Mutations in UNC-108/RAB-2 affect different neurotransmitter signaling

III Methods and materials

4.3 Mutations in UNC-108/RAB-2 affect different neurotransmitter signaling

Since the aldicarb test is specific for cholinergic signaling, we wanted to see whether other neurotransmitter systems are also affected in unc-108 mutants.

Two of these system, GABAergic and serotonergic systems are involved in

controlling defecation and egg laying, the processes that are stereotyped and fully described in C. elegans (Branicky and Hekimi, 2006; Schafer, 2006;

Wolinsky and Way, 1990).

percentage of expulsion failure

wild type

percentage of expulsion failure

wild type

percentage of expulsion failure

wild type

percentage of expulsion failure

wild type

Figure 18. unc-108 mutants have defects in GABAergic signaling.

(A) Defecation cycle is a highly stereotyped process in C. elegans. The cycle starts with the contraction of the posterior side (pBoc) when the intestinal content is pushed towards the head. Then the anterior part of the body contracts pushing food back to the tail. The last step is expulsion. The entire cycle occurs every 50 seconds in wild type. (Jorgensen, 2005)

(B) The defecation cycle is controlled by GABAergic neurons AVL and DVB neurons. The enteric muscle consists of anal depressor (AD), sphincter and two intestinal muscles. The muscles are connected via gap junctions and send muscle arms to the AVL and DVB neurons. The release of GABA leads to contraction of AD and IM (pBoc step) and relaxation of sphincter muscle (expulsion). (Adapted from Jorgensen, 2005)

(C) Each animal was followed for 10 minutes period and the expulsion failures were counted.

In unc-108 mutants the expulsion occurs in 60-70% of the cycles (number of animals analyzed is indicated in the graph bars, error bar=SEM).

Defecation in C. elegans is achieved by cyclical stereotyped motorprogram (Fig. 18A). The first step is the contraction of the posterior side (pBoc), which pushes the intestinal contents towards the head. Then the contraction of the anterior part of the body occurs (aBoc), which pushes the content back to the tail and is followed by expulsion step. The entire program is highly stereotyped and occurs every 50 seconds in wild type. The expulsion step is controlled by GABAergic neurons, which excite and contract the enteric muscles.

While in wild type the expulsion step occurs in every defecation cycle, in unc-108 mutants the expulsion step is missing in 30-40% of the cycles (Fig. 19C).

However, the phenotype is not as severe as the one observed in other synaptic proteins, synaptotagmin 1, snt-1 and aex-3, a RAB-3 and RAB-27 guanine exchange factor (GEF).

One of the best studied behaviors in C. elegans is egg-laying. Egg-laying involves a simple motor program involving a small network of motorneurons and specialized muscle cells. One of the key modulators of the egg-laying program is serotonin. Thus, this behavior assay is commonly used for screening for defects in the serotonergic signaling. Serotonin is the activator of hermaphrodite-specific neuron (HSN) which forms synapses with vulva muscles. After fertilization, the eggs remain in uterus for about 2 to 3 hours and are then expelled from the worm through the vulval opening. Most of the eggs laid by worms are still in the blastula stage, continuing with gastrulation and further development in external environment (Fig. 19A). If the signal does not reach the vulva muscle, the eggs are retained in the body cavity, thus

once the laying event occurs, the embryos are in the later developmental stages (Egl-egg laying defective phenotype).