Neural Optical and Electrical Stimulation

Neural Optical and Electrical Stimulation Application
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While neural cultures are often spontaneously active, electrical and optical stimulation offers enhanced control of neural activity. Electrical stimulation can be used to study network connectivity, evaluate excitability, and reduce variability between replicates. Optogenetics can be used to further customize an experiment with the ability to stimulate or suppress activity within specific subpopulations of a culture.

The Maestro MEA platform provides noninvasive electrical activity recordings across each neural network, while the Lumos optical stimulation system delivers precisely controlled light. Together, these devices seamlessly pair to unlock entirely new assay capabilities, in a high-throughput format.

 

Evoked neural activity for seizurogenic screening

Electrical stimulation enables the computation of evoked activity metrics. For each electrode key parameters of the stimulus-evoked response can be calculated and used to assess seizurogenic activity. As an example, a “paired stimulus” assay can assess the excitatory-inhibitory balance of a network. The assay measures the ratio of the evoked response between two stimuli, with increasing delay between them. Compounds that decrease inhibition in the network, like picrotoxin, create a leftward shift in the plot.

A paired stimulus response assay ban assess the excitatory-inhibitory balance of a network
The ratio of the response for two stimuli, with increasing delay between them, is highly sensitive to compounds, like picrotoxin, that decrease inhibition in the network.

 

Chronos enables high frequency responses without adaptation

High-frequency blue light stimulation (2 ms pulses at 40 Hz for 1 s) was applied with the Lumos to channelrhodopsin-expressing (ChR2) primary rat neurons. The light-evoked neural response showed adaptation with reduced responses to later pulses in the train. In contrast, the opsin Chronos has faster kinetics which enabled a consistent response to each pulse in the high-frequency train.

 
ChR2+ expressing primary rat neurons showed light-evoked neural responses
Opsin Chronos has faster kinetics and responded to a higher frequency train
Evoked spike count showed consistent results for ChR2 and Chronos

 

High-frequency stimulation to probe excitatory-inhibitory balance

ChR2 and Chronos neurons were dosed with either a proconvulsant (picrotoxin,10µM), antiepileptic (carbamazepine, 30µM). Neural networks dosed with picrotoxin showed a sustained response to high frequency blue light stimulation (2ms pulses at 40Hz for 1s) in which firing did not return to zero between each pulse, resulting in a higher cumulative spike count across the 40 pulses. In contrast, carbamazepine reduced the response, resulting in a lower cumulative spike count.

 
Chronos baseline response to optical stimulation
Chronos with picrotoxin added showed variability in response to light stimulus
Evoked spike count showed a decreast in spike count after the application of carbamazepine

 

 

Neuro electrical pacing
Neuro optical pacing

Getting started with Maestro Pro and Edge couldn't be easier. Culture your neurons in an Axion multiwell MEA plate (Day 1). For optogenetic experiments, using adeno-associated virus-based vectors, add the virus to the cell suspension at the time of plating. Allow 7-14 days for opsin expression.  When transfecting neurons with mRNA-based opsins, transfect at peak neural activity and allow 2 days for opsin expression. Load the MEA plate into the Maestro MEA system at the desired recording times and begin recording. Perform optical stimulation experiments using the Lumos optical stimulator, or electrical stimulation studies using any microelectrode in the MEA plate (Day 14). Analyze the neural activity with AxIS Navigator software. 

 

Maestro MEA user

 

The advantage of measuring electrically or optically evoked neural activity on the Maestro Pro and Edge systems:

  • Electrical stimulation – Axion’s multiwell plates bring flexibility to your experimental design. Each MEA electrode is dual-purpose, capable of recording or stimulating. The AxIs Navigator makes stimulation simple yet customizable, while optimized artifact elimination and automated detection of electrophysiological features make analysis easy, efficient, and reproducible.

  • Optical stimulation – The patented Lumos optical stimulation system seamlessly pairs with Maestro Pro and Edge, offering precise control over light intensity and duration of four stimulation wavelengths in each well. The Lumos allows researchers to simultaneously direct and record functional neural network activity.

