Worm Breeder's Gazette 10(1): 89

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.

The Amphid Sensory Neuron ASH Mediates Osmotic Avoidance

J. Thomas and B. Horvitz

Figure 1

When a worm encounters a region of high osmotic strength with the 
tip of its nose, it responds by backing up, typically several body 
lengths, and usually turning around.  Genetic analysis of this 
behavior should be powerful, since selection of animals that fail to 
respond to osmotic strength is simple.  To determine which sensory 
neurons mediate the osmotic avoidance response (hereafter called the 
Osm phenotype), we used a laser to kill neurons in early L1 larvae, 
and performed single-animal Osm assays on the animals as adults.  For 
a discussion of various concerns about the validity of laser-killing 
experiments on neurons, see the accompanying newsletter entry (Thomas, 
Garriga, Avery, and Horvitz).
Our assay is a modification of that used by Culotti and Russell (
1978).  About 15 l of 4 M fructose (plus Congo Red, a dye that worms 
do not respond to) is applied to a dry NG plate in a ring with an 
inner diameter of about 0.8 cm and allowed to soak into the agar.  A 
single animal is moved without bacteria onto the test plate inside the 
osmotic ring and allowed to run for 15 minutes, with visual checks at 
least once per minute (tracks are observed at the end of the assay to 
be sure that a transient crossing did not occur).  The time the animal 
takes to cross the osmotic barrier is recorded, rounded to the nearest 
minute.  The minimum score is one minute.  Notes on individual 
encounters are usually made as well.  A wild-type animal usually 
encounters the fructose ring in fewer than 30 seconds, responds 
normally, and heads off in a new direction.  In a typical assay the 
animal will encounter the ring about 20 times in 15 minutes.  Wild-
type animals very rarely cross the ring, and then only very late in 
the assay (when the osmotic barrier has diffused considerably).  When 
a mutant such as osm-3(p802) is run in this assay, the animal 
typically crosses the barrier in less than one minute, without any 
response [See Figure 1].  From the results of several such tests we 
calculate an osmotic avoidance index (OAI) - 10/14 (t - 1), where t is 
the mean time to cross.  This produces a range of OAI from 0 to 10.  
The OAI for N2 is very close to 10 and that of osm-3(p802) is 0.2 (osm-
3 animals never respond to the osmotic barrier, but sometimes take 
longer than one minute to cross).
What sensory neurons mediate this behavior?  We knew the sensing was 
likely to be in the head, and the only known head defect of osm-3 
animals is ultrastructural abnormalities in the amphid sensory endings.
Therefore we killed the amphid sheath cell (both sides) to test 
whether the Osm response was eliminated.  It was (see Table for a 
summary of all of the cell killing data).  Since the sheath acts as a 
glial-like sheet around all eight of the amphid chemosensory endings, 
it seemed likely that one or more of these neurons was mediating Osm.  
We FITC-filled six of the amphid chemosensory neurons (ADF, ASH, ASI, 
ASJ, ASK, ADL), by the method of Hedgecock et al.  (1985), and 
bleached the dye using a fluorescein filter set and an epifluorescence 
light source.  This treatment also eliminated the Osm response (see 
Table for data and controls).  With the laser we killed seven of the 
eight amphid chemosensory neuron types individually and tested each 
for osmotic avoidance (the eighth neuron, ASG, has not been done 
because it is more difficult to identify with certainty and is not 
FITC-filled.) In addition, the amphid interneurons AIB and PVQ were 
killed.  Only when ASH was killed were animals significantly less 
responsive than normal.  ASH animals frequently failed to respond to 
the osmotic barrier at all, but sometimes responded and crossed only 
later in the assay.  Even when an ASH animal responded it was usually 
deeper in the osmotic barrier than normal, suggesting a less sensitive 
or slower response.  Killing ASH reduces the efficacy of the Osm 
response, but it is not the whole story.
Any other sensory neuron involved in the Osm response should also be 
in the amphid, and probably one of the FITC-filled cells, because of 
the amphid sheath and FITC-filling results.  We searched for a second 
neuron by doing double kills with ASH (both sides) and a second amphid 
sensory neuron (both sides).  Of the four neurons tested, only killing 
ASJ effectively eliminated the residual Osm response (we are not yet 
sure whether the apparent reduction by killing ASI is significant; we 
have not yet tested ADF).  ASJ has output only to PVQ, an interneuron. 
Killing PVQ and ASH also eliminated the residual Osm sensitivity, 
suggesting that ASJ acts through PVQ.  We should emphasize that the 
assignment of ASJ and PVQ to the Osm response is tentative; we must 
kill these cells together with ASH in more animals to be certain of 
this relatively marginal effect.
We anticipate that further application of the laser to the Osm 
behavior will clarify the rest of the neuronal pathway from sensory 
input to motor response.  Identification of the cells in this pathway 
will assist in the interpretation of mutants defective in the Osm 

Figure 1