required for proper motor neuron and sensory neuron circuit organization. Loss of astrocyte-encoded Sema3a led to dysregulated α−motor neuron axon initial segment orientation, markedly abnormal synaptic inputs, and selective death of α−but not of adjacent γ−motor neurons. Additionally, a subset of TrkA+ sensory afferents projected to ectopic ventral positions. These findings demonstrate that stable maintenance of a positional cue by developing astrocytes influences multiple aspects of sensorimotor circuit formation. More generally, they suggest that regional astrocyte heterogeneity may help to coordinate postnatal neural circuit refinement. 12 total samples consisting of three biological replicates each of flow sorted postnatal day 7 dorsal spinal cord astrocytes, ventral spinal cord astrocytes, dorsal SC non astrocytes, and ventral SC non astrocytes
Project description:Growing evidence implicates the importance of glia, particularly astrocytes, in neurological and psychiatric diseases. Here, we describe a rapid and robust method for the differentiation of highly pure populations of astrocytes from human induced pluripotent stem cells (hiPSCs), via a neural progenitor cell (NPC) intermediate. Using this method, we generated hiPSC-derived astrocyte populations (hiPSC-astrocytes) from 42 NPC lines (derived from 30 individuals) with an average of ~90% S100β-positive cells. Transcriptomic analysis demonstrated that the hiPSC-astrocytes are highly similar to primary human fetal astrocytes and characteristic of a non-reactive state. hiPSC-astrocytes respond to inflammatory stimulants, display phagocytic capacity and enhance microglial phagocytosis. hiPSC-astrocytes also possess spontaneous calcium transient activity. Our novel protocol is a reproducible, straightforward (single media) and rapid (<30 days) method to generate homogenous populations of hiPSC-astrocytes that can be used for neuron-astrocyte and microglia-astrocyte co-cultures for the study of neuropsychiatric disorders.
Project description:We co-cultured P0 TdTomato-expressing hippocampal neurons with wild type cortical astrocytes for 2, 4 or 7 days in vitro and then separated the populations using FACS. We then compared transcripts from co-cultured with those from astrocytes cultured alone