Effects of aging on habituation in the nematode Caenorhabditis elegans

Effects of aging on habituation in the nematode Caenorhabditis elegans

Behavioural Processes, 28 (1993) 145-164 0 1993 Elsevier Science Publishers B.V. AIf rights reserved 0376-6357/93/$06.00 145 BEPROC 00450 Effects o...

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Behavioural Processes, 28 (1993) 145-164 0 1993 Elsevier Science Publishers B.V. AIf rights reserved 0376-6357/93/$06.00

145

BEPROC 00450

Effects of aging on habituation Caenorhabditis C.D.O. Department

in the nematode

elegans

Beck and C.H. Rankin

of Psychology, University of British Columbia, British Columbia, Canada

Vancouver,

(Accepted 6 August 1992)

Abstract

The effects of aging on spontaneous locomotor behavior and habituation in a mechanosensory reflex were examined in the nematode Caenorhabditis elegans. Worms were tested at 4 days (at the peak of egg laying), at 7 days (when egg laying ends) and at 12 days post-hatching. Both spontaneous and reflexive movements were smaller in older worms than in younger worms. In addition the magnitude of these movements was related to life span; the shorter an animal’s life span, the smaller its reversal movements while still young. Worms at all ages expressed habituation and dishabituation at a 10 s interstimulus interval (151); thus even aged worms were capable of non-associative learning. However, older worms showed greater habituation than did 4-day-old worms to stimuli delivered at a 60 s ISI. There was also an age-related change in the recovery from habituation. At days 4 and 7, worms had recovered from habituation by 30 min after training; however, responses of day 12 worms were still significantly smaller than baseline at 30 min after training. Further behavioral tests with normal and mutant worms may help elucidate the nature of the age-related changes in the learning and memory processes of C. elegans and the genetic mechanisms which underlie them.

Key words:

Aging; Habituation;

Learning; Nematoda

Correspondence to: C.H. Rankin, Department of Psychology, 2136 West Mall, Columbia,

Vancouver,

BC, Canada,

V6T 124.

University

of British

146

Introduction The effects of aging on the relationship

between behavior and biology can be studied

through the use of model systems. One such system is the marine mollusc Aplysia californica, in which the neurophysiological function of identified neurons in circuits controlling the behaviors of gill and siphon withdrawal are known. Studies of aging in Aplysia have shown that these simple animals display losses in learning and memory similar to those found in vertebrates. Rattan and Peretz (1981) found that the threshold for a behavioral response to gill stimulation was significantly higher in old Aplysia than in young, mature animals. Furthermore they found that old Aplysia habituated more rapidly than the younger animals. In addition, these old animals did not exhibit dishabituation when a neuron (L7), identified as one that produces dishabituation in younger animals, was stimulated. The studies with Aplysia have advanced our understanding of how age-related neurophysiological changes in identified neurons may be reflected in behavior. However, if we are to understand the role of the genome in behavioral aging, we should use a model system in which both the nervous system and the genetics may be readily understood. These experiments were designed to study the effects of aging on behavioral plasticity in such a model system, the nematode Caenorhabditis elegans. This work will provide a basis for investigations of the role of the genome in the effects of aging on the nervous system of this simple organism. Caenorhabditis elegans, a small free-living soil nematode, has been widely used as a genetic model in the study of anatomy, development and behavior. A map of over 90% of the C. elegans genome (which is half the size of the genome of Drosophila) has been established (Coulson et al., 1986; Hodgkin et al., 1988). The short reproductive cycle (about 3 days) makes C. elegans a convenient subject for genetic analysis. The genetics of C. elegans have been extensively studied and many mutant strains have been isolated. Because the neuroanatomy of C. elegans is simple, containing only 302 neurons, and the cell lineage of each somatic cell is known, the relationship between gene, neuron and behavior may be narrowly determined. The neuroanatomical map of this nematode has been described completely (White et al., 1986; Chalfie, 1984). Furthermore the functional elements of neural circuits underlying behaviors such as touch withdrawal circuit have been demonstrated, establishing the link between behavior and anatomy (Chalfie et al., 1985; Chalfie and Au, 1989). Recent work on the behavior of this organism has shown that C. elegans has a rich repertoire of behavioral plasticity (Rankin and Chiba, 1988; Rankin et al., 1990; Kumar et al., 1989). Rankin et al. (1990) showed that the tap withdrawal reflex in C. elegans exhibits the major forms of non-associative learning: habituation, dishabituation and sensitization. An advantage of working with the tap withdrawal reflex is that the neural circuit for touch withdrawal, described by Chalfie et al. (1985), has been shown to underlie the tap withdrawal reflex as well (Rankin and Chalfie, 1989; Wicks and Rankin, 1991). Thus C. elegans possesses a range of behavioral plasticity, making it a promising candidate for the investigation of the biological mechanisms underlying age-related changes in behavioral plasticity. The short life cycle of C. elegans makes studies of behavior across life span convenient. In this paper, we have focused on changes in behavior during post-reproductive development. The average life span of an individual worm is 14 days under the conditions maintained in our laboratory. In these experiments worms were