  • Measure what matters – In contrast to regularly-used indirect measures of excitability, the Maestro MEA system directly measures the action potentials. Indirect measures of excitability, such as calcium imaging, are unable to capture important but subtle changes to neural network signaling, and protein expression levels often poorly correlate with cell model performance. The Maestro MEA system tracks cell excitability in real-time, allowing you to answer the questions that matter.

  • Analyze cell activity label-free – 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 – Other higher throughput platforms (e.g. automated patch clamp, flow cytometry) often require cell samples to be transferred into a single-cell suspension before testing. This is far from ideal since excitable cells exist as a functional network of inter-linked cells. In addition, the cell harvesting process requires numerous handling steps. The Maestro MEA system captures neural excitability while preserving the morphological complexity of your neural cell model.

  • 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!

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.

 

 

Optogenetics Technology

Optogenetics

 

Optogenetics: Using light to control cells

Optogenetics is a technique that involves the use of light to control cell function. Cells are first genetically modified to express light-sensitive ion channels, called opsins. Then, light can be used to activate the opsin. The most well-known opsins are light-gated ion channels that can control the excitability of the cell membrane. When activated by the opsin-specific wavelength of light, the channels open allowing ions to flow across the cell membrane to either excite or inhibit the cell. Optogenetics enables precise control over a targeted cell population.

 

Many opsins, many options

Since the first microbial opsin was introduced into mammalian neurons in 2005, many different opsins have been used to control the excitability of electroactive cells such as neurons and cardiomyocytes. Each opsin is sensitive to a specific wavelength range, or color of light and induces a precise biological event.

Opsins activated channel

Channelrhodopsin (ChR2), for example, is activated by blue light. When ChR2 opens, positive cations (like sodium and calcium), flow into the cell, depolarizing or exciting the cell. In contrast, halorhodopsin and archaerhodopsin both inhibit cell excitability by hyperpolarizing the cell in response to yellow or green light, respectively. With optogenetics, you can turn on and off cells like a light switch.

The timing of these light-activated events is fast, facilitating highly precise control. First generation opsins, such as channelrhodopsin, open and close in milliseconds, ideal for kicking off an action potential. Second generation opsins have fined tuned kinetics for even faster, more precise control or slower, longer-lasting inhibition. For example, step-function opsins stay open until another pulse of light switches them off.

Optogenetic silencing of target cell
Optogentic activation of blue light neuron only

Optogenetics can precisely control which cells are turned on or off by employing different gene promoters for opsin expression. Opsins can be expressed in all neurons or used to control specific subpopulations. Unlike electrical stimulation, which excites all nearby cells, optical stimulation can be finely targeted to the cells expressing the opsins responsive to a narrow band of light wavelengths.

ChR2 Neural excitation
ArchT inhibition
ChR2+ Rat cortical neurons (Div 14)

 

In summary, optogenetics is a powerful toolbox for precise control over targeted cell populations at fast time scales. Superior spatial and temporal control, reversibility, and easy stimulus delivery make exploring complex biology simpler than ever before.

 

More than just ion channels

As the field has advanced, opsins have been used to control more than just ion flow. Light-activated gene expression with light-inducible transcription factors can control the proteins made by cells. The combination of optogenetics with CRISPR provides even greater control over CRISPR/Cas9 gene editing.

Opsins have also been incorporated into many biochemical and intracellular signaling pathways to control key protein functions. MAPK and PI3K pathways, Rho family GTPase activation, apoptosis, and protein trafficking can now all be precisely controlled by light.

Opsins in cell membrane

 

Shining light in vitro

Sophisticated biology like optogenetics demands sophisticated technology to explore it. In vitro technology relied on single wavelength lasers and custom lab-specific tools while many in vivo technologies were quickly developed for optical stimulation. The Lumos Optical Stimulation system is the first-of-its-kind multiwell optical stimulator with the ability to deliver up to four wavelengths of light per well with microsecond precision. From controlling the excitability of your neurons to pacing the beating of your cardiomyocytes, discover how the Lumos and optogenetics can revolutionize your assay.

Lumos system