Behavioral Ncurosciencc Copyright 2000 by the American Psychological Association, Inc.

2000, Vol. 114, No. 3, 496-505 0735-7044/00/S5.00 DOI: I0.1037//0735-7044.I14.3.496

Context Conditioning in Habituation in the Nematode

Caenorhabditis elegans

Catharine H. Rankin
University of British Columbia

Habituation has traditionally been considered a nonassociative form of learning. However, recent
research suggests that retention of this nonassociative form of learning may be aided by associations
formed during training. An example of this is context conditioning, in which animals that are trained and
tested in the presence of a contextual cue show greater retention than animals trained and tested in
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different environments. This article reports context conditioning in habituation in the nematode Caeno-
This document is copyrighted by the American Psychological Association or one of its allied publishers.

rhabditis elegant. The results showed that retention of habituation to tap at both 10- and 60-s
interstimulus intervals was significantly greater if training and testing occurred in the presence of the
same chemosensory cue (NaCH3COO). This context conditioning showed both extinction and latent
inhibition, demonstrating that these simple worms with only 302 neurons are capable of associative
context conditioning.

Habituation, a decrease in responding as a result of repeated
stimulation, is a member of a class of learning phenomena termed
"nonassociative" (Groves & Thompson, 1970). This classification
was based on the belief that the response decrement seen in
habituation is governed solely by the parameters of the presenta-
tion of the unconditioned stimulus (US: Marlin & Miller, 1981).
However, recent studies in several different organisms have sug-
gested that an organism may make associations during habituation
training that may affect later performance. The most common
associative learning phenomenon reported with habituation is con-
text conditioning. In context conditioning some aspect of the
training environment—the chamber itself, the experimenter, the
subject's state, or the subject's circadian rhythms—may be asso-
ciated in some way with the US presentation and come to influence
later behavior. In particular, context cues may play a role in
memory retrieval of earlier training. This notion was suggested by
Wagner (1976, 1978, 1979), who hypothesi?£d that short-term
habituation was nonassociative in nature but that memory for
earlier habituation training results from associative processes. He
argued that an association is formed between the US and the
context in which it is delivered. When an animal is later reexposed
to the same context, a retrieval-generated representation of the US
is produced in short-term memory. This representation of the US
then attenuates the response to the US when it is presented. Thus,
context has become the conditioned stimulus (CS), predicting the
delivery of the US.
The role of context in habituation has been explored in a number
of different organisms. In some experiments, context appears to be
important for habituation (i.e., Evans & Hammond, 1983), whereas
in other cases it does not (i.e., Marlin & Miller, 1981). One of the
difficulties with studying context conditioning is to determine the
situational cues that are salient to the animal. This may be one
explanation for the sometimes elusive nature of the phenomenon.
Support for the notion of context conditioning in habituation
comes from studies of differential generalization of habituation
across contexts (Evans & Hammond, 1983). Evans and Hammond
discussed a number of studies in rats, fish, and birds, in which
generalization of habituation was a function of contextual similar-
ity—this translates to greater habituation expressed in contexts
most similar to training. In addition they suggested that the more
biologically relevant a stimulus is, the more likely it is to be a
context cue for earlier learning.
Several recent publications on habituation to a visual stimulus in
the crab Chasmagnathus have shown that context plays a role in
long-term memory for habituation in this animal. Tomsic, Mas-

This work was supported by Natural Sciences and Engineering Research
Council and Human Frontiers of Science operating grants. The original
nematode strain used in mis work was provided by the Caenorhabditis
Genetics Center, which is funded by the National Institutes of Health
National Center for Research Resources. We thank Jewel Sarnowski,
Marion Buday, Megan Tolbert, Tracy Tanchuk. and Susan Sangha for
running experiments; Richard Faber, Sylvia Chen, Carina Fu, and Anthony
Chau for scoring videotapes and entering data into the computer; and Liisa
Galea, Don Wilkie, and Stan B. Floresco for statistical consulting. We also
thank Norman White for comments on previous versions of this article.
Correspondence concerning this article should be addressed to Catharine
H. Rankin, Department of Psychology, University of British Columbia,
Vancouver, British Columbia, Canada V6T 1ZH. Electronic mail may be
sent to crankin@cortex.psych.ubc.ca.
soni, and Maldonado (1993) showed that long-term habituation in
Chasmagnathus was abolished by multimodal changes in context
between training and testing. Saraco and Maldonado (1995) dem-
onstrated that ethanol injection before training did not affect re-
tention of habituation but did impair contextual memory. Tomsic,
Pedriera, Romano, Hermitte, and Maldonado (1998) investigated
the nature of the context-US association in habituation in the crab
and showed context memory for a visual cue as well as latent
inhibition and extinction of context.
One of the reasons for using model systems for the study of
learning and memory is the hope that information can be learned
about the cellular mechanisms underlying various forms of learn-
ing. By understanding the biological processes underlying types of

