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Activity changes in neuron-astrocyte networks in culture under the effect of norepinephrine.

ABSTRACT: The concerted activity of neuron-glia networks is responsible for the fascinating dynamics of brain functions. Although these networks have been extensively investigated using a variety of experimental (in vivo and in vitro) and theoretical models, the manner by which neuron-glia networks interact is not fully understood. In particular, how neuromodulators influence network-level signaling between neurons and astrocytes was poorly addressed. In this work, we investigated global effects of the neuromodulator norepinephrine (NE) on neuron-astrocyte network communication in co-cultures of neurons and astrocytes and in isolated astrocyte networks. Electrical stimulation was used to activate the neuron-astrocyte glutamate-mediated pathway. Our results showed dramatic changes in network activity under applied global perturbations. Under neuromodulation, there was a marked rise in calcium signaling in astrocytes, neuronal spontaneous activity was reduced, and the communication between neuron-astrocyte networks was perturbed. Moreover, in the presence of NE, we observed two astrocyte behaviors based on their coupling to neurons. There were also morphological changes in astrocytes upon application of NE, suggesting a physical cause underlies the change in signaling. Our results shed light on the role of NE in controlling sleep-wake cycles.

SUBMITTER: Bar El Y

PROVIDER: S-EPMC6192555 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Activity changes in neuron-astrocyte networks in culture under the effect of norepinephrine.

Bar El Yasmin Y Kanner Sivan S Barzilai Ari A Hanein Yael Y

PloS one 20181017 10

The concerted activity of neuron-glia networks is responsible for the fascinating dynamics of brain functions. Although these networks have been extensively investigated using a variety of experimental (in vivo and in vitro) and theoretical models, the manner by which neuron-glia networks interact is not fully understood. In particular, how neuromodulators influence network-level signaling between neurons and astrocytes was poorly addressed. In this work, we investigated global effects of the ne ...[more]

PMID: 30332429

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Project description:Background:Monitoring the activity and morphology of neuron-astrocyte networks in culture is a powerful tool for studying dynamics, structure, and communication in neuron-astrocyte networks independently or as a model of the sub-brain network. These cultures are known to produce stereotypical patterns of activity, e.g., highly synchronized network bursts resembling sleep or seizure states, thus it enables the exploration of behaviors that can relate to brain function and disease. High-resolution microscopy of calcium imaging combined with simultaneous electrical recording provides a comprehensive overview on the network's dynamics. This setup makes it possible to apply global perturbations of electrical and chemical stimulation on the cultures during the recording task and to record the effects on network activity on-line. Morphological changes in the cultures can be obtained to have a complete dataset for structure-function study of neuron-astrocyte networks in vitro. Findings:The 4 TB of data presented here was recorded and imaged as part of an accompanying study looking at in vitro structure-function of neuron-astrocyte networks. Simultaneous optical (calcium imaging) and electrical (micro-electrode array) recordings lasted 5-12 minutes and included spontaneous activity recording, electrical and chemical stimulation of neuron-astrocyte, and isolated astrocyte cultures. The data include activity recordings of 58 different cultures, with 1-2 regions of interest recorded for each culture. Production procedures, experimental protocols, and reuse options are included. The data have been suitable to reveal changes in the activity and morphology of the cultures and enabled observation and analysis of neuron-astrocyte and isolated astrocyte culture behaviors under the applied perturbations. Conclusions:Our dataset is sufficient to show significant changes in activity and morphology of neuron-astrocyte networks in culture under the applied stimulations. More than 100 recordings of 58 different cultures give insight of the observation's significance and led to conclusions about astrocyte activity and neuron-astrocyte network communication. Making it available here will allow others to test new tools for calcium imaging analysis and extracellular neuronal voltage recordings.

