Neural Co-culture and Population Dynamics

Neural Applications
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Neural co-cultures are cultures that contain more than one type of cell. In vivo, a wide variety of neurons and other cells exist and interact together and the composition of a neural culture can alter development and diseases progression. A host of glial cells, like astrocytes, play a supporting role and are crucial to neural function. Microglia, which compose much of the immune response in the central nervous system, have been implicated in many neurodegenerative diseases, like Alzheimer’s. While these glial cells don’t directly fire action potentials, their influence on a co-culture’s activity can be dramatic.

 

Different neuronal compositions display unique patterns of activity

How different neuronal compositions influence activity can easily be seen in how cultures from different regions in the brain have distinct patterns of electrical activity. Each region has a unique blend of neuronal and glial subtypes. Primary murine neuronal cell cultures compared on the Maestro MEA demonstrate these distinct patterns in vitro.

 

Network spike train patterns of brain-region specific primary cell cultures from embryonic murine tissue
Neuro activity characterization of brain region in neural culture
Network spike train patterns of midbrain and frontal cortex specific primary cell cultures from embryonic murine tissue

Raster plots of primary cell cultures derived from embryonic murine tissue of the frontal cortex, spinal cord (with dorsal root ganglia), hippocampus, and midbrain (co-cultured with frontal cortex). Activity at 28 days in vitro is plotted for 60 seconds and shows distinguishable patterns based on the brain region of origin.  Data courtesy of Neuroproof GMBH, taken from Voss et al. 2014 presented at SfN2014.

 

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

The interaction of excitation and inhibition define the activity of neural circuits. Different regions of the brain contain different populations of neuronal subtypes and their activity is marked by distinct network activity. With iPSC technology it is possible to customize the composition of your neural co-cultures by mixing known neural subtypes together. In this example, glutamatergic and GABAergic neurons are cultured for two weeks at different proportions. Cultures with a predominately GABAergic populations showed activity that peaked around 2 weeks but lacked coordinated network activity. The large inhibitory population prevented synchronous bursts of network activity. Network bursting was seen when the ratios were reversed and excitatory glutamatergic neurons increased activity. With the addition of astrocytes, a 52/22/26% mixture (glutamatergic, GABAergic, and astrocyte) had 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

Raster plots of neurons at different ratios of glutamatergic neurons, GABAergic neurons, and astrocytes. (B) Firing rate of each co-culture over 4 weeks. Data provided by Neucyte

 

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

 

Reveal the role of astrocytes and microglia in inflammation and disease

Glial cells make up the majority of cells in the brain. They play a host of roles from neuron support, development, immune regulation, signal transmission, and synapse formation and function. While glia like astrocytes do not fire action potentials, their function is critical to proper network development.

When co-cultured with astrocytes, iPSC-derived glutamatergic neurons showed a 40% increase in network bursting (n=10 wells). After the addition of 100 nM picrotoxin, the difference increased to 84%. No activity was seen with astrocytes alone (data not shown) suggesting the astrocytes were 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.

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

 

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.
  • 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.
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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.