Figure 1. C. elegans An adult worm showing the pharynx (green), intestine (yellow),eggs (large blue ovals), neuronal cell bodies (small blue ovals), synapse-rich process bundles (red), commissural tracts (black). The major synapse-rich regions are the nerve ring (red surrounding the pharynx, the ventral nerve cord (red line with neuronal cell bodies on ventral side of animal), the dorsal nerve cord (red line on the dorsal side of the animal). For reference the worm is approximately 1 mm long.

Caenorhabditis elegans (see-no-rab-DITE-iss Eh-Leh-GANZ) is a small soil nematode which feeds on bacteria and is found all over the world. Adults are about 1 mm in length and have a reproductive cycle of about 3 days when grown at room temperature. There are two sexes: hermaphrodites and males. In the laboratory, we usually grow them on agar plates covered with bacteria. Individual worms are manipulated under a dissecting microscope using a small platinum wire.
Several features of worm biology make them ideal for genetic analysis. First, worms are easy to maintain in the laboratory. They are small and have a short generation time and produce many progeny (figure 2). Thus, it is feasible to maintain and propagate large number of strains over many generations in a small space. Another great feature of worms is that stocks can also be frozen so one can create large numbers of mutants strains without having to constantly maintain them. C. elegans is also a self-fertile hermaphrodite. The hermaphrodite goes through a brief period when it produces sperm then switches to oocytes production (figure 2). The sperm are stored in the hermaphrodite spermatheca allowing a lone hermaphrodite to produce approximately 300 progeny. As a result many genetic manipulations that are difficult to perform in mice or even flies, are quick and efficient in C. elegans. For example, since a single animal acts a both mom and dad to breed a mutation to homozygosity in a single generation. This permits one to easily perform genetic screens for recessive mutations much more easily than in other genetically tractable metazoans. And strains with severe neuronal dysfunction can easily be maintained in the homozygous state because the ability to mate is not obligatory for propagation. Another advantage is that our laboratory stocks are highly inbred and essentially homozygous at all loci. This reduces confounding 'genetic background' issues which commonly hamper behavioral genetic analysis and also has enabled the development of very powerful gene mapping strategies based on single nucleotide polymorphism (SNP) detection. Lastly, the worm is clear allowing one to easily view internal structures using green fluorescent protein (GFP) and differential interference contrast (DIC) in live animals.

Figure 2 Lifecycle and timing of developmental events of C. elegans grown at 25°C. Under favorable conditions C. elegans goes through four larval stages in about 35 hr before molting to an adult. When conditions are unfavorable (low food or crowding) an the alternate dauer developmental pathway insues. Dauer larvae are capable of living for months in poor conditions and exit the dauer stage and matures to adulthood when conditions are more favorable.Graph from M. Blaxter and diagram from Jorgensen and Mango Nature Reviews Genetics 3:356-369

In the lab we commonly use behavioral analysis as a surrogate to assess neuronal development and function. The behaviors we examine include locomotion, feeding (or pharyngeal pumping), defecation, as well as response to touch. To address more directly whether synapses have formed and are functioning property we visualize synaptic terminals using GFP tags, immunohistochemistry and EM, and analyze synaptic efficacy using both pharmacological assays and electrophysiology.

Figure 2 Cross-section through the midbody of C. elegans This ultrastructural cross-section through C. elegans shows the location and morphology of a variety of anatomical structures. A thin tough cuticle surrounds the animal. This cuticle has specialized structures called alae. The syncyticial epidermis (or hypodermis; labeled H) is very thin where body wall muscle (M) is present, but becomes thicker in lateral regions near the alae and in proximity to the ventral and dorsal nerve cords. Several different processes bundles of the nervous system are visible including the ventral nerve cord, the dorsal nerve cord, the ventral and dorsal sublateral cords (DSC and VSC) and the mechanosensory process ALM. Four distinct quadrants of body wall muscle (M) are seen, two in ventral and two in dorsal sublateral positions. The intestine and the gonad fill most of the pseudocoelomic cavity. The microvilli brush border of the intestine is clearly visible in the intestine. This image was generously provided by Dr. David Hall. The diameter of an adult is approximately 60 nm. Click on image for a larger version

