Neural Co-culture and Glial Interaction

Neural Applications
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A neural co-culture is simply an in vitro culture that includes more than one type of cell. Because neurons co-exist with other types of cells in the central nervous system, such as microglia and astrocytes, neural co-cultures can better mimic the complex interactions between cells and provide a more robust platform for studying neurodevelopment, disease progression, and neural function. Dysfunction of microglia, a key component of the nervous system’s innate immune system, has been implicated in many neuropsychiatric and neurodegenerative diseases, including bipolar disorder, depression, Alzheimer’s disease, and Parkinson’s disease.

To explore neuro-immune interactions and how the Maestro is being used to investigate their impact in health and disease, download our Publication Highlights review.

 

Investigate microglial and astrocyte regulation of neural activity

Microglia continuously monitor their microenvironment and respond to pathological changes. They play a critical role in protecting the brain from runaway excitation by regulating local neural activity and acting as the resident macrophage scavengers.

By recording from co-cultures of primary cortical neurons and microglia, the Maestro MEA platform reveals the mechanisms underlying microglial control of neural activity. Read Badimon et al, Nature 2020 for more details.

microglial regulation of neural activity

Microglia regulate neuronal activity in an adenosine receptor-dependent manner. When neurons are active, they release adenosine triphosphate (ATP), which nearby microglia convert to the neural activity inhibitor adenosine. Microglial suppression of glutamate-induced activity is blocked by an adenosine receptor antagonist. Figure modified from Badimon et al, Nature 2020.

Astroctye-neuron interactions also support homeostatic regulation of neural network activity. When co-cultured with astrocytes, iPSC-derived glutamatergic neurons show a 40% increase in network bursting (n=10 wells). After the addition of 100 nM picrotoxin, the difference increases to 84%. No activity is seen with astrocytes alone (data not shown), suggesting the astrocytes are not responsible for the activity directly, but through their influence on the glutamatergic neurons.

Neural co-culture raster plot from MEA system
Neural co-culture activity in raster plot from Maestro MEA
Neural co-culture activity compared over time

(A) A raster plot of glutamatergic neurons and (B) glutamatergic neurons with astrocytes. (C) The response of network burst frequency in both cultures to 100 nM picrotoxin.

 

publication highlights

Publication Highlights: Neuroimmune

The neuroimmune field is always evolving. Download highlights from four publications by researchers using the Maestro platform to understand the neuronal and immune cell effects on network activity.  

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Develop complex models of neuro-immune organoid network development

Neuroimmune organoids offer a powerful model for studying the role of neuron-microglia interactions in neurogenesis and network development. Microglia not only protect neurons from stress and damage, they also accelerate network development. In Popova et al, Cell Stem Cell 2021 the Maestro MEA platform showed how transplanted microglia increased synchronous, oscillatory network activity of cortical neuron organoids.

Neural organoid with neuroimmune

Transplanted microglia promote and accelerated the development of synchronous network activity in cortical neural organoids. Figure from Popova et al, Cell Stem Cell, 2021.

 

Reveal the role of astrocytes and microglia in neuroinflammation and disease

Chronic neuroinflammation can impair glial function, leading to neurodegenerative disease. In Alzheimer’s disease, inflammation reduces microglial clearance of amyloid beta (Aβ42), resulting in synaptic damage and pruning.

In Li et al, Frontiers in Immunology 2020, researchers used the Maestro MEA platform to understand how amyloid beta accumulation impacts cortical neuron function. Exposure to Aβ42 oligomers leads to hyperexcitability in vitro after only 24 hrs, mirroring the phenotype observed in vivo. Changes in biophysical properties of neurons are also confirmed using spike sorting.

Co-culture with activated, bone-derived macrophages results in synaptic preservation and phenotype rescue, again mirroring in vivo observations and suggesting a promising therapeutic.

Spike shape recorded from neurons changing with addition of immune cells

(A) Cortical neuron activity is more sensitive to Aβ42oligomers (XL-oAβ42) compared to fibrils (fAβ42) after only 24 hrs. Activity significantly increases after exposure to both fibrils and oligomers after 48 hours. (B, C) Changes in activity are accompanied by changes in extracellular spike shape, including shorter trough-to-peak width and faster repolarization. Figure modified from of Li. et al, Frontier in Immunology 2020

Observe how these interactions develop over time and how individual components can alter your disease phenotype and understanding of its progression.

 

Watch the interplay of inhibitory and excitatory neurons in neural co-cultures

The balance of excitation and inhibition define the activity of neural circuits. Different regions of the brain contain different compositions of neuronal and glial cell types and their activity is marked by distinct network phenotypes.

With iPSC technology it is possible to customize the composition of your neural co-cultures by co-culturing known neuronal subtypes. In this example, glutamatergic and GABAergic neurons are cultured for two weeks at different proportions. The activity of cultures with predominately GABAergic populations peak around 2 weeks, but lack coordinated network activity. The large inhibitory population prevents synchronous bursts of network activity.

Network bursting is seen when the ratios are reversed, with excitatory glutamatergic neurons driving increased activity. With the addition of astrocytes, a 52/22/26% mixture (glutamatergic, GABAergic, and astrocyte) exhibits similar activity levels to primary rodent cortical neurons.

