Behavioral Neuroscience Copyright 1992 by the American Psychological Association, Inc.

1992, Vol. 106. No. 2,239-249 0735-7044/92/53.00

Factors Affecting Habituation and Recovery From Habituation in the

Nematode Caenorhabditis elegans
Catharine H. Rankin and Brett S. Broster
University of British Columbia
Vancouver, British Columbia, Canada

In four experiments, the factors that affect the rate of habituation, the degree of habituation, and
the rate of recovery from habituation in a simple reflex circuit in Caenorhabditis elegans were
investigated. The results showed that habituation was more pronounced and faster, and that
recovery from habituation was more rapid, with short interstimulus intervals (ISIs) than with
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longer ISIs. Rate of recovery differed in animals that had reached asymptotic response levels when
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compared with animals still in the descending portion of the habituation curve. Once animals
reached asymptotic response levels, rate of recovery appeared to be determined by ISI and not by
additional stimuli.

Habituation, the decrement in responsiveness resulting
from repeated exposure to a stimulus, has been studied in
many different organisms using a wide variety of types of
stimuli and protocols (Groves & Thompson, 1970). One of the
defining characteristics of habituation is that it will dissipate in
the absence of stimulation; this has been termed spontaneous
recovery (Thompson & Spencer, 1966). Although many behav-
ioral aspects of habituation have been studied in extensive
detail, little systematic research has been done on the ways in
which factors that affect the process of habituation also
influence spontaneous recovery from habituation. By studying
how spontaneous recovery from habituation is affected by a
variety of different factors, it may be possible to gain valuable
insights into the process of habituation and perhaps into its
underlying mechanisms.
Although habituation is often described as the simplest form
of learning, the current understanding of the mechanisms
underlying habituation is incomplete. The most detailed analy-
sis of these mechanisms is from the research of Kandel and
colleagues on the defensive gill- and siphon-withdrawal reflex
in the marine slug Aplysia califomica (for review see Hawkins,
1988). From this research, it has been hypothesized that
habituation is the result of two processes: a decreased Ca ++
current in the sensory neuron leading to a decrease in
neurotransmitter release (Klein, Shapiro, & Kandel, 1980) and
a depletion of neurotransmitter at the sensory axon terminal
(Bailey & Chen, 1988; Gingrich & Byrne, 1985). However, the
The results of these experiments were presented in part at the
Meeting for the Society for Neuroscience in St. Louis, Missouri,
November 1990 (Rankin & Broster, 1990).
This research was supported by grants from the National Science
and Engineering Research Council of Canada.
We gratefully acknowledge the editorial assistance of Catherine
Chiba, Christine Beck, Stephen Wicks, and Shannon Cerniuk.
Correspondence concerning this article should be addressed to
Catharine Rankin, Department of Psychology, University of British
Columbia, 2136 West Mall, Vancouver, British Columbia, Canada
V6T 1Z4.
mechanism for the reduced Ca ++ current is unknown, as-is the
nature of the transmitter depletion. In addition, it is unclear
whether these two mechanisms are independent or related. At
this point, our understanding of the molecular mechanisms of
habituation is far from complete.
The nematode Caenorhabditis elegans is an attractive subject
for research on learning and underlying mechanisms of learn-
ing because the worm's genetics, neuroanatomical develop-
ment, and general physiology are well understood. Such
extensive knowledge provides a foundation for an investigation
of the connection between neural events and observed behav-
ior. This tiny (1-mm) soil-dwelling worm has a simple nervous
system consisting of only 302 neurons. By studying such a
simple nervous system, it should be possible to determine the
role of every cell in learning. However, before moving to a
cellular analysis, a thorough understanding of a phenomenon
at a behavioral level is necessary.
The tap withdrawal reflex (swimming backward in response
to a mechanical tap to the side of a petri dish in which the
subject is held) of C. elegans shows forms of nonassociative
learning such as habituation, dishabituation, and sensitization,
as well as both long- and short-term memory (Rankin, Beck, &
Chiba, 1990; Rankin & Chiba, 1988). In this study, we used the
tap withdrawal reflex to focus on a number of factors that
might affect habituation and spontaneous recovery from habit-
uation. These experiments were specifically designed to study
the effects of frequency of stimulation and number of stimuli
on habituation and the effect of both these factors, as well as
the degree of response decrement, on the process of recovery
from habituation.
Genera] Method
Subjects
A total of 180 hermaphroditic adult Caenorhabditis elegans Bristol
(strain N2) were used. All subjects were maintained at 20 °C in 5-cm
petri plates that were filled with 10 ml nematode growth medium
(NGM) agar and streaked with Escherichia coll (strain OP50; Brenner,
1974).