496
 CONTEXT CONDITIONING IN C ELEGANS
learning such as habituation, context conditioning, and classical
conditioning, we can better understand the relationships between
these processes. These studies are designed to examine the role of
context in habituation in the nematode worm Caenorhabditis el-
egans, a self-fertilizing hermaphroditic nematode that has been the
target of intensive developmental, anatomical, and genetic analy-
sis. The nervous system of C. elegans consists of 302 identified
neurons that have been mapped at the electron microscope level,
with all morphologically distinct electrical and chemical synaptic
zones recorded (White, Southgate, & Durbin, 1988; White, South-
gate, Thomson, & Brenner, 1986). This information has allowed
the construction of a morphological wiring diagram of the nervous
system. The detailed mapping of the nervous system has allowed
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the development of laser microsurgery of individual identified
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neurons, for the purpose of mapping the neural circuits that un-
derlie specific behaviors (Avery & Horvitz, 1989; Chalfie, Sulston,
White, Southgate, Thomson, & Brenner, 1985).
C. elegans has been shown to be capable of both short- and
long-term habituation of the tap withdrawal response (Beck &
Rankin, 1997; Rankin, Beck, & Chiba, 1990). The tap withdrawal
response is seen when a worm swims backward for some distance
in response to the vibrations produced by a mechanical tap to the
side of the Petri dish in which it swims (termed a "reversal"
response). A detailed circuit analysis has identified the neurons
involved in the behavior (Wicks & Rankin, 1995) and the role of
those neurons in habituation (Wicks & Rankin, 1996). C. elegans
has also been shown capable of discriminative classical condition-
ing in experiments in which a chemosensory cue is paired with the
presence of food and a second chemosensory cue is paired with the
absence of food (Wen et al., 1997). In these experiments, worms
given a choice between the two chemosensory cues choose the cue
associated with food. Because C. elegans is capable of both
habituation and classical conditioning, it is possible that an asso-
ciative learning component might play a role in habituation.
The experiments presented here were designed to determine (a)
whether context conditioning plays a role in habituation in C.
elegans and (b) whether context mediates its effect by means of a
context-US association.
General Method
Subjects
Three hundred sixty 4-day-old adult hermaphroditic nematodes (C
elegans, strain Bristol N2) were used in these experiments. They were
maintained at 20 °C on 5-cm Petri plates filled with 10 ml nematode
growth medium (NGM) agar and streaked with E. coli (strain OP50;
Brenner, 1974).
Apparatus
Worms were viewed with a stereomicroscope (Wild M32, Wild Leitz
Canada; Leica, Willowdale, Ontario, Canada), and their behavior was
recorded with a Panasonic D5000 high-resolution video camera attached to
a Panasonic AG1960 videocassette recorder and a 13-in. NEC monitor. A
Panasonic 814 time-date generator superimposed a stopwatch on the
recorded image to allow for accurate delivery of stimuli at the appropriate
interstimulus interval (ISI). Individual worms were observed on Petri plates
placed in a holder that was constructed from the lid of a Petri plate mounted
onto a plastic rod. A micromanipulator attached to the rod allowed the plate
to be moved smoothly to keep the worm in the camera field. Also attached
497
to the holder was a mechanical tapper consisting of a wire arm and an
electromagnetic relay, which was connected to a Grass 88 stimulator
(Grass Instruments, Quincy, MA) to regulate stimulus delivery. Stimuli
consisted of trains of taps (six 25-ms pulses with a total duration of 600 ms)
