Worm Breeder's Gazette 10(1): 91
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
We have recently been attempting to assign functions to various neurons, many of which are located in the head ganglia near the nerve ring. We use a Laser Sciences Inc. nitrogen-pumped laser (described in WBG 9(2), 1986, p. 110) to kill specific neurons identified on the basis of position in L1 worms, then let the animals grow to adulthood and test behavior. In combination with the complete wiring diagram ( White et al., 1986), this method provides a powerful approach to understanding the function of the nervous system. We can envision at least four potential pitfalls with this approach. First, we may fail to eliminate functionally the neuron that is killed. Second, we may cause damage to neighboring cells or processes, especially those located near the site of the laser strike. Third, the cell that we identify on the basis of position might not invariably be the same cell; for example, it might occasionally be switched with some neighboring cell. Fourth, the cell in a particular position might not be correctly correlated with the wiring diagram. Of these pitfalls, we have collected some information about the first three. The fourth problem is more difficult to address, and largely depends on the assignments by Sulston et al. (1983). The evidence so far suggests that killing a neuron with the LSI laser results in functional elimination of the target cell. First, when we kill a neuron (by this we mean all members of a class of neurons) with a previously described function, we reliably obtain the expected phenotype for loss of the neuron's function (based on genetic studies and/or embryonic kills; Chalfie et al., 1986; M. Chalfie, C. Desai, personal communications). Such cells include AVA (backward Unc; 6), RIP (loss of Mec inhibition of pumping; 2), HSN (egg-laying defective; 3), PLM (tail Mec; 5), ALM (reduced head touch sensitivity; 3), and PVC (tail Mec; 3). (Parenthetical remarks after each neuron indicate the behavioral phenotype and the number of animals analyzed.) In other cases, we have observed a behavioral defect associated with killing a particular neuron. When we kill this neuron in more than one animal we consistently observe the same behavioral defect. These cells include ASH (12), ASJ (2), PVQ (3), and M4 (150). (For the functions of ASH, ASJ, and PVQ see the accompanying Thomas and Horvitz newsletter entry.) A related concern is how fast a killed neuron loses function. The best evidence for this time course is for the pharyngeal motorneuron M4. This neuron is required for peristalsis of the pharyngeal isthmus (Avery and Horvitz, WBG 9(2), 1986, p. 57), a function that can be assayed at any stage of development. There is a pronounced, but not always complete, deficit in M4 function 5 hours after laser killing in young L1 larvae, and M4 is invariably nonfunctional after 24 hours. Similarly, when PVC (3), AVA (6), or PLM (5) is killed during the early L1 stage, animals acquire the loss- of-neuron-function phenotype within 24 hours (the earliest time tested) . These results suggest that laser killing in the early L1 typically eliminates neuron function well before adulthood. We also have evidence that laser damage to cells or processes near the target cell is not generally a problem. Even when we kill neurons in the head ganglia, where neuron cell bodies and processes are closely packed, we do not observe effects on other behaviors. For example, no kill (other than AVA) has caused a backward Unc phenotype, characteristic of AVA animals. The most extensive analysis has been done for the cell ASH, which is required for normal osmotic avoidance ( see accompanying Thomas and Horvitz newsletter entry). Neurons with cell bodies that surround ASH on all sides (ADF (2), AWC (3), AUA (2), AIB (1), and ASE (3)) have been killed with no effect on osmotic avoidance. In addition, many neurons with a process that runs alongside the ASH process in the amphid bundle (ADL (2), AWC (3), and ASK (4)) or in the nerve ring (AIB (l) and ADF (2)) or both (ASE (3)) have been killed with no discernible effect on ASH function. These results should be taken with a grain of salt, since most C. elegans neurons (including AVA and ASH) are bilateral. Since probably only one of the pair need function for normal behavior, we might require damage of both cells to see a phenotype. However, this redundancy also works to our advantage, since only damage to both sides will produce spurious results. The same set of laser experiments show that, for several neurons in the head, position in the early L1 is sufficient to identify the same cell in different animals. These cells are RIP (2), ASH (12), ASJ (2), and AVA (6). For each of these cases, when the cells in these positions are killed in different animals they cause the same behavioral defects in the adult. We have also tested cells in the tail ganglia (PLM (5), PVC (3), and PVQ (3)) with the same result. This evidence indicates that the same functional cell type occupies the same position during the L1 stage in different animals. This rule may not apply to all neurons, but as more cells are assigned invariant positions, the potential variability of the remaining neurons becomes more restricted. Although we cannot directly address the final possible pitfall ( correlation of the killed neuron with the wiring diagram), we can point out that the behavioral phenotype observed for each kill fits well with the target cell's assigned connectivity. Finally, we offer some general comments on the usefulness of this method. The position of most neurons in the head ganglia and elsewhere can be learned fairly easily by careful inspection of a number of animals and frequent comparison with the excellent diagrams found in Sulston et al. (1983). Some neurons are easier to identify than others, and a few (around the posterior bulb of the pharynx) may not be possible to identify because of their variable positions (as noted in Sulston et al., 1983). However, the general impression one gets, after sufficient observation time to learn the patterns, is the remarkable reproducibility of the relative positions of most neurons. It is quite reasonable, for a cell one knows well, to kill a particular type of neuron and confirm the kill in more than 30 animals in one day.