A fundamental question for neuroscientists is how the activity in neuronal circuits generates behaviour. The nematode worm Caenhorhabditis elegans is an excellent model organism for studying the neural basis of behaviour, because it is small, transparent, and has a simple nervous system consisting of only 302 neurons.
Typically, an organic glue is used to permanently immobilize the worm on an agar plate, and specific cells of the nervous system are stimulated with microelectrodes. This method has its limitations, however. As it is restricted, the worm’s muscles and nervous system cannot function properly, and the organism can therefore generate only a very limited number of behaviours. Furthermore, it is unclear whether or not the glue is toxic, or if it interferes with the function of nerve cells.
Researchers from the Howard Hughes Medical Institute now appear to have overcome some these difficulties. In the journal Nature Methods, Nikos Chronis and his colleagues report that they have developed microfluidics chips for investigating the relationship between neuronal activity and behaviour of the nematode worm.
The movements of nematodes are generated sequential muscular contractions that lead to sinusoidal waves which travel along the length of the body. Forward movements occur as a result of waves travelling from the front to the back of the worm’s body, while backward movements are the result of forward-moving waves.
The microfluidics devices developed by Chronis et al are made of silicon elastomer attached to a glass coverslip, and are microfabricated using a technique called soft lithography. Two types of chip were made, each for a different set of experiments.
Each chip contains a worm trap 1.2 mm long and 70 microns (thousandths of a millimeter) wide. The chips are only slightly than the worm itself – a young adult nematode is 1 mm long and 40 microns wide. The chips therefore constitute a well-controlled microscopic environment within which the neamtode can be manipulated – the worms are trapped, but at the same time can move freely in all directions.
The researchers created transgenic worms for their experiments, which synthesize, in two specific cell types, a protein that fluoresces when it binds calcium. Because an increase of calcium ion concentration is an indicator of neuronal activity, the responses of those cells, either in response to a stimulus, or correlated to a behaviour, can be visualized using fluorescence microscopy.
For one set of experiments (using the “behaviour chip”), the worms were loaded into the trap through a hole at one end. In the trap, the worms were slightly compressed at the thickest region of their body, so that their vertical movements were restricted. This compression kept the cell bodies of the neurons being investigated within the focal plane of the microscope.
First, the researchers imaged the changes of calcium ion concentrations in cells called AVA interneurons. These cells regulate the backward movements of the worm, and are believed to elicit an escape response. They receive inputs from sensory neurons that are sensitive to mechanical pressure and chemicals in the surroundings, and send outputs to motor neurons that activate muscles in the worm’s body wall.
It was found that when the worms switched from a backward- to a forward-moving wave (that is, from moving forwards to moving backwards) there was an increase in calcium ion concentration in the AVA interneurons. In all cases, the timing of interneuron activation corresponded exactly to the initiation and duration of forward-travelling body wave.
In the second set of experiments, the “olfactory chip” was used. This chip integrates a worm trap with a mcrofluidics system that can deliver streams of solutions. The end of the trap was designed to match precisely the shape and size of the worm’s head, so that the end of the nose protrudes into a microchannel through which the solutions flowed (above).
The olfactory chip was used to investigate the responses of ASH sensory neurons. These cells are polymodal, i.e. they are sensitive to different kinds of stimuli – chemical, mechanical and osmotic. (An osmotic stimulus is the pressure produced by the different concentrations, on either side of the membrane, of something dissolved in water.)
Using the olfactory chip, the researchers exposed the worms’ noses to streams of highly osmotic solutions (solutions containing high concentrations of chemicals). It had previously been shown that ASH neurons are activated in response to the onset of osmotic stimulus. The authors found that the cells also respond to the offset of an osmotic stimulus, with a transient increase in calcium ion concentration.
The authors suggest that the response of ASH neurons to the offset of osmotic stimuli had previously been masked because of the way the worms are immobilized in agar. They also say that the chips can easliy be modified to investigate other behaviours.
For example, chips containing moveable parts could be used to look at worms’ responses to mechanical stimuli; chips with heated elements could be used to investigate thermoreception; and a combination of the behavioural and olfactory chips could be developed to investigate more complex stimuli.
This study demonstrates the usefulness of microfluidics devices for the manipulation of small organisms like the nematode worm. The anatomy of the nematode nervous system is very well characterized; using such devices, researchers will be able to superimpose a functional map onto the anatomical one.
Chronis, N., et al. (2007). Microfluidics for in vivo imaging of neuronal and behavioral activity in Caenorhabditis elegans. Nat. Methods 19 doi: 10.1038/nmeth1075.