Inflammatory cytokine-induced changes in neural network activity measured by waveform analysis of high-content calcium imaging in murine cortical neurons

Benjamin D S Clarkson,Charles L Howe,Christina B Mccarthy,Robert J Kahoud

doi:10.1038/s41598-017-09182-5

Benjamin D S Clarkson, Charles L Howe + Show 2 more

Open Access

https://doi.org/10.1038/s41598-017-09182-5

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Journal: Scientific Reports	Publication Date: Aug 22, 2017
Citations: 34	License type: open-access

Affiliation: Mayo Clinic, Winneshiek Medical Center

Abstract

During acute neuroinflammation, increased levels of cytokines within the brain may contribute to synaptic reorganization that results in long-term changes in network hyperexcitability. Indeed, inflammatory cytokines are implicated in synaptic dysfunction in epilepsy and in an array of degenerative and autoimmune diseases of the central nervous system. Current tools for studying the impact of inflammatory factors on neural networks are either insufficiently fast and sensitive or require complicated and costly experimental rigs. Calcium imaging offers a reasonable surrogate for direct measurement of neuronal network activity, but traditional imaging paradigms are confounded by cellular heterogeneity and cannot readily distinguish between glial and neuronal calcium transients. While the establishment of pure neuron cultures is possible, the removal of glial cells ignores physiologically relevant cell-cell interactions that may be critical for circuit level disruptions induced by inflammatory factors. To overcome these issues, we provide techniques and algorithms for image processing and waveform feature extraction using automated analysis of spontaneous and evoked calcium transients in primary murine cortical neuron cultures transduced with an adeno-associated viral vector driving the GCaMP6f reporter behind a synapsin promoter. Using this system, we provide evidence of network perturbations induced by the inflammatory cytokines TNFα, IL1β, and IFNγ.

Highlights

Immune mediators and inflammatory cytokines have been implicated in synaptic dysfunction in an array of CNS inflammatory and autoimmune diseases, including multiple sclerosis[1,2,3], autoimmune epilepsy, limbic encephalopathy[4], febrile infection-related epilepsy syndrome[5, 6], and post-traumatic epilepsy[7, 8]
In addition to analysis of activity evolution in the neurons through time in vitro, we provide evidence of network perturbations induced by the inflammatory cytokines tumor necrosis factor alpha (TNFα), interleukin-1 beta (IL1β), and interferon gamma (IFNγ)
The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate receptor antagonists CNQX (3 μM; Fig. 4F,K) and NBQX (1.3 μM; Fig. 4K) both reduced the amplitude and network synchronization of calcium signals in the neurons but did not change the pulse width and only modestly decreased the frequency of the signals (Fig. 4K). These findings indicate that both NMDA and AMPA/kainate receptors are necessary for high-amplitude, synchronized spontaneous calcium transients in the network, but NMDA receptors drive the frequency response independently of AMPA/kainate receptors

Summary

Introduction

Immune mediators and inflammatory cytokines have been implicated in synaptic dysfunction in an array of CNS inflammatory and autoimmune diseases, including multiple sclerosis[1,2,3], autoimmune epilepsy, limbic encephalopathy[4], febrile infection-related epilepsy syndrome[5, 6], and post-traumatic epilepsy[7, 8]. Current strategies for medium-to-high throughput screening of potentially neuroactive compounds largely rely on multi-well microelectrode arrays Such arrays use non-invasive detection of extracellular field potentials in dissociated neuronal cultures to quantify spike frequencies and voltage waveforms and to measure neuronal synchronicity based on cross-correlation of spike events[13,14,15,16,17,18]. Recent advances in genetically encoded calcium indicators (GECIs) provide a means for stable cell-specific expression of calcium indicators with high signal-to-noise ratios and rapid fluorescence kinetics[27] These indicators allow repeated measures of calcium in neurons over substantially longer time frames than conventional fluorescent dye indicators that must be loaded into cells at the time of imaging and which have poor toxicity profiles. In addition to analysis of activity evolution in the neurons through time in vitro, we provide evidence of network perturbations induced by the inflammatory cytokines tumor necrosis factor alpha (TNFα), interleukin-1 beta (IL1β), and interferon gamma (IFNγ)

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Conclusion