to the side of the Petri plate.
Procedure
In each of the experiments, individual worms were tested in 5-cm Petri
plales filled with 10 ml NGM agar. The plain context was produced by
using Petri plates containing 10 ml unadulterated NGM agar. The sodium
acetate (NaCH-,COO) context was obtained by mixing fresh solutions
of 0.4 M NaCH3CGO every testing day; 5-cm Petri plates filled with 10-ml
NGM agar were washed with 400 microliters of this solution, excess was
poured off, and the plates were allowed to dry for 1 hr before testing began.
Worms were preplated for at least 1 hr on Petri plates containing NGM
agar streaked with a small amount of E. coli. All transfers between plates
were made in M9 buffer solution by using a 20-microliter mouth pipette.
Worms were moved to the training and testing plates 3.5 min before the
beginning of the stimulation. In the context studies (Experiments 2-5)
worms were administered 30 trains of taps in a specific context (either
plain agar or NaCH3COO-treated plates) during habituation training, trans-
ferred to another plate for a 60-min rest interval, and then moved to a new
testing plate (either plain or NaCH-jCOO context) where another 30 trains
of taps were administered. Both training and testing were videotaped for
later scoring. To score the data, the reversal response (the distance traveled
backward) to each tap for each worm was traced onto an acetate sheet by
stop-frame video analysis; the acetate sheets were then scanned onto a
Macintosh computer. The magnitude of the reversals was measured with
the public-domain NIH Image program (developed at the United States
National Institutes of Health LNIHJ and available from the Internet by
anonymous FTP from zippy.nimh.nih.gov). Previous research (Wicks &
Rankin, 1996) has shown that reversals and accelerations are mediated by
different subsets of neurons and have different habituation kinetics. In this
study, the focus was on reversals, and, therefore, accelerations were des-
ignated as missing data points (approximately 10% of the data), whereas
pauses and no responses represented the absence of a reversal and were
included in the analysis as zeros.
In Experiments 2-5, the effect of context on habituation was most
evident early in the testing block and was confounded later in the block by
a floor effect (as can be seen in Figures 2 and 4); therefore, statistical
analyses involved comparison of only the average response to the first two
trains of taps for the training block and for the testing block. Two-tailed
paired t tests on the mean of the first two responses in each block were used
to test for differences in retention of habituation from training to testing.
Because there were multiple independent comparisons (2-8 / tests) made
in each of the experiments, the alpha level for significance was reduced
with a Bonferroni correction that divided alpha level (set at .05) by the
number of comparisons made in that experiment.
The experimenter sometimes mistimed a tap and delivered it while the
worm was already reversing—this too was considered a missing data point.
Of the 360 worms tested, 29 were not used in the analysis: Some were
discarded for technical reasons because the worm was already reversing
when one or both of the first two taps was delivered during testing. If only
one was an "already reversing," and that worm's response to the other
stimulus was an acceleration, then there were two missing data points. In
addition, some were discarded because both responses in the test block
were accelerations and thus were considered missing data points. The
number of worms discarded in each group is reported in each experiment.
Experiment 1: Effect of NaCH3COO
The habituation protocol used in C elegans involves a mech-
anosensory stimulus; therefore, to find a stimulus to use as a salient
 498 RANKIN

