Using a genetic and molecular biology based approach, we have cloned and characterized a gene,
nmr-1, which encodes a putative ionotropic glutamate receptor subunit in C. elegans.
nmr-1 shows highest identity to vertebrate NMDA-type glutamate receptors and is expressed in a small subset of neurons in hermaphrodites, including AVA, AVB, AVD, AVE, AVG and PVC. Excepting AVG, these neurons comprise the "command" interneurons first described by Chalfie, et al., who used laser ablation analysis to show that they are required for both coordination and modulation of locomotion. In addition, work by Wicks and Rankin suggests that these neurons form part of the neural circuit required for the response to tap. To determine how
nmr-1 contributes to worm behavior, we have generated two Tc1 insertion mutations in
nmr-1 coding sequences, and one deletion mutation. The 1.9 kb deletion of
nmr-1 genomic sequence removes three of the four putative transmembrane domains, including regions thought to line the pore of the ion channel. Deletion mutants show no obvious defects in locomotion, mechanosensation, osmotic avoidance, or chemotaxis. However, they do show defects in the timing of locomotory activity. On a food free plate, a wild-type worm will change direction from forward movement to a brief backward movement with clock-like regularity, where the average duration of forward and backward movements is approximately 20 seconds and 3 seconds, respectively. The deletion mutation in
nmr-1 appears to affect the clock by increasing the duration of both forward and backward movements to approximately 39 seconds and 4 seconds, respectively. This suggests that Nmr-1 may regulate a higher order central pattern generator in C. elegans. How does
nmr-1 contribute to the clock-like regularity of spontaneous behavior in C. elegans?
nmr-1 expressing interneurons are known to be required for coordination and modulation of both forward (AVB and PVC) and backward (AVA and AVD) locomotion. Neurons in the forward circuit receive synaptic input from neurons in the backward circuit and vice versa. Our results suggest that Nmr-1 may affect the frequency of spontaneous reversals by mediating interactions between the two opposing circuits. In addition to
nmr-1, nine genes encoding putative ionotropic glutamate receptor subunits have been identified in C. elegans. We have made deletion mutations in a number of these genes, including
nmr-2 and
glr-1, which show highest identity to vertebrate NMDA and non-NMDA-type glutamate receptors, respectively.
nmr-1 and
nmr-2 are expressed in the same subset of interneurons, suggesting that the subunits they encode may interact to form functional ionotropic glutamate receptors in vivo. In addition,
nmr-1 and
nmr-2 expressing neurons also express
glr-1. We will determine the genetic interactions between these three genes by characterizing worms that have deletion mutations in two or more of the genes. Perhaps worms change their direction of movement at regular intervals as a way of exploring their environment. If so, we would expect this behavior to be modifiable. Does the frequency of reversals change in the presence of chemical attractants or repellents? And is the change more pronounced with experience? We are currently developing paradigms that will address these questions. In addition, we are undertaking an electrophysiological analysis of Xenopous oocytes that express
nmr-1 cRNA.