147

tested at four days of age which is at the peak of egg laying, at seven days, when egg laying has ended, and at 12 days, just prior to the end of the 14day

Materials

life span.

and Methods

Subjects Mature hermaphroditic

C. elegans

(N2)

were used throughout these studies. Subjects

were maintained on solid media at 20°C. Under these conditions the average life span observed was 14 days post-hatching. Tests of behavior were performed at 4, 7 and 12 days post-hatching.

Materials Worms were maintained on Nematode Growth Medium (NCM; Brenner, 1974) agarfilled Petri plates (5 cm diameter) and fed Escherichia co/i (strain OP50). Behavioral observations were made through a stereomicroscope with attached videorecording equipment. Mechanical and electrical stimulation were controlled by a Grass S88 stimulator. Vibrational stimuli were produced by a mechanical tapper controlled by an electromagnetic relay. The mechanical tapper tapped the side of the Petri plate holding the worm. Electrical shocks were produced using a spanning electrode with the two wires placed one on either side of the animal on the surface of the agar (approximately 2 mm apart; individual C. elegans are about 0.05 to 0.1 mm in diameter and 1 mm in length). Each shock stimulus consisted of a 600 ms train of 6-l 0 ms pulses delivered at IO Hz.

Procedure The

hermaphroditic

C. elegans

that were used in these studies were hatched syn-

chronously. To ensure synchrony, mature egg-laying worms were selected from the general population and were placed on an agar plate streaked with E. co/i. These worms were permitted to lay eggs for 3-4 h and were then removed from the plate. The eggs hatched in 9-l 1 h. At 3 days post-hatching (72-84 h) the maturing worms were placed individually on numbered agar plates spotted with E. co/i. In Experiment 1, in which individual worms were followed throughout post-reproductive development, the worms were tested at intervals 4, 7 and 12 days after hatching. Tests were performed on plates without food. After each test, the worms were placed onto new plates with fresh E. co/i to ensure a consistent food source and to prevent confusing the subjects with their offspring. In Experiments 2 to 4, in which worms were tested at only one of the test ages, worms were plated individually and raised to that test age. Worms were replated approximately every two days to maintain isolation. Behavioral responses were scored in several ways depending on the experiment. The reversal response (swimming backward) to vibrational stimuli (taps> was the chief behavioral response considered in these experiments. In order to be considered a reversal to tap, the response must have occurred within 1 s after the stimulus. Both the frequency and the

148

magnitude of these reversals were scored. The magnitude of the reversal response was quantified by tracing the path of the reversal onto an acetate sheet. These tracings were then digitized and measured using a digitizing tablet, MacMeasure software and a Macintosh computer. Statistical analyses of repeated-measures proportion data were performed with Cochran Q tests. Magnitude data were analyzed using ANOVAs with Fisher’s post-hoc comparisons when statistical significance was achieved. The level of significance was set at LY = 0.05

unless the performance of multiple tests made it necessary to adjust the (Y level

downward.

