Tuesday, May 1, 2012

Virtual Reality for Worms

How do you build a virtual environment for a worm?

The Nematode C. Elegans with glowing neurons (source)

Using a little optogenetic trickery, you can directly activate specific worm neurons with light.  If you know your worm neurons, you can stimulate ones that make it think it has suddenly touched something with its nose or that the environment is suddenly very salty. 

Before we dive into worm VR, let's back up and discuss this specific worm.

The Magnificent C. Elegans
C. Elegans is a surprisingly popular subject of study in neuroscience. It has a simple and well defined nervous system that contains only 302 neurons (in the hermaphrodite, the rare males have a few extra neurons).  All the neurons and even all the connections between the neurons have been pretty well characterized.  They are small (hundreds can fit on a standard sized petri dish) and they reproduce quickly.  And it that wasn't enough to make C. elegans a desirable subject for study, they can be genetically altered with relative ease, and exhibit rudimentary learning skills. 

A recent technological development has made clever use of genetic tools that allow calcium influx (an indicator of neural activity) to be visualized in neurons and allow neurons to be activated by light.
Faumont et al., (2011) have created a worm tracking system that uses the fluorescence from a genetically altered neuron to locate the worm and recenter the microscope on the worm in real time. This allows for completely non-invasive visualization of neuronal calcium/activity in the awake behaving animal. 

The recent paper in PLoS One, describes exactly how they got the microscope to track the worm in real time without blurring of the signal or messing up the calcium imaging. The paper is open access, so you can go read the details for free.

To see this larger and more clearly, you can download this video and their 4 other supplementary videos here
In this video, you can see the animal moving around in the top left, the path it follows in the top right, the calcium fluorescence signal in the bottom left (notice the calcium neuron is always in the field of view), and the activity of this particular neuron when the worm is traveling either forward (blue) or backward (red). 

The "Dedicated Circuit" Hypothesis
The neuron imaged in this video is called AVB, and it is a 'command neuron'.  Faumont et al. show that it increases in activity when the worm is moving forward and decreases when the worm moves backwards.  A similar command neuron, AVA, does just the opposite, increasing when the worm moves backward and decreasing when it moves forward.  These data support what is called the "dedicated circuit hypothesis" which says that the worm uses one set of neurons to go forward and a completely different set of neurons to move backwards.

While Faumont et al. shows that the dedicated circuit hypothesis is supported for command neurons, they find that the activity of the actual motor neurons (the neurons on the body wall that control contraction of the muscles) does not support this hypothesis.  If the dedicated circuit hypothesis was true, the A-type motor neurons should only be active and oscillating during backward movement, and the B-type motor neurons should only be active during forward movement.  They found that this wasn't true, that both were active and oscillating during both forward and backward motion. 

Virtual Reality for Worms
Now back to virtual reality.  This Faumont et al. paper is a showcase of new tools that can be used to study C. Elegans in a simultaneously macroscopic and microscopic way.  One of the new techniques the introduce is the optogenetic stimulation of specific neurons in specific places to create and 'environment' for the worm. 

Faumont et al., 2011 Figure 2
When they genetically express channel rhodopsin, the channel which activates neurons when exposed to blue light in the ASH neuron (a neuron sensitive to osmolarity, or saltiness, changes), they can activate that neuron whenever they want by turning on the blue light.  They create a virtual environment by tracking the worm as it travels in a field, and activating the blue light when it reaches a certain xy coordinate.  In the figure above they activate the neuron when the worm's nose is within the outer ring (traces turn blue).  This makes the worm 'think' that the ring is full of saltier liquid than the rest of the area. 

This virtual environment takes away all the technical difficulties of actually creating a ring of salty water in a pool of less salty water, and the VR environment can be quickly and easily changed into any shape or size, when desired. 
This new tracking method, in combination with calcium imaging and optogenetics, represents a leap forward in cellular scale neuroscience, to non-invasively visualize neuronal activity, activate neurons, and record the coinciding behavior is a combination mammalian neuroscientists can only dream about.

Note: there are ways to image calcium in the neurons of moving mice, but even this requires installing a 'window' into the skull and mounting a mini-microscope on the mouse's head. In addition, the neurons visualized are limited to the ones closest to the surface of the brain.
© TheCellularScale

ResearchBlogging.org Faumont S, Rondeau G, Thiele TR, Lawton KJ, McCormick KE, Sottile M, Griesbeck O, Heckscher ES, Roberts WM, Doe CQ, & Lockery SR (2011). An image-free opto-mechanical system for creating virtual environments and imaging neuronal activity in freely moving Caenorhabditis elegans. PloS one, 6 (9) PMID: 21969859

1 comment:

  1. This is precisely the kind of small circuit neuroscience I was talking about! (sorry for not getting back to you soon!). But this is along the lines of what I thought: c. elegans locomotion, olfaction, thermotaxis (Bargmann and Sengupta labs in particular). Also, Drosophila olfactory, auditory processing, gustatory (Vosshall lab, Carlson lab, Laurent lab, just from the top of my head). In my opinion, Brandeis University has a lot of great people doing small circuit neuroscience in invertebrates (Garrity, Sengupta, Marder and emeritus faculty members like Jeff Hall).