Neural activity raster plot of neural co-culture
Firing rate of neural co-culture over 4 weeks

(A) A raster plot of glutamatergic neurons and (B) glutamatergic neurons with astrocytes. (C) The response of network burst frequency in both cultures to 100 nM picrotoxin.

How does your culture compare? What types of cells are present and how are they influencing your results?

 

Model complex neural co-culture interactions with the Maestro Pro

  • Understand how the composition of your culture influences its activity and optimize for more of an in vivo phenotype.
  • Discover the role of microglia and astrocytes in neurodegenerative and neuropsychiatric disease.
  • Chart network development and disease progression simultaneously across 96 cultures.
  • Selectively target specific neuronal subpopulations with the Lumos optogenetic stimulator or electrically stimulate side-by-side cultures within the same well using the Ibidi well insert.

 

Neural co-culture assay protocol

Getting started with Maestro Pro and Edge couldn't be easier. Culture your neurons in an Axion multiwell MEA plate (Day 0).  Load the MEA plate into the Maestro MEA system at the desired recording times and begin recording.  Analyze the neural activity in the MEA plate label-free and in real-time with AxIS Navigator Neural Module software. 

Note: Glial cells proliferate. When choosing a starting density, it is best to start low with glial cells so they do not overrun the culture by the time you would like to record.

 

 

multiwell microelectrode array (MEA) system in lab

 

The advantage of measuring the neural activity of neurodisease models on the Maestro Pro and Edge systems:

  • Measure what matters – The Maestro Pro and Edge MEA systems directly measure neuronal action potentials. Indirect measurements like calcium imaging are unable to capture important but subtle changes to neural network signaling while gene and protein expression are insufficient to characterize function. The Maestro MEA platforms track activity in real-time, enabling you to answer the question that matters: Do your neurons fire as expected?

  • Analyze cell activity label-free – The Maestro MEA system performs noninvasive electrical measurements from the cultured neural population, circumventing the use of dyes/reporters that can perturb your cell model and confound results. Track activity over hours, weeks, and months from the same population of cells.

  • Probe cell models in the same plate they were cultured in – Neurons exist as a functional network of inter-linked cells. The Maestro MEA platforms preserve the complex functionality of your neural models. Platforms that require single-cell suspensions (automated patch clamp, flow cytometry), require more sample handling and destroy the networks that define the functionality of these neural cultures.

  • It's easy – You don't have to be an electrophysiologist to use the Maestro MEA system. Just culture your neurons in an MEA plate, load your plate into the Maestro MEA system, and record your neural data. Axion's data analysis tools will do the rest, even generating the publication-ready graphs you need.

Neural MEA technology

Neural MEA

 

What is a microelectrode array (MEA)?

Microelectrode arrays (MEA), also known as multielectrode arrays, contain a grid of tightly spaced electrodes embedded in the culture surface of the well. Electrically active cells, such as neurons, are plated and cultured over the electrodes. When neurons fire action potentials, the electrodes measure the extracellular voltage on a microsecond timescale. As the neurons attach and network with one another, an MEA can simultaneously sample from many locations across the culture to detect propagation and synchronization of neural activity across the cell network.

That’s it, an electrode and your cells. Since the electrodes are extracellular, the recording is noninvasive and does not alter the electrophysiology of the cells - you can measure the activity of your culture for minutes, days, or even months!

 


Watch the full video and discover if an MEA assay is right for your research.
Watch the full MEA video now
CytoView well bottom

An MEA of 64 electrodes embedded in the substate at the bottom of a well.

Rendering of cells growing over the electrodes at the bottom of the well

Neurons attach to the array and form a network. The microelectrodes detect the action potentials fired as well as their propagation across the network.

 

 

 

Brain waves in a dish

Neurons communicate with other cells via electrochemical signals. Many neural cell types form cellular networks, and MEAs allow us to capture and record the electrical activity that propagates through these networks.

Neurons fire action potentials that are detected by adjacent electrodes as extracellular spikes. As the network matures, neurons often synchronize their electrical activity and may exhibit network bursts, where neurons repeatedly fire groups of spikes over a short period of time.

The MEA detects each cell's activity, as well as the propagation of the activity across the network, with spatial and temporal precision. Patterns as complex as EEG-like waveforms, or "brain waves in a dish", can be observed. Axion's MEA assay captures key features of neural network behavior as functional endpoints - activity, synchrony, and network oscillations.

Action potentials recorded from electrodes

Action potentials are the defining feature of neuron function. High values indicate frequent action potential firing and low values indicate the neurons may have impaired function.

Synchrony reflects the prevalence and strength of synaptic connections, and thus how likely neurons are to generate action potentials simultaneously

Synapses are functional connections between neurons. Synchrony reflects the prevalence and strength of synaptic connections, and thus how likely neurons are to generate action potentials simultaneously on millisecond time scales.

Network oscillations, or network bursting, are defined by alternating periods of high and low activity

Network oscillations, or network bursting, as defined by alternating periods of high and low activity, are a hallmark of functional networks with excitatory and inhibitory neurons. Oscillation is a measure of how the spikes from all of the neurons are organized in time.

 

Do more with multiwell

Axion BioSystems offers multiwell plates, ranging from 6 to 96 wells, with an MEA embedded in the bottom of each well. Multiwell MEA plates allow you to study complex neural biology in a dish, from a single cell firing to network activity, across many conditions and cell types at once.