Experiment

1: Baseline

behavioral

measures

In order to determine whether there were changes in the baseline activity during aging that might affect the expression of plasticity by that reflex, the level of spontaneous locomotor activity of the worms during late (post-reproductive) development was measured by recording the time spent active over IO min, and the number and magnitude of any spontaneous reversals (swimming backward) that occurred within the 10 min observation period. In addition to spontaneous reversals, worms also show reflexive reversals in response to a variety of stimuli. In previous experiments we have shown that, in 4-day-old adults, a tap to the dish elicits a backward swimming withdrawal response called a reversal (Rankin et al., 1990). In this experiment, age-related changes in the tap withdrawal reflex were examined. The number of worms responding to taps with reversals, and the magnitude of the reversal responses to tap were measured. In addition the occurrence of other types of response to tap (accelerations forward and pauses) were scored. Finally, each worm’s response to a light touch to the head with a hair was tested in order to determine whether the worm was capable of swimming backward (as all healthy worms reverse to head-touch; Chalfie et al., 1985). If a worm did not respond to a head-touch with a reversal, it was assumed to be incapacitated and was not included in the results. The magnitude of the reversal to head-touch was not scored because the strength of the hand-delivered headtouch could not be accurately controlled.

Subjects The same 21 hermaphroditic

worms were tested for spontaneous activity three times,

once at each of the test ages: 4, 7 and 12 days after hatching. Since some worms died before 12 days of age, 45 worms had to be tested at day 4 (35 at day 7) to obtain data for 21 animals on day 12. These data permitted an investigation of behavioral differences between worms that died young (between day 4 and day 7 or between day 7 and day 12) and those that survived 12 days. Following the recording of spontaneous activity, 20 worms at each age were tested for their response to tap and head-touch.

Procedure Spontaneous locomotor activity was observed over a IO min period during which the time active was determined by measuring the amount of time the worm spent swimming

149 (either forward or backward). The number of spontaneous reversals (swimming

backward

in the absence of an obvious external stimulus) was scored over the IO min period. The magnitude (mm) of these spontaneous reversals was determined by video stop-frame analysis and computer-aided digitizing of reversal length. A single tap and a head-touch were administered immediately following the observation period for spontaneous activity with a 3 min interval between tap and head touch. Head-touch was administered with a fine hair to the region of the worm’s pharynx. The magnitudes of the reversals to tap were determined computer-aided digitizing of reversal length.

by video stop-frame

analysis and

Results and Discussion Some of the baseline behavioral measures changed with

age, others

did not. No

sjgnificant differences were found in time spent active among the test ages (day 4: X k S.E. = 96.048 + 3.152; day 7: 99.889 f 0.084; day 12: 91.563 IE 2.085; F(2,38) = 2.67, n.s.). The number of spontaneous reversals during the 10 min observation period did not change with age (F(2,38) = 1.63, n.s.). However, as seen in Fig. IA, the mean magnitude (mm) of spontaneous reversals at day 12 was significantly smaller than at day 4 or 7 (f(2,38) = 15.43, P = 0.0001). To examine the worms’ response to tap across the three test ages both the occurrence and the magnitude of reversal responses to tap were scored. When the occurrence of reversal responses was analyzed with a Cochran Q test, no change with age was evident (Q(2) = 2.8, P > 0.05). However, as seen in Fig. 1 B, the mean magnitude (mm) of reversal responses to tap was significantly smaller at day 12 than at day 4 (F(2,38) = 7.48, P = 0.002). This is reminiscent of the observation that spontaneous reversals were smaller at day 12. In both cases the magnitude of the reversals changed while the occurrence of reversals did not. The decrease in the magnitude of spontaneous and reflexive reversals at day 12 may have been related to these worms’ approaching deaths. To determine whether life span affected the spontaneous and reflexive activity of the worms, the behavior at day 4 of worms that survived to 12 days was compared with the behavior of those that died between day 4 and day 7 and the behavior of those that died between day 7 and day 12. In addition, the behavior at day 7 of worms that survived to 12 days was compared with the behavior of those that died between day 7 and day 12. During the course of the experiment 22% of the worms run died between day 4 and 7 and 31% died between day 7 and 12. Only those behaviors that changed with age (the mean magnitude of spontaneous and reflexive reversals to tap) were examined. In both cases, the magnitude of the swimming reversals was smaller in worms that were to die earlier. As seen in Fig. IC, the mean magnitude of spontaneous reversals measured on day 7 was related to lifespan while it was not when measured on day 4 (day 4: F(2,42) = 0.302, ns.; day 7: t(33) = 2.352, P = 0.025). The magnitude of the swimming reversal response to tap (Fig. 1 D) was related to lifespan when measured on day 4 but not when measured on day 7 (day 4: F(2,42) = 3.69, P = 0.0336; day 7: t(33) = 0.51, n.s.). Thus, the magnitude of spontaneous and reflexive reversals may reflect the worms’ lifespan. Further investigations of this sort may help to define this relationship. A possible explanation for the decrease in response magnitude in 12 day old worms is that there is a change in sensory ability with increased age. An examination of the magnitude of the reversal reflex in response to a graded series of taps (beginning with a very weak stimulus) at each of the three test ages was used to test this hypothesis.