239
 240 CATHARINE H. RANKIN AND BRETT S. BROSTER
Apparatus
Worms were observed individually on NGM agar using a stereomi-
croscope (Wild Leitz, Canada, Ltd., model M3Z) and attached video
tape recording equipment (Panasonic camera D5000, Panasonic 1950
VCR, NEC color monitor). A time-date generator (Panasonic 814)
superimposed a stopwatch onto the image for timing stimulus delivery
and length of trials. For testing, 5-cm petri plates that contained a
single worm on NGM agar were held by a holder made from a petri
plate lid mounted onto a plastic rod. The rod was attached to a
Marzhauser micromanipulator (model MM33) so that the plate could
be moved smoothly while keeping the worm within the camera field. A
mechanical tapper was also mounted on the holder. This tapper
consisted of an electromagnetic relay and wire arm with the relay
connected to a Grass stimulator (model S88) to regulate stimulus
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delivery. The arm of the tapper was positioned halfway up the side wall
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of the dish to deliver the the vibratory stimulus that, when transmitted
through the dish and agar, stimulated the worm. For single taps, the
stimulator was set to deliver a 25-ms pulse to the relay. For trains of
taps, the stimulator was set to deliver six 25-ms pulses at a rate of 8.5
pulses per second (600 ms total).
General Procedure
In each of the four experiments, individual worms were observed in
5-cm petri plates that were filled with 10 ml NGM agar. A worm was
transferred to the plate 2 min before testing. For the experiments that
involved habituation, trains of taps were delivered at a designated
interstimulus interval (ISI). For the experiments that involved recov-
ery from habituation, a train of taps was delivered 30 s, 10 min, 20 min,
and 30 min after the last habituation stimulus. In these experiments,
we have expressed response magnitude as a percentage of the initial
habituation response. Because of this standardization, an initial
response criterion was implemented to prevent an initial response that
was a nonreversal (scored as 0) or a very small reversal from biasing
the response values. Therefore, to qualify as a subject, each worm had
to respond to the first stimulus in the habituation series with a reversal
of half a body length or more and at least one of the next two responses
had to be a reversal. Approximately 75% of worms tested met this
criterion.
Specific procedural details for each experiment are described in
their respective Results section.
Scoring
An animal was deemed to show a reversal if the video tape recording
showed that it moved backward within 1 s of the vibrational stimulus.
The magnitude of these reversal responses was scored by reviewing
videotapes using stop-frame video to trace the path of each reversal
onto an acetate sheet. Greater than 75% of the data was scored by an
individual who was unaware of the subjects' treatment. The tracings
were then digitized using a digitizing tablet (Summagraphics Bit Pad
Plus) that was interfaced with a Macintosh SE microcomputer and
MacMeasure software.
Experiment 1: Role of ISI in Habituation and Recovery
From Habituation
In a variety of studies with many other organisms, short ISIs
have been found to produce more rapid habituation and a
greater amount of response decrement than do long ISIs (e.g.,
Askew, 1970; Groves & Thompson, 1970). However, the role
of ISI in the rate of spontaneous recovery has not been studied
extensively. A number of studies have used a single posthabitu-
ation test to show greater retention of habituation after long
ISIs when compared with retention after short ISIs (e.g.,
Davis, 1970). In a computer simulation of habituation (based
on findings in Aplysia), Gingrich and Byrne (1985) suggested
that long ISIs might result in recovery that is slower than that
produced by short ISIs. The hypothesis that the rate of delivery
of stimuli also influences behavior after the end of habituation
training was tested in this 1st experiment in which habituation
and spontaneous recovery after habituation were examined for
four different ISIs (2, 10, 30, and 60 s).
Method
Habituation. There were four experimental groups; each consisted
of 20 worms. All worms were given a series of 60 consecutive trains of
taps at one of the four specified ISIs: 2, 10, 30, and 60 s. To assess the
rate of response decrement, the responses to the first 30 stimuli were
scored. To assess the asymptotic levels of habituation, the 30th and the
60th responses were scored.
Recovery from habituation. Immediately after habituation (after 60
stimuli), trains of taps were delivered at 30 s, 10 min, 20 min, and 30
min to test for recovery. To assess recovery from habituation, the first
response and the mean of the last three responses of the habituation
series were compared with each of the four recovery responses.
Results and Discussion
Habituation. The rate of decrement was analyzed for the
first 30 stimuli for the 10-, 30-, and 60-s ISI groups. (The 2-s ISI
was too short for responses to be completed before the next
stimulus, therefore only the initial response and the last three
responses of the habituation series were scored.) The rate of
habituation appeared to be slower in the 30- and 60-s ISI
groups than in the 30-s ISI group (Figure 1). An analysis of the
slopes of the regression lines for the responses to the first 5
stimuli confirmed this and showed that the mean slope for
habituation for the 10-s group was significantly greater than
the mean slopes for the 30- and 60-s groups, F(2, 59) = 4.16,
p = .02.
The habituation curves all showed the classic habituation
pattern of an initial, sharply descending slope followed by a
more gradual, almost flat, slope that is known as the asymp-
tote. In all groups except the 30-s ISI group, the response level
did not decrease between the 30th and 60th stimuli (Figure 2).
For the 2-, 10-, and 60-s ISI groups, the point at which a stable
minimum level of response, which we call the "asymptotic
response level" or the "final response level," was achieved
either at or before the 30th stimulus. For the rest of the
experiments, the asymptotic response level for the 10- and 60-s
ISI groups is defined as that seen in this experiment at the 30th
and 60th stimuli (see Figure 2).
These asymptotic response levels were dependent on ISI;
there was a significant difference between groups, with the 2-,
10-, and 30-s ISI groups having significantly smaller responses
to the 60th stimulus than had the 60-s ISI group, F(3, 78) =
5.09, p = .0029. The 30-s ISI group showed a significant drop in
response magnitude between 30 and 60 stimuli, which suggests
that for some reason a 30-s ISI takes longer to produce
asymptotic response levels, F(l, 75) = 5.33,p = .02.
 HABITUATION RECOVERY IN C. ELEGANS 241

This division of the habituation curve into two components

suggests that habituation might not be the result of a single
process but rather two separate processes: one associated
more with the descending portion of the curve and one
associated more with the asymptotic portion of the curve.
Recovery from habituation. In addition to studying the
habituation process itself, studies of recovery from habituation
under a variety of conditions may provide clues to the
mechanisms underlying habituation. In the present experi-
ment, the ISI of the habituating stimuli was different for each
of the four groups (2-, 10-, 30-, and 60-s ISIs), whereas the tests
60S
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Figure 2. Responses to the 30th and 60th stimuli plotted as mean