Project description:BACKGROUND:Interactions between neurons, astrocytes, and microglia critically influence neuroinflammatory responses to insult in the central nervous system. In vitro astrocyte and microglia cultures are powerful tools to study specific molecular pathways involved in neuroinflammation; however, in order to better understand the influence of cellular crosstalk on neuroinflammation, new multicellular culture models are required. METHODS:Primary cortical cells taken from neonatal rats were cultured in a serum-free "tri-culture" medium formulated to support neurons, astrocytes, and microglia, or a "co-culture" medium formulated to support only neurons and astrocytes. Caspase 3/7 activity and morphological changes were used to quantify the response of the two culture types to different neuroinflammatory stimuli mimicking sterile bacterial infection (lipopolysaccharide (LPS) exposure), mechanical injury (scratch), and seizure activity (glutamate-induced excitotoxicity). The secreted cytokine profile of control and LPS-exposed co- and tri-cultures were also compared. RESULTS:The tri-culture maintained a physiologically relevant representation of neurons, astrocytes, and microglia for 14?days in vitro, while the co-cultures maintained a similar population of neurons and astrocytes, but lacked microglia. The continuous presence of microglia did not negatively impact the overall health of the neurons in the tri-culture, which showed reduced caspase 3/7 activity and similar neurite outgrowth as the co-cultures, along with an increase in the microglia-secreted neurotrophic factor IGF-1 and a significantly reduced concentration of CX3CL1 in the conditioned media. LPS-exposed tri-cultures showed significant astrocyte hypertrophy, increase in caspase 3/7 activity, and the secretion of a number of pro-inflammatory cytokines (e.g., TNF, IL-1?, IL-1?, and IL-6), none of which were observed in LPS-exposed co-cultures. Following mechanical trauma, the tri-culture showed increased caspase 3/7 activity, as compared to the co-culture, along with increased astrocyte migration towards the source of injury. Finally, the microglia in the tri-culture played a significant neuroprotective role during glutamate-induced excitotoxicity, with significantly reduced neuron loss and astrocyte hypertrophy in the tri-culture. CONCLUSIONS:The tri-culture consisting of neurons, astrocytes, and microglia more faithfully mimics in vivo neuroinflammatory responses than standard mono- and co-cultures. This tri-culture can be a useful tool to study neuroinflammation in vitro with improved accuracy in predicting in vivo neuroinflammatory phenomena.

Project description:Pathological hallmarks of Alzheimer's disease (AD) include deposition and accumulation of amyloid- β (Aβ), neurofibrillary tangle formation, and neuronal loss. Pathogenesis of presymptomatic disease stages remains elusive, although studies suggest that the early structural and functional alterations likely occur at neuronal dendritic spines. Presymptomatic alterations may also affect different CNS cell types. However, specific contributions of these cell types as cause or consequence of pathology are difficult to study in vivo. There is a shortage of relatively simple, well-defined, and validated in vitro models that allow a straightforward interpretation of results and recapitulate aspects of pathophysiology. For instance, dissecting the AD-related processes (e.g., neurotoxicity vs. synaptotoxicity) may be difficult with the common cell-based systems such as neuronal cell lines or primary neurons. To investigate and characterize the impact of reactive astrocytes on neuronal morphology in the context of AD-related cues, we modified an in vitro co-culture assay of primary mouse neurons and primary mouse astrocytes based on the so-called Banker "sandwich" co-culture assay. Here, we provide a simple and modular assay with fully differentiated primary mouse neurons to study the paracrine interactions between the neurons and the astrocytes in the co-culture setting. Readouts were obtained from both cell types in our assay. Astrocyte feeder cells were pre-exposed to neuroinflammatory conditions by means of Aβ42, Aβ40, or lipopolysaccharide (LPS). Non-cell autonomous toxic effects of reactive astrocytes on neurons were assessed using the Sholl analysis to evaluate the dendritic complexity, whereas synaptic puncta served as a readout of synaptotoxicity. Here, we show that astrocytes actively contribute to the phenotype of the primary neurons in an AD-specific context, emphasizing the role of different cell types in AD pathology. The cytokine expression pattern was significantly altered in the treated astrocytes. Of note, the impact of reactive astrocytes on neurons was highly dependent on the defined cell ratios. Our co-culture system is modular, of low cost, and allows us to probe aspects of neurodegeneration and neuroinflammation between the two major CNS cell types, neurons, and astrocytes, under well-defined experimental conditions. Our easy-to-follow protocol, including work-flow figures, may also provide a methodological outline to study the interactions of astrocytes and neurons in the context of other diseases in the future.

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Activity changes in neuron-astrocyte networks in culture under the effect of norepinephrine.

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Activity changes in neuron-astrocyte networks in culture under the effect of norepinephrine.

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