150 A

SPONTANEOUS

IN

1

w

SPONTANEOUS

C

MAGNITUDE

(A) The

REVERSAL

RELATED

TO

TO

TAP

3

RESPONSE

D

RELATED

LIFE-SPAN

mean magnitude of spontaneous

magnitude of spontaneous stimuli)

MAGNITUDE

RESPONSE

eo

20

Fig. I,

REVERSAL

B

REVERSALS

reversals (swimming

during the 10 min observation

reversals (mm)

TO TO

was smaller

TAP

LIFE-SPAN

in old worms.

The

backward in the absence of any obvious external

period at each of the three test ages, 4, 7 and 12 days

post-hatching (mean number of spontaneous

reversals

by each worm f S.E.M.

Day 4: 34.7 k 3.2;

Day 7: 29.4 i 1.5; Day 12: 34.9 F 2.1). (B) The magnitude of the reversal response to tap (mm) was smallest

in older

worms.

The

responded with a reversal;

magnitude of reversals

number of worms

to tap (including

reversing Day 4: 20;

only

those worms

Day 7: 17;

Day 12:

that

18). (0

Life-span was retdted to the mean magnitude of spontaneous reversals at day 7. The mean magnitude of spontaneous

reversals of worms that survived until day 12

that of worms

that died between day 4 and day 7 (D4-D7;

(D7-D12;

n = 14).

04

TEST=

velocity at day 4; D7 TEST=

( > D12; R = 21) was compared with n = IO)

and day 7 and day 12

velocity at day 7. (D)

Life-span

was

related to the magnitude of reversal responses to tap at day 4. The magnitude of reversals of worms that survived until day 12 ( > D12;

n = 21) was compared with that of worms that died between day

4 and day 7 (D44D7;

Experiment In this were during

n = IO) and ddy 7 and day 12 (D7-D12;

n = 14).

2: Graded response

experiment,

examined.

The

pilot studies,

the effects tap intensities induced

of aging on the responses chosen

reversals

(weak,

of different

moderate

to taps of different and strong)

magnitudes

intensities

were ones which,

in 4-day-old

worms.

Subjects Twenty

naive worms

total of 60 worms. before

stimulation.

were tested with

A further

20 worms

taps of three different

intensities

were tested at day 12 to examine

at each age for a the effect of delay

151

Procedure Worms were placed individually on test plates with a small amount of E. co/i (strain OP50) 24 h before testing. Tap intensity was altered by changing the voltage from the Grass S-88 stimulator to the electromagnetic relay which controlled the tapper. The lowest voltage (38 V) produced a weak tap, while the highest voltage (60 V> produced a strong tap and was the intensity used in all other experiments. An intermediate voltage of 40 V produced a tap of moderate strength. The taps were administered individually to worms at IO-min intervals. In studies of recovery from habituation, Rankin and Broster (1992) showed that taps administered at IO-min intervals did not produce significant response decrement in worms that were 4 days old. Of the 20 worms tested at each age (4, 7 and 12 days post-hatching), IO worms received the stimuli in ascending order of intensity (weak, moderate intensity (strong,