context, other sensory systems were considered. In addition to

mechanosensation, C. elegans displays thermosensation and che-
mosensation and is capable of both olfaction of volatile odors and
taste of dissolved compounds (Bargmann, 1993; Bargmann,
Thomas, & Horvitz, 1990; Ward, 1973). For these experiments, a
dissolved chemical cue, NaCH3COO, was used as a context cue.
C. elegans is attracted to both sodium and acetate (Ward, 1973;
Wicks & Plasterk, 1997). The concentration chosen for these
experiments (0.4 M) has been effectively used in classical condi-
tioning studies with worms (Wen et al., 1997). It is important that
a cue used to set context in a habituation experiment does not have
any sedating or arousing properties of its own. Any stimulus used
as a context cue should not lead to any great change in response
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levels; that is, there should be neither an increase nor a decrease

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from baseline response levels as a result of exposure to the cue.

Experiment 1 was designed to assess the effects of NaCH3COO on
die baseline response levels to tap and its effect on the habituated
response to tap.

Method

Subjects. A total of 119 adult 4-day-old hermaphroditic C. elegans

(Bristol N2) were used in this experiment.
Procedures. To assess the effects of NaCH3COO on the tap with-
drawal response, plates were prepared as described in the General Method.
Worms were placed on either NaCH3COO-treated plates (« = 40) or plain
NGM plates (n = 39) and were allowed to recover from transfer for 3-4
min. Worms were then given a single train of taps to assess the baseline
response levels on each type of plate.
To determine whether NaCHjCOO affected habituation to tap in any Initial Hab NaCH3COO
way, worms (n = 20) were placed on plain NGM agar plates and given 30
Figure 1. NaCH3COO has no effect on the response to tap, or habituation
trains of taps at a 60-s ISI. A small camel hair paint brush was then used
of the response. A: Comparison of mean response magnitude (in pixels) of
to brush a small amount of 0.4 M NaCH3COO onto the agar in front of the
worms given a train of taps on either plain nematode growth medium
worm 30 s after the last train of taps. The NaCH3COO was quickly
(NGM) agar plates or NaCH3COO-treated plates. There were no differ-
absorbed into the agar and did not impede the worm's movement over the
ences in response magnitude. B: Mean response magnitude for the initial
agar. Thirty seconds after the delivery of NaCH^COO, another train of taps
response, the 30th response (Hab) and the first response (NaCH3COO)
was given to test for effects of the NaCH3COO.
after exposure to NaCH3COO. The drop in magnitude from the initial
response to the 30th response represents habituation to tap. The lack of
Results and Discussion change in responding after exposure to NaCH3COO shows that this com-
pound had no effect on the habituated response.
The results showed that 0.4 M NaCH3COO has no significant
effect on the baseline response to tap. When the response to tap
was tested on NaCH3COO versus NGM plates, there was no
significant difference in response magnitude between the two either the same environment as training or a different environment.
environments, ;(77) = 0.36, ns (see Figure 1A). Other experiments have found that short ISIs (i.e., 10 s) sometimes
When NaCHjCOO was presented to the worm immediately produce different results than do long ISIs (i.e., 60 s). For example,
during habituation training, it had no effect on response magni- worms habituate more quickly and recover more rapidly with short
tude. Thus, there was no evidence that brief exposure to ISIs than with long ISIs (Rankin & Broster, 1992); however,
NaCH3COO disrupted habituation to tap in any way, r(19) = 0.76, worms show 24-hr retention of habituation training when trained
ns (see Figure IB). with a 60-s ISI but not when trained with a 10-s ISI (Beck &
Thus, 0.4 M NaCH3COO does not appear to have any arousing Rankin, 1997). Therefore, groups of worms were tested at these
or sedating effects on the response to tap that would confound its two different ISIs to investigate whether context conditioning was
use as a contextual cue in the ensuing experiments. sensitive to ISI. The hypothesis was that worms tested in the same
context as their habituation training would display greater reten-
Experiment 2: Context and Habituation tion of habituation than worms tested in a context different from
that of training.
In Experiment 1, NaCH3COO was shown to neither arouse nor
sedate worms, and so it was deemed an appropriate cue to use for
context. In this experiment, worms were given habituation training
Method
(30 trains of taps) in one of two environments (NaCH3COO or Subjects. A total of 160 adult 4-day-old hermaphroditic C. elegans
plain NGM plates) and were tested (30 trains of taps) 1 hr later in (Bristol; N2) were used in this experiment.
 CONTEXT CONDITIONING IN C. ELEGANS
Procedure. There were four experimental groups for each of two ISIs:
10-s and 60-s ISIs; each group comprised 20 worms. Each group re-
ceived 30 stimuli at the designated ISI in the training block and 30 stimuli
at the designated ISI in the testing block. Worms were preplated individ-
ually for 1 hr on plain plates. Group NaPl was trained in the presence of
NaCH3COO and then tested on a plain agar plate; Group NaNa was trained
in NaCH3COO and then tested in that same context; Group PINa was
trained in a plain context and then tested in the presence of NaCH3COO;
and Group P1P1 was trained in a plain context and tested in the same
context. During the 1-hr interval between training and testing blocks,
worms were placed on plain NGM plates with a small amount of E. coli.
To be included in the study, a worm had to reverse at least half of its body
length in response to the first stimulus of training. Of the 160 worms