The nervous system of an adult hermaphrodite C. elegans consists of only 302 neurons that form approximately 7000 synapses. The major process tracts of the worm are the nerve ring, the ventral nerve cord and the dorsal nerve cord. A cross section through the worm viewed by electron microscopy reveals the structure and location of muscle and process bundles in the worm (Figure 2). The vast majority of synapses are found in these tracts (shown in red in Figure 1). Like most other nervous systems, the C. elegans nervous system is composed of sensory neurons, interneurons, and motor neurons (Figure 3). The cell bodies (small blue ovals in Figure 1) of most sensory and interneurons in C. elegans are found in ganglia that reside just anterior and posterior of the nerve ring and a set of ganglia in the tail. Most motor neuron cell bodies reside in the ventral nerve cord. Most of the sensory neurons have dendrite that extend into the nose, a cell body in a nerve ring ganglia and then extend a process into the nerve ring where the receive and make synaptic connections with other neurons. Most interneurons have a cell body in the nerve ring ganglia and extend a process into the nerve ring where they make contacts with sensory and command interneurons. Motor output is regulated by a series of interneurons called the command neurons. They receive innervation from a variety of interneurons in the nerve ring and synapse onto motor neurons. Command neurons have cell bodies in the nerve ring ganglia and send a process into the nerve ring and down the ventral nerve cord where they synapse onto motor neurons. Distinct sets of command neurons control forwards and backwards locomotion. Muscle in C. elegans is divided into ventral dorsal quandrants which are intervated by different neurons. Ventral and dorsal muscles are each innervated by both excitatory cholinergic neurons and inhibitory GABAergic neurons. The innervation pattern is such that while ventral muscle is being excited by cholinergic neurons, the dorsal side is being inhibited by GABAergic signaling thus facilitating the sinusoidal nature of C. elegans locomotion.

Figure 3 Organization of the nervous system in C. elegans. C. elegans neuronal organization is similar to most other metazoans. It consists of sensory neurons, local interneurons, interneurons, and motor neurons. The vast majority of axons, synapses, and neuronal soma are organized into ganglia which surround the synapse-rich nerve ring (See figure 1). Most sensory neurons, such as ASI, of C. elegans have a soma positioned in the "brain of the animal"; the nerve ring and ganglia that surround it. These sensory neurons extend processes into the nose where they have ciliated ending often exposed to the environment. Sensory neurons synapse onto interneurons, such as RIS, in the synapse-rich nerve ring. Interneurons in the nerve ring synapse onto secondary interneurons such as the command interneuron AVD which is involved in the regulation of motor output (locomotion). This interneuron also has its soma in the nerve ring ganglia. AVD extends a process through the nerve ring and down the ventral nerve cord. AVD receives synaptic input in the nerve ring and synapses onto motor neurons in the ventral cord. Motor neurons such as DA3 have their cell bodies located int the ventral nerve cord. Motor neurons like DA3 extend axons out of the ventral nerve cord and into the dorsal nerve cord where they synapse onto body wall muscle. Images of neurons from the WormAtlas, an excellent resource for both worm anatomy and neuroanatomy.

Sensory Neuron ASI in pink (pair of neurons)  Primary Interneuron RIS in orange Secondary Interneuron  AVD in red (pair of neurons)  Motor Neuron DA3 in purple

We know a great deal about the organization and synaptic connections of the C. elegans nervous system because of the extensive ultrastructural studies of the worm performed by John White and colleagues. The neurons in C. elegans are named by 2 or 3 letter designations. Many neuronal types consist of very similar bilateral partners which have mirror image projections. Details about individual neuron morphology and synaptic contacts can be found in several places including the WormAtlas.

Figure 4. Organization of mechano- sensory neurons in C. elegans. ALMs and PLMs are the major sensory neurons controling sensation of soft touch in the anterior and posterior half of the animal. Anterior and posterior touch causes animals moving forwards and backwards, respectively, to reverse direction. The ALM and PLM processes are filled with large diameter microtubules which are required for mechanosensation. These neurons for both electrical and chemical synapses onto the command interneurons which control locomotion.

In my lab we study the mechanosensory neurons. These six neurons sense gentile touch. PLMs and ALMs send processes on the side of the animal where receptors likely composed of degenerin family of ion channels are localized and thought to be involved sensing mechanosensory forces. These neurons extend synaptic branches into the ventral nerve cord (PLMs) or the nerve ring (ALMs) where they synapse onto interneurons including command interneurons like AVD.

Figure 5 Structure of the SAB motor neurons A) a diagram showing the position of the three SAB neurons cell bodies and their axonal projections. B) SAB neuons run over the surface of head muscle in each quandrant. C) Double labeling of wild type animals with synapotagmin antibodies (a-SNT-1 in green) and muscle myosin antibodies (a-MYO-3 in red). Ten to twelve synaptic varicosities are formed by each SAB neuron. D) An overlay of a Nomarski image and synaptobrevin-GFP labeled SAB varicosities running over the surface of head muscle.

We also study the SAB motor neurons which innervate head muscle (Figure 5). These are distinct from most other motor neurons in C. elegans because the synapses are formed over muscle are isolated from other synapses. Thus, these synapses are very easy to visualize. In addition, they are of interest since they change morphology and synaptic specializations in response to changes in neuronal activity.