and strong) and 10 worms received the stimuli moderate and weak). To test for an interaction

in descending order between the order

of of

presentation and the response to stimuli of different intensities at each age, the responses of worms that received the stimuli in ascending and descending orders were compared with a two-factor repeated-measures ANOVA. The magnitude (mm) of the reversals at each age for different intensities were analyzed with a repeated-measures ANOVA. Non-reversals (pauses, accelerations forward and no response), which made up approximately 5% of the data, were excluded from the analysis of reversal magnitude and were replaced with the mean of the group for the purposes of the statistical analysis. At each test age, 4, 7 and 12 days post-hatching, the numbers were analyzed with a Cochran Q test.

Results

of reversals to taps of different

intensities

and Discussion

To determine whether the data from the groups which received the stimuli in different orders could be pooled together, the mean response magnitudes from worms that received the stimuli in ascending order were compared with the mean response magnitudes from those that received the stimuli in descending order across the three stimulus intensities at each alpha (three tests,

test age (4, 7 and 12 days post-hatching). To control experiment-wise error level (0.05) was divided by the number of tests performed on the data from tests: one initial comparison of the orders of stimulation, and possibly two one for each stimulus order; alpha = 0.05/3 = 0.016). At day 4 and day 7,

rate, the each age follow-up there was

an effect of stimulus intensity, but no effect of stimulus order nor any interaction between stimulus order and intensity (day 4: Order: F(1,18) = 2.16, ns.; Intensity: F(2,36) = 5.51, P = 0.008; Order x Intensity: F(2,36) = 0.66, ns.; day 7: Order: F(1,18) = 0.14, ns.; Intensity: f(2,36) = 5.39, P = 0.009; Order X Intensity: F(2,36) = 0.091, n.s.1. However, at 12 days of age the order of stimulation interacted with stimulus intensity in addition to a significant effect of intensity (day 12: Order: F(1,18) = 5.759, P = ns.; intensity: F(2,36) = 11.78, P = 0.0001; Order X Intensity: f(2,36) = 10.26, P = 0.0003). Therefore, while data from worms run in ascending and descending orders from day 4 and 7 worms could be pooled, the data from day 12 could not. Response occurrence At days 4 and 7, stimulus intensity did not affect the number of worms responding (day 7: Q(2) = 2.4; n.s.>. At day 12, the data from worms that received the stimuli in ascending

152 DAY

A

WEAK

4

MODERATE

s

DAY

WEAK

STRONG

7

MODERATE

C

DAY

WEAK

STRONG

12

MODERATE

STRONG

Fig. 2. The reversal response to tap (mm) was graded as a function of stimulus tested, The magnitude of reversal responses+S.E.M.

days post-hatching (n = 20 at each age). At day 4 (A) and day 7 (B), received the stimuli

in ascending order (ASCENDING;

worms that received the stimuli in descending order (DESCENDING;

descending

number Q(2)

orders

of worms

= 2.0,

at all ages

at 4, 7 and 12

the data from worms

that

in ascending and descending orders were pooled, while at day 12 (C) the data

from worms that received the stimuli

and

intensity

to taps of different intensities

were

analyzed

responding

in either

separately. group

Stimulus

(ascending:

n = 10) and the data from

n = IO) are shown separately.

intensity Q(2)

did

= 1.2,

not

n.s.;

affect

the

descending:

n.s.).

Response magnitude As shown weak (Fig.

in Fig.

2B),

worms or weak

received

the stimuli

(Fig.

did not show

day 4 worms a moderate

responded

moderate worms

2A,

tap than to either

with

tap (F(2,38)

a graded

significantly = 5.66,

in ascending

2C) that received

responded or strong

larger

P = 0.007).

and descending

stimulation

response

with

to stimuli

significantly

tap (F(2,38) reversals

orders

of different

were order

analyzed

to a

At day 7

tap than either

the data from

(weak,

intensities

reversals

P = 0.007).

to a strong

At day 12,

in ascending

smaller

= 5.61,

worms

separately.

moderate

(F(2,18)

a

that

Day 12

and strong)

= 0.086,

n.s.>.