tested, 10 were not used in the analysis: 6 (1 each from NaNa and P1P1, 2
from NaPl, and 2 from PINa) were discarded for technical reasons (see
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General Method) and 4 because both responses in the test block were
accelerations and so were missing data points (2 worms from PINa, 1 worm
from NaPl, and 1 from NaNa).
Results and Discussion
The results demonstrate that C. elegans shows an effect of
context specificity in habituation when trained and tested in the
presence of NaCH3COO, at both the 10-s and the 60-s ISI. For
both ISIs, two-tailed paired t tests on the mean of the first two
responses in each block were used to test for differences in
retention of habituation from training to testing. Because there
were multiple independent comparisons (eight t tests) made, the
alpha level for significance was reduced using a Bonferroni cor-
rection to p ^ .006.
The habituation curves for training and for testing at the 10-s ISI
can be seen in Figure 2. At the 10-s ISI, there was significant
retention of habituation between training blocks for only Group
NaNa, ;(18) = 3.67, p = .002 (see Figure 3A). Groups NaPl, PINa,
and NaNa did not show a significant difference between the mean
of the first two responses for training and testing, NaPl;
r(18) = 1-26, ns; PINa; ((15) = 1.21, ns; P1P1: ((IS) = 1.65, ns.
Similar results are evident at the 60-s ISI and can be seen in
Figure 4. Two-tailed paired (tests comparing the average response
to the first two trains of taps for Block 1 to the average response
to the first two trains of taps for Block 2 showed that there was
significant retention of habituation between training blocks for
only Group NaNa, 1(18) = 4.35, p = .0004 (see Figure 3A).
Groups NaPl, PINa, and P1P1 did not show a significant difference
between the mean of the first two responses for training and
testing, NaPl: «17) = 1.10, ns; PINa: f(19) = 1.28, ns; P1P1:
((19) = 2.44, ns (see Figure 3B). The original hypothesis was that
both Groups NaNa and P1P1, which were trained and tested in the
same environments, would show context; however, only the NaNa
group did. The lack of context conditioning in the PIP1 group is
further investigated in Experiment 5.
Experiment 3: Context and Extinction
In Experiment 2, NaCH3COO was used as an effective context
cue for the retention of earlier habituation training. If context
conditioning is an associative phenomena, then exposure to the
environmental cue (the conditioned stimulus [CS]) in the absence
of the taps (US) should produce extinction of the context effect. In
this experiment, extinction was investigated by training and testing
worms on NaCH3COO plates; however, they spent the hour be-
499
tween training and testing on either a blank plate with a small
amount of E. coli (the context condition) or a NaCH3COO plate
with a small amount of E. coli (the extinction condition). It was
hypothesized that exposure to the CS cue in the absence of taps
would lead to a decrease in the amount of retention from training
to testing, even though both occurred in the presence of the CS
cue.
Method
Subjects. A total of 40 adult 4-day-old hermaphroditic C. elegans
(Bristol; N2) were used in this experiment.
Procedure. There were two experimental groups, each with 20 worms:
a context group and an extinction group. The method was generally similar
to that of Experiment I, with a few variations. All worms were preplated
for 1 hr on plain agar plates before being administered 30-train stimuli at
a 10-s ISI on a NaCH3COO plate during the training block. During the 1-hr
rest stage, they were moved to a NaCH3COO (extinction) or a plain agar
(context group) plate streaked with a small amount of E. coli. Testing
consisted of 10 trains of taps at a 10-s ISI on a NaCHjCOO plate. Thus, the
context group was habituated in the presence of NaCH3COO, transferred
for 1 hr to a plain agar plate for the resting stage, and then tested for
retention of habituation training on a NaCH3COO plate; the extinction
group was habituated in the presence of NaCH3COO, transferred to a
NaCH3COO plate for the 1-hr rest interval, and then transferred to a new
NaCHjCOO plate and tested in that same context.
Of the 40 worms tested, data from 5 worms were not used: 2 (1 from
each group) were discarded for technical reasons (see General Method)
and 3 because both responses in the test block were accelerations and so
were missing data points (2 worms from the context group, 1 worm from
the extinction group). Because there were two t tests run, the alpha level for
significance was reduced to p ^ .025.
Results
The results (see Figure 5) revealed that when the worms were
exposed to the CS (the context) in the absence of the US (the taps),
extinction to the CS occurred and no context conditioning was
evident. Two-tailed paired ( tests showed that for the context
group, the responses during testing were significantly smaller than
the responses during training, ((16) = 7.14, p < .0001, whereas
there were no significant differences between training and testing
for the extinction group, ((17) = 1.06, ns. These results then
showed that the context cue could undergo extinction when pre-
sented, after the initial pairing, for 1 hr in the absence of taps.
Experiment 4: Context and Latent Inhibition
In Experiments 2 and 3, NaCH3COO was used as an effective
context cue for the retention of earlier habituation training, and
exposure to it between training and testing produced extinction of
context conditioning. If context conditioning is associative in
nature, then exposure to the environmental cue (the CS) for an
extended period of time before the taps (US) should reduce or
eliminate the context effect through a process termed "latent
inhibition." In this experiment, an attempt was made to produce
latent inhibition by having worms placed on NaCH3COO plates
(latent inhibition condition) or on plain NGM plates (context
 500 RANKIN

