Pain

Pain in vitro on multielectrode array
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Dorsal root ganglion (DRG) cultures exhibit chemical and thermal sensitivities representative of those observed in vivo and can be cultured in vitro in Axion’s multiwell microelectrode array (MEA) plates, enabling simultaneous recording of electrical activity from numerous test conditions. In combination, DRG neurons and the Maestro multiwell MEA system constitute a high-throughput in vitro platform for chronic neuropathic pain research.

The sensation of pain is transmitted from sensory nerve endings to the central nervous system by axons of peripheral neurons whose cell bodies reside in the dorsal root ganglion (DRG). Damage to these primary afferent neurons, or defects in the proteins underlying their electrical or sensing function, can cause neuropathic pain, a persistent sensation of pain associated with increased spontaneous firing of DRG neurons

 

MEA recordings capture the transient and persistent increases in firing rate of DRG neurons induced by capsaicin

Sensitivity of DRG neurons to noxious stimuli is largely caused by the TRP family of ionotropic receptors, which display differing sensitivity profiles for stimuli including chemical agents, noxious heat and cold, and changes in pH due to inflammation. Of particular relevance is the TRPV1 channel, a primary receptor for noxious heat and pH implicated in hyperthermia and inflammatory pain. Capsaicin is an active component of chili peppers, and is a chemical irritant for humans, producing a sensation of burning in any tissue with which it comes into contact. Activation of DRG neurons was conducted using capsaicin, an agonist of the TRPV1 receptor. The effect of 100 nM capsaicin on mean firing rate (MFR) of DRG neurons (1x104 cells per well) was measured on DIV 7 with Axion's MEA system.

Pain DRG recordings on multiwell MEA system

A) The raw voltage trace shows the increased firing resulting from capsaicin addition, with the waveform of each detected spike plotted to the right (gray), along with the mean spike waveform (black). B) Three separate stages of the capsaicin response are apparent in the plot of well-wide firing rate (N=6 wells, mean – black, +/- standard error of the mean – gray): baseline spontaneous firing, transient capsaicin-induced firing, and a persistent elevated firing that lasts for tens of minutes following the capsaicin addition. C) The bar chart represents an average over a 3 minute period for both the baseline and the persistent phases of the capsaicin response, where the firing rate was higher in the persistent phase (N=6, p = 0.0313, Wilcoxon Signed Rank Test, error bars represent standard error of the mean).

In summary, Axion's multiwell MEA system with the Neural Module recorded the significant well-wide increase in DRG firing rate after the addition of capsaicin.

 

TRPV1 antagonists modulate the effect of capsaicin on DRG neuron activity

Robust in vitro activation of DRG neurons by capsaicin sets the stage for screening assays in which this response is blocked by potential pain therapeutics. Two wells of DRG neurons (1x104 cells per well) cultured in MEA plates were treated with 100 nM capsaicin at 7 minutes, causing an increase in firing rate. After 20 minutes, DRG neurons were treated with 10 mM dehydroandrosterone (DHEA), a TRPV1 competitive inhibitor. The DHEA treated DRGs exhibited a significant reduction in mean firing rate compared to the control well, consistent with previous whole cell patch clamp studies on DRG neurons. After 10 minutes of reduced firing due to DHEA exposure, the effect was markedly reversed by addition of 1 mM capsaicin, demonstrating continued sensitivity of the TRPV1 receptors.

 

Pain DRG recordings modulated on multielectrode array

The histograms represent the well-wide firing rate normalized by the capsaicin induced activity (bin size of 60 secs). Addition of a higher dose of capsaicin (1mM) rescued the firing activity suppressed by DHEA (dark gray).

Modulation of DRG neuron activity, suppression and rescue of firing activity, was recorded across a microelectrode array well. 

 

Thermal sensitivity of DRG neurons recorded on an MEA system

Noxious heat is a modulator of TRPV1 channel conformation. Temperatures in excess of 43°C lower its activation threshold, causing increased excitability of the neuron. The thermal response of DRG neurons can be evaluated on the Maestro MEA system using the integrated temperature control, which directly heats the MEA plates. The responses of 5 individual DRG neurons exposed to increasing temperature were analyzed by first spike sorting in the Plexon Offline Sorter and then loading the sorted spike trains into the Neural Metric Tool.

 

Pain DRG neurons respond to changes in temperature on MEA

Spike raster plots for 5 different DRG neurons recorded over time as the temperature was ramped from 24°C to 45°C. B) MFR for Cell 3 (green) at different temperatures with a Gaussian curve fit (bin size = 60 secs). C) Normalized firing rate for the 5 cells in A, color-coded with Gaussian curve fits to show temperature sensitivity ranges.

Maestro MEA system's ability to change the plate temperature through integrated temperature control showed correlated temperature sensitivity ranges in the firing of DRG neurons.

 

 

Burning Man Syndrome neurological activity increases as temperature increases

Neurons expressing a specific mutation associated with inherited erythromelagia, a chronic pain syndrome characterized by a burning sensation in response to heat, were cultured on the MEA and treated with carbamazepine which reduced their sensitivity to temperature increases. Two patients with the same mutation were then treated with carbamazepine and reported a reduction in pain duration when compared to a placebo [Mis et al. 2018].

Neural activity map of burning man pain syndrome on MEA plate

Maestro microelectrode arrays recorded the change in mean firing frequency of the burning man syndrome neurons in response to suppression treatment as the multiwell MEA plate environment increased in temperature.   

DRG neurons for pain assay protocol steps on multiwell MEA system

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

 

multiwell microelectrode array (MEA) system in lab

 

The advantage of modeling pain and measuring the neural activity of DRG neurons on the Maestro Pro and Edge multielectrode array 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 multiwell 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!

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.