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10 SECOND ISI
percentage of initial (INIT) response, for worms given 60 stimuli at 2-,
Ul 140- 30-, 30-, and 60-s interstimulus intervals (ISIs). (In all groups except
W
z for the 30-s ISI, the animals reached by the 30th stimulus an
o asymptotic level of responding that remained unchanged up to 60
a. 100- stimuli. This response level was highly ISI dependent, that is, shorter
to
ui ISIs produced more decrement than did longer ISIs.)
EC 8 0 -
60 -
for recovery were at the same intervals (30 s, 10 min, 20 min,
40- and 30 min posthabituation) regardless of the habituating ISI.
z 20-
This approach allowed the comparison of a common posthabit-
< uation frame of reference for animals habituated with different
UJ
0
ISIs.
10 20 30
STIMULI
In the analysis of the relationship between habituation and
spontaneous recovery from habituation, two aspects of recov-
ery were examined: (a) whether there was recovery above the
habituated level and (b) whether there was recovery back to
B 30 SECOND ISI the initial response level. An analysis of variance (ANOVA)
140-
followed by Fisher's least significant difference planned com-
parisons on the initial response, the mean habituated response
z 120- (the mean of the last three habituation responses), and the
o
Q. four recovery tests was done for each of the ISIs. The results
100-
W
UJ
8O-
indicated that in each ISI condition there was significant
IT
habituation (the initial response and the mean habituated
60- response were significantly different) and significant recovery
40- above the habituated level at some point during the 30-min test
period (Figure 3).
20-
< For the 2-s ISI group, there was no significant recovery at 30
Ul 0 s; but at 10 min (as well as at 20 and 30 min), recovery was not
10 20 30
only significantly above the habituated level, but it also did not
STIMULI
differ significantly from initial response levels, F(19, 120) =
9.00,/? = .0001 (Figure 3A).
For the 10-s ISI group, there was significant recovery above
C 60 SECOND ISI habituated levels at 10, 20, and 30 min posthabituation, but
UI 140- response levels remained significantly below initial response
levels, F(19, 100) = 20.23,p = .0001 (Figure 3B).
o 120 '
ui 100-
Ul
EC 80- Figure 1 (left). A: Habituation of response amplitude expressed
as mean percentage of initial (INIT) response (±SE) for 30 stimuli
| 60-
delivered at 10-s interstimulus intervals (ISIs; n = 20). B: Habituation
5? 4 °: of response amplitude expressed as mean percentage of initial re-
sponse (±SE) for 30 stimuli delivered at 30-s ISIs (n = 20). C:
"~ 20-
< Habituation of response amplitude expressed as mean percentage of
ui 0 initial response (±SE) for 30 stimuli delivered at 60-s ISIs (« = 20).
10 20 30 (The shortest ISI [10 s] produces greater and more rapid decrement
STIMULI than the longest ISI [60 s].)
 242 CATHARINE H. RANKIN AND BRETT S. BROSTER

2 SECOND ISI B 10 SECOND ISI

01 140-
CO
z 1 20-
o
Q.
CO 1 00
UJ
tr 80 -

60 -

40 -

20 -
<
LJJ 0
Xhab 30s 10m 20m 30m Xhab 30s 10m 20m 30m
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This document is copyrighted by the American Psychological Association or one of its allied publishers.

30 SECOND ISI 60 SECOND ISI

UJ 140 g 1401
CO
Z
120- 120-
O O
Q.
(0 100 CO 1 00
UJ UJ
EC 80 - tr 80 -

60 -
z
40- 40 -
5?
Z 20- 20 -
UJ 0 UJ 0
Xhab 30s 10m 20m 30m s Xhab 30s 10m 20m 30m
Figure 3. Spontaneous recovery from habituation plotted as mean percentage of initial (INIT) response
(±SE) for worms tested for recovery 30 s, 10 min, 20 min, and 30 min after habituation to 60 stimuli
(n = 20 per interstimulus interval [ISI]). (A: 2-s ISI; B: 10-s ISI; C: 30-s ISI; and D: 60-s ISI. The
horizontal line represents the initial response level of 100%. Recovery was highly dependent on ISI. A
shorter ISI [i.e., 2 s] resulted in more rapid recovery, whereas a longer ISI [i.e., 60 s] resulted in slower
recovery. Xhab = mean habituated response [the mean of the last three habituation responses; Mhah in
text], m = minute.)

For the 30-s ISI group, there was significant recovery above
habituated levels at 10, 20, and 30 min posthabituation, and
response levels at 30 min were significantly larger than re-
sponse levels at 10 min posthabituation. However, response
levels during the recovery period were again significantly lower
than initial response levels, F(19, 100) = 15.92, p = .0001
(Figure 3C).
For the 60-s ISI group, there was significant recovery from
habituation at only the 20-min test. All recovery response
levels were significantly smaller than initial response levels,
F(19, 100) = 7.09, p = .0001 (Figure 3D).
The recovery rates for the different ISIs were difficult to
compare directly because they began from different levels of
habituation. Therefore, to more easily compare the recovery
rates for the different ISIs, the recovery curves were standard-
ized by subtracting each animal's mean percent habituated
level from each of its percent recovery scores to produce
standardized percent recovery scores (Figure 4). These data
were then used to calculate the slope of the regression line for
the recovery of each animal at each of the ISIs. An ANOVA,
with planned comparisons, was then used to determine whether
the slopes for recovery after different ISIs were significantly
different. The analysis showed that the slopes of the recovery
for the 2- and 10-s ISIs were significantly steeper than the slope
for recovery after the 60-s ISI, F(3, 76) = 4.47, p = .006.
This standardized percent recovery data can also be used to
compare the amount of recovery at each of the test times
across the four ISIs. ANOVAs and planned comparisons on
the data for the percentage of recovery showed there were
significant differences in the recovery level at 10 and 30 min. At
10 min, the worms that had been habituated with the 2-s ISI
showed significantly greater recovery than did worms that had
been habituated at the 30- and 60-s ISIs, and worms that had
been habituated at the 10-s ISI showed significantly more
recovery than did worms that had been habituated at the 60-s
ISI, F(3, 79) = 3.84, p = .0129. At 30 min posthabituation,
worms that had been habituated at the 2-s ISI showed
significantly greater recovery than any of the other worms, and
 HABITUATION RECOVERY IN C. ELEGANS 243

worms that had been habituated at either the 10-s ISI or the
30-s ISI showed significantly greater recovery than worms that
were habituated at the 60-s ISI, F(3, 79) = 6.74, p = .0004.
The results indicated that the 2-s ISI group recovered to
initial response levels within 10 min; however, the longer ISI
groups had not recovered to initial response levels within the
30-min test period. The 60-s ISI group was the slowest to begin
to recover and showed significant recovery at only 20 min
posthabituation. Thus, the data suggest that, with the same
number of stimuli, shorter ISIs produce more rapid and
complete recovery than do longer ISIs.
To ensure that the test stimuli at 10, 20, and 30 min
posthabituation were not themselves producing habituation or STIM 1 STIM 2 STIM 3
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slowing recovery from habituation, two control groups were

