Neuromuscular Junction

Neuromuscular Junction
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Neuromuscular junctions are specialized synapses between a motor neuron and a muscle fiber.  Motor neurons transmit signals to muscle fibers and initiate muscle contraction through the release of acetylcholine (ACh). The pathophysiology of neuromuscular disorders can be diverse. Physiological function of the junction may be interrupted by an autoimmune response, such as the case with myasthenia gravis, or through toxins like botulinum toxin or many insecticides.

The Maestro Pro and Edge MEA systems have the ability to measure from both neurons and skeletal muscle simultaneously. Combined with the selective optical stimulation capabilities of the Lumos, an easy-to-use, scalable model of neuromuscular junction function can be achieved.


Controlling neuromuscular junction-mediated contractions with light

The failure of the neuromuscular junction (NMJ) is a key component of degenerative neuromuscular disease, yet how NMJs degenerate in disease is unclear. With the ability to create relevant cell types from any genetic background, human induced pluripotent stem cells (hiPSCs) are a useful tool for disease modeling.  Here Swartz et al. present a scalable, hiPSC-derived co-culture system composed of independently derived spinal motor neurons (MNs) and skeletal myotubes (sKMs). In a model of C9orf72-associated disease, co-cultures form functional NMJs that can be manipulated through optical stimulation. Channel rhodopsin expressing motor neurons were able to elicit muscle contractions in the co-cultured sKMs when illuminated with blue light using the Lumos. Utilization of this co-culture model as a tunable, patient-derived system may offer significant insights into NMJ formation, maturation, repair, or pathogenic mechanisms that underlie NMJ dysfunction in disease.

Co-culture on a 4x4 array of electrodes

(A-B) Co-culture on a 4x4 array of electrodes. (C) Raster plot of the well shown in A-B during paced stimulation. Tick marks on the x-axis indicate light stimulation. Pink boxes represent evoked spike timeframe with bursts in blue. Electrode coordinates are labeled on the y-axis. (D) Quantification of electrode activity and network activity (E) during baseline and stimulated recordings. Dots represent individual wells. **, p < .01; ***, p < .001. (F) Decreased evoked spike counts following treatment with the gap junction blocker, heptanol treatment, which could be partially recovered with decreased concentration or washout (recovery). (G-H) Evoked spike counts were abolished with neuromuscular antagonists, decamethonium bromide and vecuronium. Data represented as the percent change per active electrode at baseline (untreated). (I) Model depicting co-cultures on MEAs. sKMs are plated on top of protein substrate and exist in an extracellular protein matrix [Swartz et al. (2020), Establishment of a Human Induced Pluripotent Stem Cell-Derived Neuromuscular Co-Culture Under Optogenetic Control. bioRxiv].


Formation of Functional Spinal Cord Networks with CPG-like Activity in NMOs

Neuromuscular networks assemble during early human embryonic development and are essential for the control of body movement. Previous neuromuscular junction modeling efforts relied on the generation of either spinal cord neurons or skeletal muscles from hPSCs and their co-culture. Here, Martins et al. used hPSC-derived axial stem cells, the building blocks of the posterior body, to simultaneously generate spinal cord neurons and skeletal muscle cells that self-organize to generate human neuromuscular organoids (NMOs). NMOs contain functional neuromuscular junctions supported by terminal Schwann cells, contract and can be maintained in culture for several months.

NMJ from Martins et al

(A) Bright-field imaging of day 50 NMO in a CytoView MEA 6-well plate. (B) Plot showing the average spike frequency before (spontaneous) and after the application of 50 µM NMDA and 40 µM 5-HT in days 30 and 50 NMOs. Day 50 NMOs show central pattern generator (CPG)-like activity. (C) Network graph showing the connectivity between electrodes recording spontaneous (left) or 50 µM NMDA/40 µM 5-HT at day 30 (middle) or 50 µM NMDA/ 40 µM 5-HT at day 50 stimulated electrical activity. Line thickness represents the number of times the paired firing occurred between electrodes, while node size shows the number of spikes captured by each single electrode during a recording. The stimulation of neurons at day 50 with NMDA/5-HT showed increased single firings and an increase in paired electrode spikes. The position of the individual NMOs in the MEA grid is shown with a dashed line. The Maestro Edge MEA system was used to perform functional analysis of NMOs and strikingly demonstrated the formation of central pattern generator (CPG) like neuronal circuits. The NMO activity recorded from single electrodes in combination with a Matlab script were used to plot network graphs that highlight the formation of neuronal circuits  [Faustino Martins et al. (2020), Self-Organizing 3D Human Trunk Neuromuscular Organoids. Cell Stem Cell 26: 1-15].

NMJ protocol

Protocol taken from Swartz et al. 2020.  Previously frozen vial of iPSC- skeletal muscle (sKM) cells (day 79, isoA80) was thawed and plated onto a Lumos MEA 48 plate (Day 1).  iPSC-sKMs were grown for 3 days in N2 medium and on Day 4, live iPSC-MN spheres (day 40, Opto-isoA80-8) were plated on top, creating co-cultures. Co-cultures were grown in NDM5 and ¾ medium was replaced every 48 hours with NDM6 containing recombinant human Agrin (50 ng/µL).  At the time of recording, the co-culture was 7 days old (Day 9), with the last medium change occurring 48 hours prior. Optogenetic stimulation was performed with the Lumos 48 system using 475 nm light at 50% power with 500 ms pulses at 0.25 Hz. Electrophysiological activity was recorded using the Maestro Pro, and analyzed with the AxIS Navigator Neural Module software.



Maestro MEA user


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

  • Optical stimulation – The 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 NMJ activity.

  • Measure what matters – The Maestro Pro and Edge MEA systems directly measure neuronal and skeletal muscle action potentials. Indirect measurements like calcium imaging are unable to capture important but subtle changes to NMJ 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 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 NMJ excitability while preserving the morphological complexity of your NMJ model.

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