153

However,

day 12 worms

that received

the stimuli

in descending

order

(strong,

moderate

and weak) responded with significantly larger reversals to the strong tap than to either the weak or moderate taps (F(2,18)= 13.79, P = 0.0002). Furthermore, when the first stimuli of the ascending and descending order series were compared, the first tap of the ascending series (weak intensity) evoked a reversal response that was significantly smaller than the response to the first stimulus of the descending series (strong intensity) (two-tailed t(l8) = - 3.61; P = 0.002). This finding shows that day 12 worms, like day 4 and 7 worms, are capable of responding to stimuli of different intensities with graded responses, but that unresponsiveness produced by repeated stimulation (habituation) even when the stimuli are administered at IO-min intervals.

may

mask the effect

An alternate explanation for the unresponsiveness of day 12 worms to stimuli in the ascending order of presentation is that treatments, other than stimulation, such as vibrational stimulation from placement on the microscope stage and the light and warmth on the microscope stage, may have caused fatigue in these older worms during the 20-min testing period. Thus the responses of an additional 20 day-12 worms were tested after a delay of 0 or 20 min (IO worms with a 0-min delay and IO worms with a 20-min delay) on the microscope stage. A two-tailed t-test showed that there was no significant difference in the reversal

magnitude

to strong tap between

worms

that received

the tap immediately

after being placed on the microscope stage and worms that received the tap after a 20.min delay (0-min delay: 2 = 0.377 + 0.165; 20 min delay: x = 0.601 f 0.305; t(l1) = -0.61, ns.). Simply being on the microscope stage did not appear to affect the day 12 worms’ responses to tap. This finding suggests that indeed the day 12 worms responded with small reversals during the tests with taps of graded intensities because of habituation to stimuli delivered 10 min apart. It is evident that even the worms tested on day 12 were sensitive to changes in stimulus intensity. Thus there is no reason to attribute the decreased responsiveness of the older worms to a loss of sensory ability. These results suggest that older worms are more susceptible to habituation training than younger worms. If so, day 12 worms might be expected to exhibit a faster rate of response decrement during habituation training.

Experiment

3: Habituation

and dishabituation

The effects of aging on habituation

to 60 stimuli

delivered

at short (IO s) or long (60 s)

interstimulus intervals in C. elegans were examined at each of the three test ages, 4 days, 7 days and 12 days post-hatching. Four-day-old worms show more rapid and greater habituation to stimuli at a 10 s ISIthan to stimuli at a 60 s ISI (Rankin and Broster, 1992). In addition the effect of aging on dishabituation was tested following habituation with 60 stimuli at a 10 s ISI.

Subjects Twenty naive worms were used at each of the three test ages (4, 7 and 12 days) for each of the two ISIS (IO and 60 s) for a total of 180 worms.

Procedure Trains of taps (1 train = 6 taps at 8.5 Hz) were the stimuli used in all of the following habituation experiments because this stimulus produces larger responses than do single

154 taps (Chiba plasticity tap

was

2.005

f

and Rankin,

be observed.

1.275

f

0.141

0.157.

In

non-associative (ISI).

(*S.E.M.)

learning

(Rankin

of

et al.,

60 trains

(6 shocks

produces

stimulus,

the

taps 1990;

mean

trains

to

used

changes

administered

our

to

taps

was

studies

of

1992).

at a 10 s or a 60 s interstimulus

The

(Rankin

of

other

after the last habituating

stimulus,

60 V train of shocks

et al.,

of taps were

in

response

a train

in

and Broster,

was administered.

more

response

been

Rankin

ten seconds

range in which

at day 4 the mean reversal

have

in 4-day-olds

12

a large response

study,

of taps were

at 10 Hz)

dishabituation

dishabituating

afford

while

trains

For the 10 s ISI group

train of shocks that

and thus

In the present

addition,

In this experiment, interval

1990)

might

1990).

