Animals continuously search their environment for food, water, and shelter. In animals with complex nervous systems, different senses aid the search for necessities. The simple microscopic worm C. elegans has only 302 neurons and no eyes or ears—it perceives its environment entirely through touch and smell. Chance observations while imaging C. elegans led researchers in the Bai Laboratory (Basic Sciences Division) to wonder whether these animals with a relatively simple nervous system could sense different physical structures in their environment. While it was clear from previous work that the worms change direction when they encounter an obstacle, it was unknown whether they could distinguish different physical patterns in their environment. In their recent publication in eLife, Bicheng Han and colleagues demonstrate that worms do have this ability and the researchers identify genes responsible for the behavior.
The scientists built tiny chambers that contained four sections ("quad chamber") with different densities of pegs in each to test whether the worms preferred more open or more confined spaces. Interestingly, the standard laboratory strain of C. elegans, the Bistol, UK N2 strain, ended up spending most of its time in the densely packed section of the chamber. In contrast, a strain of C. elegans isolated in Hawaii (HW) spent most of its time in the most open section. The scientists even measured the frequency of returning to a preferred section after crossing over to a less-preferred section. They found that worms that leave their preferred section do indeed "reverse" more frequently back into that section, more so than the reversal frequency at less preferred section boundaries. These results show that worms do indeed sense the layout of their environment. Additionally, because different strains prefer different environments, it suggests that these preferences are adaptive.
Given four different spatial patterns, the Bristol N2 strain spent the most time in the densely patterned section IV of the “quad” chamber. The scientists wondered whether the movement or lack of movement in quadrant IV would be different if the worms had never encountered other spatial patterns. Put simply, did past experience influence the worms response to the spatial environment? To test this they constructed a “uni” chamber with only pattern IV (dense pegs) and measured the speed and pause rates of worms in this “uni” pattern IV chamber vs in pattern IV of the quad chamber. Interestingly, they found that the worms in pattern IV of the quad chamber moved more slowly and paused more than those in the same pattern within the “uni” chamber. The results suggest that past experience influences the worms’ movement.
Next the scientists wanted to uncover which genes were responsible for inducing this behavior. Given previous evidence from behavioral studies in C. elegans, they suspected that dopamine signaling might be required. To test this, they analyzed the behavior of mutant worms lacking the ability to synthesize dopamine. They found that these worms showed a reduced preference for pattern IV in the quad chamber and that ectopic addition of dopamine restored wild-type like (normal) pattern preference for section IV. The scientists examined the role of 4 dopamine receptors in C. elegans and, mutating each of the 4 individually, they found that mutation of receptor DOP-3, but not the other receptors, reduced the worms preference for pattern IV. The DOP-3 receptor is expressed in neurons in the head, tail and ventral cord and whether a specific point-of-sensation contributes to the behavior more than others remains to be investigated.
After finding that dopamine signaling was required for generating the behavior, they wanted to determine which sensory receptors were initially receiving the spatial signals created by the different patterns. They reasoned that the spatial patterns must be sensed by mechano-sensitive receptors that are known to function in neurons that also generate dopamine (dopaminergic neurons). TRP-4 is one such receptor and mutation of this receptor did reduce the N2 worms’ preference for pattern IV. Double mutant worms defective in TRP-4 function as well as dopamine synthesis had about the same low level of pattern preference for section IV as worms with either component mutated individually, suggesting that TRP-4 and dopamine synthesis are in the same single pathway leading to the behavior.
The scientists compared the sequence of the TRP-4 receptor in the Bristol N2 vs Hawaii HW strains of C. elegans. They found that the HW strain had three amino-acid substitutions. To determine whether these differences accounted for the difference in behavior, the scientists performed “rescue” experiments where they genetically engineered worms to express the N2 or HW TRP-4 gene in N2 worms defective in endogenous (native) trp-4 function. While expression of N2 TRP-4 fully restored pattern selectivity to the N2 worms, expression of HW TRP-4 did not. This suggested that N2 TRP-4 is responsible for the preference for pattern IV. In line with this, overexpression of N2 TRP-4 in HW worms caused HW worms to prefer pattern IV. Further genetic experiments suggest that other genes located on chromosome I may contribute to the spatial preference behavior.
“These findings link spatial pattern selectivity to a key neuromodulator that controls behavior and emotion throughout the animal kingdom,” said principal investigator Jihong Bai. “This elucidates a conserved mechanism for a basic sense of physical space in a simple nervous system.”
Han B, Dong Y, Zhang L, Liu Y, Rabinowitch I, Bai J. 2017. "Dopamine signaling tunes pattern selectivity in C. elegans." Elife. 6, e22896.
Funding for this research was provided by the National Institutes of Health, the Hartwell Innovation Fund, and a FHCRC New Development Grant.
See also the Fred Hutch News story on this work.