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run. The first control group was designed to test whether

stimuli delivered 10 min apart might have a habituating effect B
on their own. For this group, 10 subjects were given three
trains of taps at a 10-min ISI. There were no significant
differences between the response levels to any of the three
stimuli produced by the 10-min ISI, F(2, 29) = 1.24, p = .31
(Figure 5A). Thus, the 10-min ISI used in the recovery tests
was, by itself, insufficient to produce significant habituation.
However, in a study that used footshock with a spinal frog
preparation, Farel and Krasne (1972) found that although
stimuli delivered once every 0.5 hr resulted in no habituation,
the same treatment produced a slowing of recovery if subjects
had been previously habituated to the same stimulus using an
ISI of 30 s. Therefore, the second control group was designed 10 MIN ISI 30 MIN ONLY
to test whether the three 10-min recovery tests might be
Figure 5. Control tests showed that the 10-min recovery tests were
slowing recovery in a similar manner. Each of 10 subjects
not confounding recovery results. (A: Animals [n = 10] that received 3
received 60 trains of taps at a 10-s ISI, followed by a single stimuli [STIM] 10 min apart showed no significant habituation. B: A
recovery test at 30 min after the last habituating stimulus. control group [n = 20] that received only one recovery test [at 30 min]
There was no significant difference between the 30-min test in after 60 stimuli at a 10-s interstimulus interval [ISI] did not show a
this group and the 30-min test for the 10-s ISI subjects that significantly different 30-min response level from the group that
received all four recovery tests, t(26) = — .28,/> = .39 (Figure received all four recovery tests [30 s, 10 min, 20 min, and 30 min] after
5B). Thus, the recovery testing procedure used in this 1st 60 stimuli at a 10-s ISI.)

experiment appears not to have had any obvious effects on the

rate of recovery for at least the 10-s ISI groups.
From the results of the tests that used different ISIs to
produce habituation, it seems reasonable to conclude that
shorter ISIs produce more rapid recovery from habituation
than do longer ISIs. One of the more interesting aspects of this
conclusion is that the 60-s ISI group recovered much more
slowly despite the fact that it showed less decrement during
habituation than did the other ISI groups.

Experiment 2: Role of Level of Habituation

Xhab 30s 10m 20m 30m
Figure 4. Mean percentage of recovery for each animal (% initial
response for each recovery point - Xhab [the mean habituation
response; A/tob in text]) for animals that received 60 stimuli at 2-, 10-,
30-, and 60-s interstimulus intervals (ISIs; n = 20 per ISI). (When all
groups are equated for habituation level it can be seen that short ISIs
produce rapid and more complete recovery and that long ISIs produce
slower and less complete recovery, m = minute.)
Because the amount of response decrement after 60 stimuli
at the 60-s ISI was less than that with the shorter ISIs, the
differences in rate of recovery between short and long ISIs
might be due not only to the ISI of the stimulation but also to
the different response levels at the end of habituation. To test
this hypothesis, the level of habituation in two different ISI
conditions (10 and 60 s) was equated by setting a criterion level
of habituation that must be reached by all worms before the
onset of recovery tests.
 244 CATHARINE H. RANKIN AND BRETT S. BROSTER

Method
The experiment consisted of a 10-s ISI group (n = 20) and a 60-s ISI
group (n = 20). Each subject was given a series of trains of taps at the
specified ISI until the worm met the habituation criterion of four
reversal responses in a row that were equal to or less than the length of
the worm's head (from the anterior tip of the worm to the end of the
pharyngeal bulb). Once this criterion had been reached, each animal
was permitted to recover, and the test stimuli were given at 30 s, 10
min, 20 min, and 30 min after the final habituation stimulus.

Results and Discussion

With the 10-s ISI, it took worms (M ± SE) 11.75 ± 1.1
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stimuli to reach criterion, and with the 60-s ISI, it took worms
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19 ± 2.1 stimuli to reach criterion. An analysis of the recovery

curve for the 10-s ISI criterion group showed that there was
Xhab 30s 10m 20m 30m
significant habituation and significant recovery above the
habituated level at all recovery tests, as well as significant
B
recovery back to baseline levels of responding after 30 min of
recovery, F(5, 119) = 14.29, p = .0001. An analysis of the
recovery curve for the 60-s ISI criterion group showed that
there was significant habituation and significant recovery after
10 min. There was also a significant increase at 30 min of
recovery over 10 and 20 min of recovery, but recovery did not
reach baseline levels, F(5, 119) = 18.44,p = .0001.
A comparison of the recovery of these two different ISI
groups shows that after habituation to the same level, the
longer ISI again produced slower recovery (Figure 6A).
Furthermore, when we compared the 10-s ISI criterion recov-
ery curve to the recovery curve for the 10-s ISI group in the
previous experiment, we found that there was a strong resem- Xhab 30s 10m 20m 30m
blance (see Figure 6B). This is especially striking when one
considers that, on average, the criterion group received only 12
stimuli, whereas the other group received a total of 60 stimuli
before being allowed to recover. The only difference is that, UJ 120-|
with fewer stimuli, the criterion group shows significant recov- <n
ery sooner (at the 30-s test). However, this may in part reflect
the artificially low habituated level produced by the imposed QL

