Optically imaging neural network dynamics in wild-type organisms

Interconnections among billions of relatively simple neurons drive human brain complexity. Signals transmitted in neural networks seem to be the foundation for all human activity, from yanking a hand from a flame to mulling over Descartes’ epistemology. The very complexity that enables human curiosity and reason also makes the human brain difficult to examine. That’s why neurobiologists study a range of organisms with simpler nervous systems. These include wild-type organisms from throughout the animal kingdom, and transgenic organisms such as mice, Drosophila flies, and the nematode C. elegans. Professor William Frost and his colleagues at Rosalind Franklin University (Chicago, IL) are using optical imaging to tease out the dynamics of neural networks of another class of animals: marine mollusks.

A simple nervous system

Nudibranchs, a type of sea slug, are gastropod mollusks with several features that make them desirable as subjects for neural studies. To begin with, their central ganglia have only a few thousand neurons. According to Frost, that means single neurons can have outsize effects. In addition, “Their neurons are larger,” he says, “than an elephant’s or a whale’s”—up to 800 µm. Those neurons direct several different types of behavior. For example, when a nudibranch such as Tritonia diomedea detects the touch of a seastar, it flexes alternately in opposite directions in a sequenced motion called “escape swimming,” which propels the nudibranch up into the current flow and hence to safety.

That complex signal progression is, in principle, the same kind of network activity that underlies human cognitive processes. Optical methods—using light to investigate the magnitude and timing of neural signals—provide both the spatial and temporal resolution necessary to investigate interactions among neurons, but only a handful of model organisms have been optogenetically modified to emit light in conjunction with neural activity. With only a few organisms to investigate, it’s difficult to identify general behaviors and their underlying principles.

A new multicenter project is aimed at producing an optogenetically modified version of a sea slug. Part of the groundwork is demonstrating the value of these organisms, which Frost and his team do by applying a technique they developed to visualize neural activity in wild-type gastropod mollusks such as T. diomedea and Aplysia californica (see figure).¹

One hundred and fifty in a single acquisition

The nervous systems in organisms such as T. diomedea remain functional even when almost completely excised from the body. This makes it possible to image the pedal ganglia, the brain region connected to the “foot” in these organisms. Frost uses voltage-sensitive dyes (VSDs) that rapidly change their optical properties in response to voltage changes. The VSDs chosen for this work—RH155 and RH482—increase their absorption at long wavelengths in response to a neural voltage spike. The VSDs exhibit a small absorption change from 650 to 750 nm, so an LED centered at 735 nm is used as the illumination source.

The imaging system can either be a CMOS-based camera or a photodiode array. The photodiode array is AC-coupled, making it sensitive only to fluctuations in light level. The hexagonal 464-element photodiode array (PDA) used in this work is capable of simultaneously acquiring signals from all of the 150 or so ganglion neurons. The PDA resolution is not high enough to resolve detail in a typical pedal ganglion, but the signals are processed to correlate the photodiodes with specific neurons—often with several photodiodes detecting a single neuron. In addition, the system registers the photodiode field to a photographic image of the neurons.

In a recent methods paper,² Frost and his colleagues described several typical experiments. Correlated photodiode outputs are processed to determine which photodiodes are associated with each given neuron. When one of the pedal sensory nerves is stimulated, simulating a predator’s touch, the photodiode array collects intensity information from each of the previously identified neurons. Signal analysis identifies interactions among individual neurons and groups of neurons, revealing details of neural network dynamics.

In addition to demonstrating the method on previously studied organisms, the group tailored the technique to analyze Berghia stephanieae, an organism new to neurobiological investigation, and now the focus of efforts to produce a transgenic version. Unlike other sea slugs, B. stephanieae is easily bred in the laboratory environment. The organism is an attractive candidate for neurological study because it has the same features as other sea slugs—large neurons, behavior triggered by single neurons, and the capability to exhibit networked neural activity.

“By recording activity from many neurons in organisms like this,” says Frost, “we can identify structures that underlie fundamental processes such as memory formation—which we have already discovered involves the allocation of additional neurons to a network.” New insight into these underlying processes can eventually guide research in addressing, for example, problems with memory formation in older people.

REFERENCES

1. W. N. Frost et al., Adv. Exp. Med. Biol., 859, 127–145 (2015) .

2. E. S. Hill et al., J. Vis. Exp., 161, e61623 (2020).