Retina

View of the back of a retina
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The retina is composed of a layer of light-sensitive cells at the back of the eye that transmit electrical signals via the optic nerve to the brain. Retinal degradation is characterized by the progressive death of these cells due to physical damage or genetic factors of which there is currently no cure. An important example of a progressive neurodegenerative disorder is retinitis pigmentosa which results in the initial loss of rod photoreceptor cells and eventually cone cells, the foundation of human vision. Current research has focused on the development of pharmacological compounds that act to reduce photoreceptor degradation.

The Maestro Pro and Edge MEA platforms provide noninvasive electrical activity recordings across neural networks, while the Lumos optical stimulation system delivers precisely controlled light. Together, these devices seamlessly pair to unlock entirely new assay capabilities for retinal applications in a high-throughput format.

 

Reveal visual contrast responses of retinal ganglion cells

Antipsychotic drugs haloperidol and clozapine have been reported to increase the sensitivity of retinal ganglion cells (RGCs) to flashes of light in the P23H rat model of retinitis pigmentosa. In order to better understand the effects of these antipsychotic drugs on the visual responses of P23H rat RGCs, Jensen (2019) examined the responses of RGCs to a drifting sinusoidal grating of various contrasts. In vitro multielectrode array recordings were made from P23H rat RGCs and healthy Sprague-Dawley (SD) rat RGCs.

Spike activity from RGCs on electrode
RGC response to a flash

Spike activity of many SD and P23H rat RGCs was modulated by the full-field drifting sinusoidal grating (spatial frequency: 1 cycle/mm, temporal frequency: 2 cycles/s). Example recordings here show a RGC response to a full-field flash of light (A) and to the drifting sinusoidal grating (B).

Retina's stimulated with drifting sinusoidal grating
Cloapine decreased the response of the rat RGCs

Most (73%) P23H rat RGCs could be categorized as either saturating or non-saturating cells. The remaining ‘uncategorized’ cells were poorly responsive to the drifting grating and were analyzed separately. Haloperidol and clozapine increased the responses of non-saturating (C2, D2) and uncategorized (C3, D3) P23H rat RGCs to most grating contrasts, including the highest contrast tested. Haloperidol and clozapine also increased the responses of saturating P23H rat RGCs to most grating contrasts but these increases were not statistically significant (C1, D1). Overall, the findings show that haloperidol and clozapine have differential effects on the contrast response functions of SD and P23H rat RGCs [Jensen et al. 2019].

Most rat RGCs could be categorized as either staturating or non-saturating cells.
Haloperidol increased the response of saturating rat RGCs

Most (73%) P23H rat RGCs could be categorized as either saturating or non-saturating cells. The remaining ‘uncategorized’ cells were poorly responsive to the drifting grating and were analyzed separately. Haloperidol and clozapine increased the responses of non-saturating (A2, B2) and uncategorized (A3, B3) P23H rat RGCs to most grating contrasts, including the highest contrast tested. Haloperidol and clozapine also increased the responses of saturating P23H rat RGCs to most grating contrasts but these increases were not statistically significant (A1, B1).

Overall, the findings show that haloperidol and clozapine have differential effects on the contrast response functions of SD and P23H rat RGCs. Data courtesy of Ralph Jensen (VA Boston Healthcare System).

Retina protocol

Getting started with Maestro Pro and Edge couldn't be easier. Following euthanasia of a rat, cut out a square piece of retina measuring approximately 2–3 mm on each and place with the ganglion cell side down onto an Axion multiwell MEA plate. Load this MEA plate into the Maestro MEA system and allow the environmental chamber to automatically equilibrate. Measure the neural activity of your retina sample. Add test compounds as required.

 

 

Maestro MEA user

 

The advantage of measuring retinal physiology on the Maestro Pro and Edge systems:

  • Optical stimulation – The patented Lumos optical stimulation system seamlessly pairs with Maestro platform, 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 cell activity.

  • Measure what matters – In contrast to 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 retinal cell 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 – 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 – The Maestro MEA system captures retinal excitability while preserving the morphological complexity of your model.

  • It's easy – You don't have to be an electrophysiologist to use the Maestro MEA system. Just culture your retinal cells in an MEA plate, load your plate into the Maestro MEA system, and record your cell activity 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