criteria. A similar finding is apparent when one compares the

rr
60-s ISI criterion group (M = 19 stimuli per subject) to the 60-s ,_ 60-
ISI group that received 60 stimuli (Figure 6C). Based on a z
comparison of response levels with the asymptotic response — 40 -
levels determined in the previous experiment, it appears that
the criterion groups (which received, on average, 12-19 stim- 20 -
<
uli) were brought to or below their asymptotic response levels UJ
0
(as defined by the previous experiment). This suggests that
Xhab 30s 10m 20m 30m
once asymptote is reached the number of stimuli has minimal
impact on recovery. Figure 6. A: The recovery rate, plotted as mean percentage of initial
(INIT) response, of animals habituated to a criterion (crit; refer to
text) at a 10-s interstimulus interval (ISI; n = 20) was more rapid than
Experiment 3: Role of Number of Stimuli that of animals habituated to the same level at a 60-s ISI (n = 20). (On
average, the 10-s ISI animals received 12 stimuli [stim], and the 60-s
In Experiments 1 and 2, the response levels for each of the ISI animals received 19 stimuli.) B: The recovery rate for the group of
groups appeared to have reached asymptotic levels for the ISIs worms habituated to criterion at a 10-s ISI exhibited a recovery rate
before the assessment of recovery. This 3rd experiment was similar to that of a group that had received 60 stimuli at the same ISI.
designed to test recovery from habituation before asymptote, C: The recovery rate for the 60-s ISI criterion group was similar to the
when animals would still be in the descending portion of the recovery rate for the 60-s ISI, 60-stimuli group (the difference in Xhab
habituation curve. Two groups were run, one at each of the [the mean habituation response; Mtob] levels is due to the artificially
ISIs (10 and 60 s) used in the previous experiment. low Xhab produced by training to criterion).
 HABITUATION RECOVERY IN C. ELEGANS 245

Method To demonstrate that this difference was due to the animals'

response level in relation to asymptote and not simply an
One group of 20 worms was given eight trains of taps at a 10-s ISI indication that, at the same ISI and regardless of the number
and then tested with the recovery protocol of trains of taps at 30 s, 10 of stimuli, less habituated animals recover more rapidly, we
min, 20 rain, and 30 min. A second group of 20 worms was given eight
reanalyzed data from the earlier experiment in which 20
trains of taps at a 60-s ISI and then tested with the recovery protocol of
trains of taps at 30 s, 10 min, 20 min, and 30 min. worms received 60 stimuli at the 10-s ISI before being tested
for recovery. When we divided the worms into two subgroups,
one consisting of worms that had a mean habituated response
Results and Discussion
level that was above the group mean habituated level (n = 6)
An examination of the performance of individual worms and one subgroup of worms that had mean responses that were
that were given 8 stimuli at the 10-s ISI showed that there were below the group mean habituated level (n = 14) in the same
individual differences in the rate of response decrement and
that some of the worms had reached asymptotic levels after 8
stimuli, whereas others were still responding at relatively high
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ill 200-1 --D-- ios/8stim

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rates. To examine recovery before asymptotic responding, the

worms were divided into two subgroups: one subgroup (Sub- CO 10s/8stim above
z
group 1) consisted of the 10 worms that had individual mean o 10s/8stim below
0.
habituated levels that were below the group mean habituated CO
level (Afhah = 1.13 ± 0.965; similar to the asymptote of UJ
rr
3.557 ± 1.29 determined in the habituafion experiment), and a 100-
second subgroup (Subgroup 2) consisted of the 10 worms that
had individual mean habituated levels that were above the
group mean habituated level (Mhab = 25.972 ± 4.737; above
asymptote, still in the descending portion of the curve; Figure <
UJ
7A). A t test on the mean habituated level for the two
subgroups showed that they were significantly different from Xhab 30s 10m 20m 30m
one another before recovery, f(19) = 26.40, p = .0001. An
analysis of the regression lines for recovery showed that the
slopes of Subgroup 2 were significantly steeper than the slopes B
of Subgroup 1, F(l, 19) = 4.87,p = .04. Thus, the worms that 10s/60stim
in
were still in the descending portion of the curve recovered CO 10s/8stim below
significantly more rapidly than the worms that had reached z ioo-
o
asymptotic response levels. The recovery of the group that Q.
80-
CO
received 8 stimuli that reached asymptote is compared with the UJ
recovery of worms that received 60 stimuli at the same ISI in rr 60 '
Figure 7B. The only difference between the groups was a delay
in the onset of recovery. With a greater number of stimuli (60), 40-
the onset of recovery is delayed, and the responses at 30 s are
significantly smaller than the responses at 30 s for the worms 20-
that received only 8 stimuli, F(l, 28) = 8.54,p = .0069.
0
Xhab 30s 10m 20m 30m

Figure 7 (right). Animals (n = 20) given 8 stimuli (stim) at a 10-s

interstimulus interval (ISI) and then recovery stimuli (30 s, 10 min, 20 - - o - - 10s/60stim
min, and 30 min). (A: The broken line represents the mean of 20 *— 10s/60stim above
worms' responses after 8 stimuli at a 10-s ISI. When these worms were Ill —*— 10s/60stim below
CO 1 00-
divided into subgroups of worms that had a mean habituated level Z
[Xhab; M^t in text] that was either above or below the mean o
habituated level for the group [n = 10 for each subgroup], the a. 80-
CO
subgroup above the mean exhibited a significantly higher recovery rate IU
DC 60 -
than did the subgroup below the mean. Data are expressed as mean
percentage of initial [INIT] response. B: Those animals that received 8
stimuli and that had a mean habituated level below the group average
exhibited a recovery curve highly similar to that of animals that had
received 60 stimuli at the same ISI. C: When the group [n = 20] that
had received 60 stimuli [broken line] was split into subgroups above
UJ
and below the group mean, there was no significant difference in s Xhab 30s 10m 20m 30m
recovery rates.)
 246 CATHARINE H. RANKIN AND BRETT S. BROSTER