A B
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This document is copyrighted by the American Psychological Association or one of its allied publishers.

10 15 20 25 10 15 20 25 30

Stimulus Stimulus

600 -i 600 -i

500 -

10 15 20 10 15 20 25
Stimulus Stimulus

Figure 2. 10-s interstimulus interval (ISI): Mean response magnitude (in pixels) for training and testing at a
10-s ISI for four groups of worms. Group NaPl (A) was trained on NaCHjCOO plates and tested on plain agar
plates; Group NaNa (B) was trained on NaCH3COO plates and tested on NaCH3COO plates; Group PINa (C)
was trained on plain agar plates and tested on NaCH3COO plates; and Group P1P1 (D) was trained on plain agar
plates and tested on plain agar plates. The test block for Group NaNa shows significant greater retention of the
earlier training block, whereas the other three groups do not.

condition) before training and testing on the same plates. All to the CS cue before the pairing of the CS with taps (US) would
worms spent the hour between training and testing on a blank plate lead to a decrease in amount of retention from training to testing,
with a small amount of E. coli. It was hypothesized that exposure even though both occurred in the presence of the CS cue.
 CONTEXT CONDITIONING IN C. ELEGANS
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NaPI NaNa PINa PIPI
Figure 3. Mean response magnitude for the first two responses of training
and testing for groups trained and tested (A) at 10-s interstimulus intervals
(ISIs) and (B) at 60-s ISls. There were four groups of worms in each
experiment. Group NaPI was trained on NaCH3COO and tested on plain
agar; Group NaNa was trained on NaCH3COO and tested on NaCH3COO;
Group PINa was trained on plain agar and tested on NaCH3COO; and
Group P1P1 was trained and tested on plain agar. At both ISIs, there was
significant retention (*/? < .006) of the training for Group NaNa when
worms were both trained and tested in the NaCH3COO context, whereas
there was not significant retention from training to testing for Groups NaPI,
PINa, and P1P1 at either ISI.
Method
Subjects. A total of 40 adult 4-day-old hermaphroditic C. eiegans
(Bristol; N2) was used in this experiment.
Procedure. There were two experimental groups of 20 worms each.
The context group was preplated for 1 hr on plain agar, habituated in the
presence of NaCH3COO, transferred for 1 hr to a plain agar plate for the
resting stage, and then tested for retention of habituation training on a
NaCH3COO plate. The latent inhibition group was preplated for 1 hr on a
NaCH3COO plate, habituated in the presence of NaCH3COO, transferred
to a plain agar plate for the 1-hr rest interval, and then transferred to a new
NaCH3COO plate and tested in that same context.
Of the 40 worms tested, data from 7 worms were not used: 2 (1 from
each group) were discarded for technical reasons (see General Method)
501
and 5 because both responses in the test block were accelerations and so
were missing data points (2 worms from the context group, 3 worms from
the latent inhibition group). Because there were two / tests run, the alpha
level for significance was p ^ .025.
Results
The results (see Figure 6) show that exposure to the context CS
without the US before training interfered with the subsequent
context conditioning; the worms demonstrated latent inhibition.
Two-tailed paired t tests showed that for the context group, the
responses during testing were significantly smaller than the re-
sponses during training, /(15) = 3.68, p — .0022, whereas there
were no significant differences between training and testing for the
latent inhibition group, ((14) = 1.30, ns. These results showed that
prior exposure to the context cue in the absence of taps interfered
with the development of the context-tap association. This is latent
inhibition.
Experiment 5: Plain Agar as Context
Experiments 3 and 4 show that when the worm is exposed to the
context for a period of time before training or between training and
testing, this exposure interferes with the formation of an associa-
tion between context and taps. A reevaluation of the groups in
Experiment 2 shows that, although it was originally hypothesized
that context might occur in groups trained and tested in the same
context (NaNa: trained and tested on NaCH3COO; P1P1: trained
and tested on plain agar), the protocol led to the NaCH3COO being
a distinct context that occurred only in conjunction with taps,
whereas in the P1P1 condition, the worms rested on plain plates
both before training (latent inhibition) and between training and
testing (extinction). The plain agar was hypothesized to be a novel
stimulus for the worms because the plates on which they are raised
were always treated with E. coli and so had the chemicals associ-
ated with E. coli growth present at all times. Experiment 5 was
designed to test whether the plain agar condition could serve as a
context if it was experienced only in conjunction with taps.
Method
Subjects. A total of 60 adult 4-day-old hermaphroditic C. elegans
(Bristol; N2) was used in this experiment.
Procedure. There were three experimental groups of 20 worms each.
The context group was preplated for 1 hr on plain agar, habituated in the
presence of NaCH3COO, transferred for 1 hr to a plain agar plate for the
resting stage, and then tested for retention of habituation training on a
NaCH3COO plate; the reverse context group was preplated for 1 hr on a
NaCH,COO plate, was habituated on a plain agar plate, transferred to a
NaCH3COO plate for the 1-hr rest interval, and then transferred to a new
plain agar plate and tested in that same context; the P1P1 group, as in
Experiment 2, was preplated for 1 hr on a plain agar plate, was habituated
on a plain agar plate, transferred to a plain agar plate for the 1-hr rest
interval, and then transferred to a new plain agar plate for testing.
Of the 60 worms tested, data from 4 worms was not used: 2(1 from each
group) were discarded for technical reasons and 2 because both responses
in the test block were accelerations and so were missing data points (1
worm from the context group, 1 worm from the PIPI group).
Two-tailed paired t tests on the mean of the first two responses in each
block were used to test for differences in retention of habituation training
from training to testing. Because there were three / tests run, the alpha level
for significance wasp ^ .017.
 502 RANKIN

A B

500-

i
This article is intended solely for the personal use of the individual user and is not to be disseminated broadly.
This document is copyrighted by the American Psychological Association or one of its allied publishers.

