Confocal microscopy reveals rhythm of neurons
Confocal immunofluorescent microscopy of 'circuitoid': a spinal cord neural circuit made entirely from stem cells.
Neuroscientists at the Salk Institute have used stem cells to generate diverse networks of self-contained spinal cord systems in a dish.
Unveiling a stunning immunofluorescent image of the networks, Professor Samuel Pfaff and colleagues are using these so-called 'circuitoids' to study the rhythmic firing patterns of neurons.
Research has revealed that some of the circuitoids exhibited spontaneous, coordinated rhythmic activity of the kind known to drive repetitive movements, such as breathing and chewing.
"It's still very difficult to contemplate how large groups of neurons with literally billions if not trillions of connections take information and process it," says Pfaff. "But we think that developing this kind of simple circuitry in a dish will allow us to extract some of the principles of how real brain circuits operate."
"With that basic information maybe we can begin to understand how things go awry in disease," he adds.
Samuel Pfaff from the Gene Expression Laboratory at the Salk Institute for Biological Sciences is also a Howard Hughes Medical Institute investigator.
To model these complex neural circuits, Pfaff and colleagues prompted embryonic stem cells from mice to grow into clusters of spinal cord neurons, which they named circuitoids.
Each circuitoid typically contained 50,000 cells in clumps just large enough to see with the naked eye, and with different ratios of neuronal subtypes.
The researchers went onto tag four key subtypes of both excitatory and inhibitory neurons vital to movement, called V1, V2a, V3 and motor neurons.
They observed the cells in the circuitoids in real time using an upright epifluorescent Olympus microscope with a Hamamatsu C9100‐13 camera and ImageJ plugin.
Analyses revealed that the circuitoids composed only of V2a or V3 excitatory neurons or excitatory motor neurons spontaneously fired rhythmically, but that circuitoids comprising only inhibitory neurons did not.
Salk researchers create synthetic brain systems called 'circuitoids' to better understand dysfunctional movements in Parkinson's, ALS and other diseases. [Salk Institute]
Interestingly, adding inhibitory neurons to V3 excitatory circuitoids sped up the firing rate, while adding them to motor circuitoids caused the neurons to form sub-networks, smaller independent circuits of neural activity within a circuitoid.
"These results suggest that varying the ratios of excitatory to inhibitory neurons within networks may be a way that real brains create complex but flexible circuits to govern rhythmic activity," says Pfaff. "Circuitoids can reveal the foundation for complex neural controls that lead to much more elaborate types of behaviors as we move through our world in a seamless kind of way."
Because these circuitoids contain neurons that are actively functioning as an interconnected network to produce patterned firing, Pfaff believes that they will more closely model a normal aspect of the brain than other kinds of cell culture systems.
Aside from more accurately studying disease processes that affect circuitry, the new technique also suggests a mechanism by which dysfunctional brain activity could be treated by altering the ratios of cell types in circuits.
Research is published in eLife.