way, we did not see the same pattern of results (Figure 1C). A t
test for the mean habituated level for the two subgroups
showed that they were significantly different from one another
before recovery, ((18) = 6.26, p = .0001. However, in this
separation there were no significant differences in the slopes of
the recovery lines for the two subgroups, t(18) = 1.26, p = .11.
Thus, once worms had achieved their own asymptotic response
levels, the actual degree of decrement did not affect rate of
recovery.
If the animals that received 8 stimuli at the 60-s ISI were
divided into the worms below the mean habituated level
(n = 11; Mhab = 18.9 ± 5.6; at or below an asymptote of
This article is intended solely for the personal use of the individual user and is not to be disseminated broadly.
30.57 ± 8.97 as determined by the earlier habituation experi-
ment) and the worms above the mean habituated level (n = 9;
M»b = 88.4 ± 10.2; above asymptote but still in the descend-
ing portion of the habituation curve), the same pattern of
results was seen (Figure 8A). The mean habituated levels for
the two subgroups were significantly different from one an-
other, t(lS) = 6.26,p = .0001. The subgroup of worms that had
reached asymptote recovered more slowly than the subgroup
that was still in the descending portion of the curve. An
analysis of the regression lines for recovery shows that the
slopes of the two subgroups were significantly different from
one another, r(15) = 2.04, p = .03. The recovery of the
subgroup that had reached asymptotic habituation is shown
with the worms that received 60 stimuli at the same ISI in

Figure 8B.
This document is copyrighted by the American Psychological Association or one of its allied publishers.

— o - - 60s/8stim
—•— 60s/8stim above If we take the group of 20 worms that received 60 stimuli at
—•— 60s/8stim below
the 60-s ISI (described in Experiment 1) and divide them into
two subgroups using the same criteria, we do not see the same
o pattern of results (Figure 8C). Although there was a significant
a.
CO 100- difference in the mean habituated levels for the two subgroups,
UJ
f(17) = 4.82, p = .0001, there was no significant difference in
the slopes of the recovery curves, f(17) = —0.81,p = .22, when
60- the subgroup that had a mean habituated level that was below
40 ' the group mean (n = 13) was compared with the subgroup that
Z had a mean habituated level that was above the group mean
< 20-
(n = 7). Therefore, as with the 10-s ISI group, number of
UJ
stimuli did not seem to play a major role in recovery once
Xhab 30s 10m 20m 30m asymptote had been reached.
A comparison of the mean percentage of recovery (recovery
scores minus Mhab) shown by the two groups of worms that
B received eight stimuli and reached asymptotic response levels
UJ 60s/60stitn (Figure 9) showed that the animals that received the shorter
100-1
CO
60s/8stim below ISI (10 s) recovered more rapidly and more completely than
Z
O the animals that received the longest ISI (60 s).
0. 80 •
CO
Thus, regardless of number of the stimuli it has received,
UJ once an animal reaches its asymptotic level of habituation for a
EC 60-
given ISI, the overall rate of recovery does not appear to
change. After reaching asymptote, the recovery 10, 20, and 30
min after habituation did not differ, regardless of whether the
3? worms received 8 stimuli or 60 stimuli (Figure 7B). This finding
Z 20 -
4 also suggests that, once asymptotic response levels have been
UJ