100-

10 15 20 25 30 10 15 20 25 30

Stimulus Stimulus

500 500

400
" 400-

300 - 300 -

200- 200

100 - 100

10 15 20 25 30 10 15 20 25 30

Stimulus Stimulus

Figure 4. 60-s interstimulus interval (ISI): Mean response magnitude (in pixels) for training and testing blocks
at a 60-s ISI for four groups of worms. Group NaPl (A) was trained on NaCH3COO plates and tested on plain
agar plates; Group NaNa (B) was trained on NaCH3COO plates and tested on NaCH3COO plates; Group PINa
(C) was trained on plain agar plates and then tested on NaCH3COO plates; and Group P1P1 (D) was trained on
plain agar plates and tested on plain agar plates. The test block for Group NaNa shows significantly greater
retention of the earlier training block, whereas the other three groups do not.
 CONTEXT CONDITIONING IN C. ELEGANS
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This document is copyrighted by the American Psychological Association or one of its allied publishers.
Context Extinction
Figure 5. Extinction: Mean response amplitude of the first two responses
of training and testing blocks for groups of worms that were both trained
and tested on NaCH3COO plates. The context group was placed on plain
agar plates for the 1-hr interval between training and testing. The extinction
group was placed on NaCH3COO plates for the 1-hr interval between
training and testing. There was significantly more retention of training
when the worms were not exposed to NaCH3COO between training and
testing.
Results
The results (see Figure 7) show that the plain agar condition
could serve as a context if it was experienced only in conjunction
with taps. Two-tailed paired t tests showed that for the context
group, the responses during testing were significantly smaller than
the responses during training, /(18) = 3.86, p = .0012, and for the
reverse context group, the responses during testing were signifi-
cantly smaller than the responses during training, ?(17) = 4.287,
p — .0005. In contrast there were no significant differences he-
Context Latent Inhibition
Figure 6. Latent inhibition: Mean response amplitude of the first two
responses for training and testing for groups of worms that were both
trained and tested on NaCH3COO plates. The context group was placed on
plain agar plates for 1 hr before training. The latent inhibition group was
placed on NaCH3COO plates for 1 hr before training. Both groups were
placed on plain agar plates for the 1 hr between training and testing. There
was significantly less retention of training when the worms were exposed
to NaCH3COO before training.
503
Context Reverse PI PI
Figure 7. Plain agar as context: Mean response amplitude of the first two
responses for training and testing. The context group was placed on plain
agar plates for the 1 hr before training and for the 1 hr in between training
and testing. The reverse context group was placed on NaCH3COO plates
for 1 hr before training and for the 1 hr in between training and testing. The
P1P1 group was preplated for 1 hr on a plain agar plate, was habituated on
a plain agar plate, was transferred to a plain agar plate for the 1-hr rest
interval, and then was transferred to a new plain agar plate for testing. The
context and the reverse context group both showed significant retention of
training, whereas the P1P1 group did not.
tween training and testing in the P1P1 group, f(17) = 2.044, ns.
These results showed that the lack of context conditioning ob-
served in Group P1P1 in Experiment 2 (worms trained and tested on
plain agar) was the result of design confounds. The original P1P1
condition (exposure to plain agar plates before training and during
the training-testing interval) was inappropriate to show context. If
the relationship between taps and plain agar is kept distinct from
exposure to either alone, then the plain agar can serve as a context
for habituation to tap.
General Discussion
Taken together, the results of these experiments show that there
is an associative component to habituation: With only 302 neurons,
the worms were able to use the information provided by the
context and taps during the testing phase in such a way that the
context acquired conditioning and affected responding in the test
phase. Obviously, the data do not suggest that a lack of contextual
cues obstructs memory. Indeed, all groups demonstrated a trend
toward retention of habituation training in the test block (seen as
more rapid rates of habituation in the test blocks at both the 10-
and 60-s ISI), but, with habituation at both ISIs, Group NaNa,
trained and tested in the same context, was able to demonstrate
significantly better retention of the habituation training after 1 hr.
The results of Experiment 5 show that associative habituation can
be made specific to at least two different contexts, NaCH3COO or
plain agar. The critical variable in determining whether context
conditioning occurred was whether the context occurred only in
conjunction with taps. These data support the notion that contex-
tual effects can influence retention of habituation, as postulated by
Wagner's (1976, 1978, 1979) comparator theory. The effects of
context on habituation were just as strong at the 10-s ISI as at the
 504 RANKIN
60-s ISI. This is an interesting finding because habituation training
at a 60-s ISI has been shown to lead to long-term habituation
