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The Fundamental Role of Pirouettes in Caenorhabditis elegans Chemotaxis
The Fundamental Role of Pirouettes in Caenorhabditis elegans
Chemotaxis
Jonathan T. Pierce-Shimomura, Thomas M. Morse, and Shawn R. Lockery
Institute of Neuroscience, University of Oregon, Eugene, Oregon 97403-1254
To investigate the behavioral mechanism of chemotaxis in Cae-
norhabditis elegans, we recorded the instantaneous position,
speed, and turning rate of single worms as a function of time
during chemotaxis in gradients of the attractants ammonium
chloride or biotin. Analysis of turning rate showed that each
worm track could be divided into periods of smooth swimming
(runs) and periods of frequent turning (pirouettes). The initiation
of pirouettes was correlated with the rate of change of concen-
tration (dC/dt) but not with absolute concentration. Pirouettes
were most likely to occur when a worm was heading down the
gradient (dC/dt
0) and least likely to occur when a worm was
heading up the gradient (dC/dt
0). Further analysis revealed
that the average direction of movement after a pirouette was up
the gradient. These observations suggest that chemotaxis is
produced by a series of pirouettes that reorient the animal to
the gradient. We tested this idea by imposing the correlation
between pirouettes and dC/dt on a stochastic point model of
worm motion. The model exhibited chemotaxis behavior in a
radial gradient and also in a novel planar gradient. Thus, the
pirouette model of C. elegans chemotaxis is sufcient and
general.
Key words: nematode; chemosensation; spatial orientation;
neural
computation;
behavioral
models;
sensorimotor
integration
The nematode Caenorhabditis elegans is an excellent experimen-
tal system for studying the neuronal mechanisms of chemotaxis.
C. elegans is a small, soil-dwelling nematode attracted by com-
pounds thought to be associated with its food source, bacteria
(Ward, 1973; Dusenbery, 1974; Bargmann et al., 1993). C. elegans
chemotaxis is studied in the laboratory by following the move-
ments of worms in gradients of attractants on agar plates (Ward,
1973). The C. elegans nervous system is easy to study for three
main reasons. First, the adult hermaphrodite has only 302 neu-
rons, each reidentiable from animal to animal. Second, nearly all
of the anatomically dened synaptic connections in the adult
hermaphrodite have been reconstructed from electron micro-
graphs (Albertson and Thomson, 1976; White et al., 1986). Third,
it is possible to study neuronal function electrophysiologically in
patch-clamp recordings (Goodman et al., 1998) from identied
neurons. Little is known, however, about the behavioral and
neuronal mechanisms of chemotaxis in C. elegans.
Spontaneous locomotion in C. elegans involves two elementary
behaviors. On a moist agar surface, a worm makes a long series of
sinusoidal-swimming movements, called a run, interrupted ap-
proximately twice a minute by a sharp turn. Turns are pro-
duced in two main ways: by an omega turn in which a worms
head curls back, touching or crossing the tail, as the animal
continues to move forward (Croll, 1975a,b) or by a reversal in
which a worm moves backward for several seconds and then
moves forward again in a new direction (Croll, 1975a,b).
Previous anatomical and behavioral observations suggest that
chemotaxis in C. elegans may be regulated by attractant concen-
tration sensed at a single point on the body (Ward, 1973; Dusen-
bery, 1980). Although C. elegans has pairs of chemosensory or-
gans on its head (amphids) and tail (phasmids), the phasmids are
not necessary for normal chemotaxis (Ward, 1973), making it
unlikely that a worm orients primarily by sensing the difference in
concentration between head and tail. Because there are two
amphid organs, it is formally possible that a worm orients by
taking the difference in concentration between them, but this is
unlikely because the amphids are only 8 m apart (Ward et al.,
1975). These observations suggest that C. elegans assesses the
gradient by making comparisons at a single point through time, a
computation that approximates the time derivative of concentra-
tion dC/dt. Although C. elegans chemotaxis could be regulated by
absolute attractant concentration alone, such a mechanism seems
unlikely. This is because C. elegans chemotaxis has been observed
in gradients that differ
1000-fold in absolute concentration
(Ward, 1973), requiring an absolute-concentration detector with
unusually high resolution. Behavioral responses consistent with a
sensitivity to dC/dt in C. elegans have been reported (Dusenbery,
1980).