Xhab 30s 10m 20m 30m

Figure 8 (left). Recovery of animals (n = 20) given 8 stimuli (stim) at

a 60-s interstimulus interval (ISI) and then recovery stimuli (30 s, 10
- - o - - 60s/60stim min, 20 min, and 30 min). (A: The broken line represents the mean of
UJ 1401
CO n— 60s/60stim above 20 worms' responses after 8 stimuli at a 60-s ISI. When worms were
Z 120 *— 60s/60stim below divided into groups of worms that had a mean habituated level [Xhab;
O Mhah in text] that was either above or below the mean habituated level
a 100-
CO for the group [n = 11 for below; n = 9 for above], the subgroup above
Ul
cc 80-
the mean exhibited significantly more rapid recovery than the sub-
group below the mean. Data are expressed as mean percentage of
60 ' initial [INIT] response. B: Those animals that received 8 stimuli and
that were below the group mean habituated level exhibited recovery
40-
curves similar to those of animals that received 60 stimuli at the same
ISI. C: When the group [n = 20] that received 60 stimuli [broken line]
< 20 '
was split into above and below Xhab subgroups [n = 6 for above;
UJ
0 n = 14 for below], there was no significant difference in recovery
Xhab 30s 10m 20m 30m rates.)
 HABITUATION RECOVERY IN C. ELEGANS
10s/8stim below
1001 60s/8stim below
Xhab 30s 10m 20m 30m
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Figure 9. Mean percentage of recovery (recovery score — Xhab [the
This document is copyrighted by the American Psychological Association or one of its allied publishers.
mean habituation response; Mtab in text]) for animals that received 8
stimuli at a 60-s interstimulus interval ([SI) and that had mean
habituated levels that were below the group habituated level com-
pared with animals that received 8 stimuli at a 10-s ISI that had mean
habituated levels that were below the group habituated level. The
more rapid recovery for the 10-s ISI group is similar to that seen in the
original 60-stimuli experiment [Figure 4].)
reached, the number of stimuli in a habituation series has
relatively little effect on overall rate of recovery.
Experiment 4: Role of Number of Missed Stimuli in
Recovery
A final possibility is that recovery is not as dependent on the
absolute time interval that has passed since the end of the
habituation stimulation as it is on the number of stimuli that
have been missed since the last habituation stimulus. For
example, 10 min after habituation with a 10-s ISI, an animal
will have missed 60 stimuli. In contrast, an animal that was
habituated with a 60-s ISI will only have missed 10 stimuli in
that same 10-min recovery period. Perhaps it takes a specific
number of missed stimuli before an animal will recover to any
appreciable extent, in which case it would be expected that a
longer ISI would result in a recovery that takes a greater
amount of time. There are two pieces of evidence to suggest
that missed stimuli are not a major factor governing recovery.
The first is that during recovery tests 10-min apart animals
often showed no change in recovery level despite large
differences in the number of missed stimuli (i.e., there was no
difference in recovery for the 2-s ISI at the 10- and 20-min tests
for which 299 and 599 stimuli, respectively, were missed; there
was no difference in recovery for the 10-s ISI at the 10-, 20-,
and 30-min tests for which 29,59, and 119 stimuli, respectively,
were missed). The second piece of evidence comes from
comparison across ISIs; an additional group of 20 worms was
given 60 stimuli at a 10-s ISI with recovery tested at 5 min
posthabituation (when worms had missed 29 stimuli). At 5 min
posthabituation, the mean (±SE) response amplitude was
77.8 ± 20.3. These recovery data were then compared with the
60-s ISI 30-min test (M = 47.74 ± 15.15), when worms also
had missed 29 stimuli. The 10-s ISI group showed significantly
greater recovery after missing 29 stimuli than did the 60-s ISI
group, F(l, 37) = 6.75,p = .01. These results suggest that the
247
number of missed stimuli does not greatly affect recovery; ISI
appears to be a more important factor.
General Discussion
In these four experiments, we examined several factors that
affect the processes of habituation and spontaneous recovery
from habituation in the nematode C. elegans. Mechanical
stimuli were delivered at four different ISIs (2,10, 30, and 60 s)
to examine the role played by ISI in the development and
maintenance of habituation. However, this alone might not
give a complete picture of the effects of ISI on habituation
because, as Davis and Wagner (1968) and Davis (1970)
pointed out, comparing the habituation curves themselves
confounds training conditions with test conditions; comparing
treatments that are not tested under the same conditions can
be misleading. To avoid this, Davis (1970) suggested that
animals that have been given habituation training under
different conditions should be tested under the same condi-
tions. We have addressed this problem by using a protocol of
recovery tests that were given at the same temporal intervals
posthabituation for all experimental groups. This protocol
permitted the evaluation of the effects of different ISIs from a
common point of reference.
In the present experiments, we observed that C. elegans
showed a sensitivity to ISI during habituation; shorter ISIs
produced more rapid and greater response decrement than did
longer ISIs. When we examined spontaneous recovery from
habituation, we also found that ISI played a major role in
determining rate and amount of recovery; shorter ISIs pro-
duced greater and more rapid recovery than did longer ISIs.
These data are remarkably similar to data reported by Byrne
(1982) on the cellular analog of habituation and spontaneous
recovery, synaptic depression and recovery from synaptic
depression. In a study of the monosynaptic excitatory postsyn-
aptic potential in the Aplysia motor neuron L7 produced by
stimulating siphon mechanosensory neurons, Byrne found that
shorter ISIs (i.e., 1 s) produced more rapid and complete
synaptic decrement than did longer ISIs (i.e., 30 s). In addition,
when he examined recovery, he found that 100 s after repeated
stimulation with the same number of stimuli recovery was
greater in preparations that received the shorter ISI than in
preparations that received longer ISIs.
A possible explanation for the differences seen in the rate of
recovery is that the final levels of habituation differed for the
different ISIs. Therefore, we investigated the role of level of
habituation and number of stimuli. A group of animals was run
in which the level of habituation was controlled by setting a
specific criterion for habituation, as opposed to delivering a set
number of stimuli. Under these conditions, recovery still
showed the same characteristics; the shorter ISI produced
more rapid and greater recovery than did the longer ISIs.
Thus, the differences seen in recovery after different ISIs
cannot be accounted for by differences in habituation levels.
In this study, we also surprisingly found that within an ISI
recovery rates were very similar, regardless of the number of
stimuli worms had received. At a 10-s ISI, worms that had
received only 12 stimuli recovered at a rate similar to that for
worms that had received 60 stimuli. The same pattern was seen
 248 CATHARINE H. RANKIN AND BRETT S. BROSTER

with the 60-s ISI worms that received 19 and 60 stimuli. In both 10 SECOND ISI
cases, the effect of an increase in stimulus number was to delay 10s/60stim
the onset of recovery. For example, those subjects given only 10s/crit
12 stimuli (on average) at a 10-s ISI showed a significant >- 100
tr 10s/8stim below
increase in responsiveness within 30 s, whereas worms given 60
stimuli at the same IS! showed no recovery after 30 s. This
delay was apparently compensated for because subsequent
recovery points showed no significant differences. These find-
ings suggested that the number of stimuli was a less important
determinant of rate of recovery than was ISI. The observation
that the number of stimuli had a small impact on the rate of
recovery from habituation is a novel finding. In studies of
habituation of a monosynaptic response in isolated frog spinal T 1 1 r
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cord, Farel, Glanzman, and Thompson (1973) found that when Xhab SOS 10M 20M 30M
This document is copyrighted by the American Psychological Association or one of its allied publishers.