(24-hr retention), whereas training at a 10-s ISI has been shown not
to produce long-term habituation in C. elegans (Beck & Rankin,
1997). This suggests that the mechanisms involved in context
conditioning and the mechanisms of long-term memory are not
isomorphic and that long-term memory requires cellular processes
not recruited by short ISIs, whereas cellular mechanisms of context
conditioning are activated by both short and long ISIs.
Extinction, a decrease in responding after presentations of a CS
without the associated US, plays an important role in associative
learning theory. If the argument is made for an associative com-
ponent to habituation, it is logical to assume that contextual effects
on habituation can be extinguished through exposure to a partic-
This article is intended solely for the personal use of the individual user and is not to be disseminated broadly.
This document is copyrighted by the American Psychological Association or one of its allied publishers.
ular context without presentation of the habituating stimulus. In-
deed, that was the case in the present study; exposure to the context
for the 1-hr rest period between training and testing eliminated the
effects of context conditioning. The finding of latent inhibition
after exposure to the context alone, before training, strengthens the
conclusion that C. elegans is capable of context-dependent learn-
ing and that this learning follows the same rules as associative
learning in higher vertebrates. Taken together, these findings of
extinction and latent inhibition support the notion that context
conditioning in habituation in C. elegans mediates its effect by
means of a context-US association.
There are several other possible nonassociative explanations for
the contextual effects of habituation that should be considered.
One possibility is that some contextual cues may enhance or
diminish attention to concurrent sensory stimuli, such as taps, thus
changing the effective intensity of the stimuli. Worms do show a
graded response to taps of different intensities (Chiba & Rankin,
1990) and so would respond with smaller reversals if the tap was
perceived as less intense in the presence of NaCH3COO. The
results of Experiment 1 showed that there was no difference in the
response magnitude of reversals to tap on plain agar and on
NaCH3COO. This suggests that the altered-intensity explanation is
not likely. Another possibility is that prolonged exposure to the cue
in the latent inhibition or extinction protocols might result in
sensory adaptation so that the effects of attention to concurrent
stimuli are decreased. Sensory adaptation should lead to worms
that no longer detect the NaCH3COO on the plate. Thus, if worms
exposed to NaCH3COO plates for 1 hr experienced sensory adap-
tation, they would find those plates to be indistinguishable from
plain plates. If that were the case, then Experiment 5 should not
have worked. In that experiment, worms spent 1 hr on NaCH3COO
plates before training and between training and testing. If they
could not detect the NaCH3COO any longer, then they should not
have shown the reverse context effect seen in this experiment. It
appears as though a sensory adaptation argument does not explain
the results obtained from these experiments. Thus far, the best
explanation for the effects observed across Experiments 2-5 is
associative context conditioning.
The observation of context conditioning in habituation in C.
elegans is exciting given that there are only 302 neurons in the
nervous system of the worm and that there are only 5 sensory
neurons and 11 interneurons in the tap withdrawal circuit. The
chemosensory neurons that detect NaCH3COO have been identi-
fied, and the neural circuit from these neurons to the interneurons
for tap withdrawal can be tracked. The most likely site of conver-
gence for the tap and chemosensory systems are the interneurons
of the tap withdrawal system. The tap withdrawal system appears
to use glutamate as its primary neurotransmitter, and it appears that
glutamate may also play a role in chemotaxis. Interestingly, the
interneurons in the tap withdrawal circuit contain a number of
types of glutamate receptors in a pattern similar to that implicated
in associative learning in mammals. These include glutamate re-
ceptors homologous to mammalian alpha-amino-3-hydroxy-5-
methylisoxazole-4-propionic acid (AMPA)-type receptors (Hart,
Sims, & Kaplan, 1995; Mariq, Peckol, Driscoll, & Bargmann,
1995) and glutamate receptors homologous to mammalian
W-methyl-D-aspartate (NMDA)-type receptors (Brockie, Madsen,
& Mariq, 1997). The NMDA-type receptors have been implicated
in the production of long-term potentiation (LTP), a physiological
process thought to be involved in Learning both in mammals
(Rogan, Staubli, & LeDoux, 1997) and in Aplysia (Lin & Glanz-
man, 1994). Recent studies in mammals have shown that genetic
manipulations that lead to an impairment in LTP also impair a
context-dependent type of learning (Jiang et al., 1998). It will be
interesting to see whether mutations in the NMDA-like receptors
in C. elegans will eliminate the context effect while leaving
habituation intact.
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Received July 12, 1999
Revision received December 28, 1999
Accepted December 30, 1999 •

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