We used a tracking system to record the position, speed, and
turning rate of individual worms in well-dened gradients of
attractant. We found no evidence that C. elegans performs che-
motaxis simply by adjusting its speed or turning rate as a function
of concentration. Instead, we found that C. elegans modulates the
probability of large, brief turns as a function of dC/dt experienced
in the recent past. A computer model showed that this mechanism
is sufcient to account for the main features of C. elegans chemo-
taxis in laboratory assays. These results dene the behavioral
Received July 15, 1999; accepted Aug. 11, 1999.
This work was supported by the National Science Foundation; the National
Institute of Mental Health; the National Heart, Lung, and Blood Institute; the Ofce
of Naval Research; The Sloan Foundation; The Searle Scholars Program; and
National Institutes of Health predoctoral fellowship Training Grant GM07257. We
thank S. Owens for assistance; T. Ferre´e for help with the diffusion equation; B.
Marcotte for tracking-system development; W. Kristan, E. Martins, W. Roberts, and
J. Weeks for comments; and M. Gallegos and C. Bargmann for sharing unpublished
results. Worms were provided by the Caenorhabditis Genetics Center, which is
funded by the National Institutes of Health National Center for Research
Resources.
Correspondence should be addressed to Dr. Shawn R. Lockery, Institute of
Neuroscience, 1254 University of Oregon, Eugene, OR 97403-1254. E-mail:
shawn@chinook.uoregon.edu.
Copyright © 1999 Society for Neuroscience 0270-6474/99/199557-13$05.00/0
The Journal of Neuroscience, November 1, 1999, 19(21):95579569 inputoutput function of the neural network for chemotaxis in
C. elegans.
MATERIALS AND METHODS
Animals. Nematodes (C. elegans; Bristol strain N2) were cultured at
19 24°C on 1.7% agar-lled plates containing nematode growth medium
seeded with the Escherichia coli strain OP50 (Brenner, 1974). Mixed-
stage worms were rinsed off culture plates with assay medium containing
(in m
M
): ammonium chloride (NH
4
Cl) 2, CaCl
2
1, MgSO
4
1, and KPO
4
25, pH
6.5. To remove bacteria and other potential chemical stimuli,
we washed the animals by pelleting them loosely in a table-top micro-
centrifuge and transferred them to unseeded holding plates (diameter
9 cm) lled with 15 ml of agar-containing assay medium. Animals
remained on holding plates for 0.52 hr before being transferred indi-
vidually to an assay plate for study. All animals were either adults or
young adults. Assay plates contained assay medium under one of three
possible conditions: (1) a spatially uniform concentration of the attract-
ant NH
4
Cl, (2) a radial Gaussian-shaped chemical gradient (Ward,
1973), or (3) a planar gradient.
Chemical gradients. Radial gradients [NH
4
Cl, 2.0 6.0 m
M
, or biotin,
4.3
10
7
to 3.0 m
M
(Ward, 1973)] were established in assay plates,
which contained the same agar as holding plates, by placing a 5 l drop
of attractant at the center of the plate at two different times (t
1
and t
2
)
before the experiment (500 m
M
NH
4
Cl, 16
t
1
22 hr; 3
t
2
4 hr;
200 m
M
biotin, 16
t
1
22 hr; 4
t
2
5 hr). For each assay plate, t
1
and t
2
were recorded for estimation of the attractant concentration
during the assay. At each time point t in the assay, the concentration of
attractant C (millimolar) at the position of a worm was estimated accord-
ing to the solution of the diffusion equation (Crank, 1956) for a point
bolus in a cylindrical, aqueous volume having the same dimensions as the
agar in the assay plate (diameter
9 cm; depth
0.264 cm).
Accordingly:
C t, r
c t, t
1
, r
c t, t
2
, r
(1)
c t, t
i
, r
10
6
N
0
4 dD t
t
i
e
r
2
/4D t t
i
,
(2)
where N
0
is the moles of attractant in the 5 l drop, d is the depth of the
agar (centimeters), r is the distance (centimeters) between the peak of
the gradient and the location of the animal, t is the time (seconds) since
the animal was placed in the assay plate, i is the drop number (1 or 2),
and D is the diffusion coefcient: NH
4
Cl, D
1.861
10
5
cm
2
sec
1
(Robinson and Stokes, 1959); biotin, D
5.0
10
6
cm
2
sec
1
. The
coefcient for biotin was approximated as the coefcient for uorescein,