two groups had all major variables equated (level of habitua-

tion, ISI, and stimulus quality) the group that had received
more stimuli recovered more slowly. Because, in our experi-
ments, the greatest amount of recovery took place between the
30-s and 10-min recovery tests, further information on the
effect of number of stimuli and ISI could be obtained by
conducting a detailed analysis of recovery within this period.
One important similarity between Experiments 1 and 2 is
that, in both cases, the response levels before the recovery tests
were at or below asymptotic levels for that ISI (as determined
in Experiment 1; see Figure 2). One possibility that we
investigated was that once animals reached asymptote the
dynamics of recovery might change. Therefore, we looked at
recovery in animals that had not yet reached the asymptotic
response levels determined in our analysis of habituation at
the 10- and 60-s ISIs. It is important to note here that the rate
at which animals reached asymptote was subject to a large
amount of variability; there were individual differences in how
rapidly animals habituated to the stimuli. Thus, of 20 worms
given 8 stimuli at a 10-s ISI, half appeared to be at asymptotic
response levels, whereas the other half were still responding
above asymptotic response levels. We found that with both the
10- and 60-s ISIs recovery after 8 stimuli was more rapid and
complete if animals had not reached asymptotic response
levels, compared with those animals that had. In fact, animals
that appeared to reach asymptote after only 8 stimuli, at both
the 10- and 60-s ISIs, had recovery curves remarkably similar
to animals that received 60 stimuli at the same ISI. Thus,
although the absolute number of stimuli played a relatively
small role in determining recovery, the achievement of asymp-
totic response levels for a given ISI appeared to be an
important factor regulating recovery from habituation.
This observation, and the differences between the appear-
ance of the descending and asymptotic portions of the curve,
suggest that the habituation curve is shaped by two sets of
processes. In the descending phase, processes that have an
impact on recovery undergo their greatest change, and respon-
siveness declines in a manner that is highly influenced by the
ISI. The asymptotic phase may represent an ISI-determined
level at which response level cannot be decremented much
further.
To summarize, in these four experiments on recovery, the
most important determinant of rate of recovery was ISI. The
total number of stimuli and the absolute level of habituation
after reaching asymptote played relatively small roles in
B 60 SECOND ISI
>- 100
DC 60s/60stim
UJ
> 80 60s/crit
O 60s/8stim below
O
UJ 60-
CC
40-
z
<
20-
Xhab SOS 10M 20M 30M
Figure 10. For each of two different interstimulus intervals (ISIs; 10 s
and 60 s), recovery, expressed as mean percentage of recovery (mean
% initial response - Xhab [the mean habituation response; Aftah in
text]), for three different groups, all of which had reached an
asymptotic level of response during habituation. (A: Three groups of
worms received stimuli [stim] at a 10-s ISI; one group [n = 20] of
animals was given 60 stimuli, another group [n = 20] was habituated to
criterion [crit; average of 12 stimuli], and another group [n = 10]
received 8 stimuli and was below the mean habituated level for their
group [n = 20]. All groups habituated with a 10-s ISI had similar
overall recovery rates, except at the 30-s point at which animals that
received fewer stimuli showed greater recovery. B: Three groups of
worms received stimuli at a 60-s ISI; one group [n = 20] of animals was
given 60 stimuli, another group [n = 20] was habituated to criterion
[average of 12 stimuli], and a third group [n = 11] received 8 stimuli
and was below the mean habituated level for their group [n = 20]. All
groups habituated with a 60-s ISI had similar overall recovery rates.)
recovery. This can be seen in Figure 10 in which the recovery of
the worms that received 60 stimuli, the worms that received
sufficient stimuli to achieve a habituation criterion, and the
worms that received only 8 stimuli and had reached asymptotic
habituation levels are plotted together for the 10- and 60-s
ISIs. Despite these different numbers of stimuli, there is
remarkable similarity in the recovery curves within each ISI.
The data from our experiments on habituation and recovery
from habituation provide evidence for the hypothesis that
there may be two phases of habituation that may have different
underlying mechanisms. A strong indication of the presence of
 HABITUATION RECOVERY IN C. ELEGANS
two different processes resides in the finding that there are two
distinct patterns of recovery and that each seems to be
associated with a distinctly different part of the habituation
curve. A more rapid recovery pattern is seen if stimulation
ceases before asymptote, that is, during the descending phase.
A slower recovery pattern is evident if the animal has been
habituated to asymptotic response levels before being permit-
ted to recover.
The present study makes two different contributions to the
study of habituation. The first contribution is the determina-
tion of the major role that ISI plays in habituation. The data
suggest that after only a very few stimuli the organism has
somehow "coded" or been changed by the ISI in a way that
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lasts for a considerable period of time after stimulation. Byrne
This document is copyrighted by the American Psychological Association or one of its allied publishers.
(1982) hypothesized that at short ISIs an accumulation of
intracellular Ca t+ might lead to a facilitatory process that
opposes habituation, thus causing more rapid recovery from
habituation. The finding that long ISIs recover more slowly
despite less overall decrement creates problems for any model
suggesting that reduced responsiveness seen during acquisi-
tion is the result of a decrease in the amount of neurotransmit-
ter available for release (e.g., Bailey & Chen, 1988). If a
shortage of neurotransmitter were the only factor involved,
then a longer ISI with less decrement during acquisition
should result in faster recovery. Because this appears not to be
the case, loss of neurotransmitter alone does not regulate
recovery, at least in C. elegans. However, the hypothesis that
inactivation of the presynaptic Ca++ current contributes to
synaptic depression (as proposed by Klein et al., 1980) might
explain the differences produced by different ISIs. During
shorter ISIs, stimuli might be delivered during the Ca ++
current inactivation, whereas during longer ISIs, sufficient
time passes between stimuli for the currents to return to
baseline. Another factor that, might differentiate short and
long ISIs is a period of sensory adaptation that at short ISIs
might protect the cell terminals from the full effects of
habituation. Longer ISIs would then provide enough time
between stimuli for the adaptation to dissipate. It is clear that
more detailed behavioral and physiological experiments will be
required before the mechanisms underlying the interaction
between ISI and habituation are fully understood.
The second contribution of this study has been to show the
value of studying recovery from habituation in order to further
understand habituation. By examining recovery from habitua-
tion, we have demonstrated that the ISI of the habituating
stimuli has effects that last at least 30 min after habituation. By
comparing recovery after the delivery of a range of numbers of
stimuli, we have shown the relative unimportance of absolute
number of stimuli and the major impact reaching asymptotic
response levels has on the rate of recovery.
249
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Received July 1,1991
Revision received September 18, 1991
Accepted September 18,1991