Within

administered

a 60 V

is a stimulus

20

s after

the

at a 10 s ISI to test for

dishabituation.

and Discussion

Results

IO s ISI

Response occurrence The

occurrence

stimulus

with

of reversals,

a reversal,

scored

showed

4, 7 and 12 days of age (see Figs. 10%

of the responses)

Q tests initial Q(2)

= 7.63,

pattern

habituation

P < 0.05;

early

This

averaged 33.67,

of worms

training

finding

across

significantly

Q(2)

4:

Q(2)

responding

and then

habituation

as missing

(fewer

at

than

data. Cochran across

P < 0.02;

the

day 7:

However,

as seen in Fig. 3, the

did appear different

at day 7. At 4 and 12

decreased

early

began responding with

evident

changes

= 10.64,

in the habituation

appeared

age; the

to stop

training

responding

again as the habituation

in a change in the proportion

trials

to each

were

forward

significant

At 7 days of age, the worms

was reflected

all 60

greater

were

P < 0.001).

training

responding

Accelerations

and treated

(day

= 21.37,

habituation

low throughout.

in habituation

continued.

from the analysis

of worms

and dishabituation

and C, and 5A).

and dishabituation

during

days of age, the number

proportion

that at each age there

day 12:

of responding

and remained

3A,B

were omitted

at each age confirmed response,

as the

that both habituation

mean

of worms

frequency

at day 7 than at day 4 or day 12 (see Table

training

responding

of response

1 for

means;

was

F(2,57)

=

P = 0.0001).

Response magnitude In order

to compare

standardized (which

because

reversals;

5B)

performed was

280.39,

mean (Class

in

response

the from

response the

0.05

was

motor

of a response

significant of

habituated P < 0.0001;

day 12:

(day

4:

reversal

were

were

response

not included

cannot

as a score

analyses (Figs.

response.

= 0.016

F(2,38)

were

that

magnitudes

initial

in the

be compared of zero

replaced

to

(Rankin

et

by the group

1984).

greater

and in addition

response

responses

was included

habituation

reversal

to 0.05/3

reversal

of the

responses

in repeated-measures

significantly

response,

ages the

Acceleration

different

and Hopkins,

magnitude

across

as a percent

on each of the three ages. To control

dishabituated than

data points

at all ages showed

reduced

initial

represent

the absence

Missing

and condition Worms

they

however,

1990).

(Fig.

magnitude

each

was set at 1000/o) for each worm.

analysis al.,

response

by expressing

and

both

F(2,38)

= 134.36,

error

Hopkins,

the

the dishabituated = 786.63,

B and C) and dishabituation

experiment-wise

(Class than

4A,

Repeated-measures 1984).

habituated

response

There

were

At all ages the

response

and

was significantly

P < 0.0001;

P < 0.0001).

ANOVAs

rate, the alpha level

day 7: were

the

greater

F(2,38)

=

no significant

155

q

DAY

7

TRIALS

DAY

Fig. 3. During decreased habituation

habituation

training

(IO

12

s ISI and 60 s ISI), the proportion

at all ages tested. The proportion

of worms

responding

of worms responding to stimuli with reversals during

training to trains of taps (IO s ISI and 60 s ISI, 30 stim; n = 20 at each age, day 4 (A), day 7 (B) and day 12 (C)j.

in the amount of dishabituation across the three ages as measured by difference scores produced by subtracting each animal‘s response magnitude for the first response

differences

following shock from its initial response magnitude (see Table 1 for means; F(2,57) = 0.66, ns.). There was no significant difference in the rate of response decrement during habituation training. The rate of decrement was analyzed by calculating the slope of the regression lines for reversal responses to the first ten stimuli in the habituation training for each worm and then taking the mean of these slopes for each age (see Table 1 for means; F(2,56) = 0.36, n.s.). Both the frequency and response magnitude data demonstrate that worms of all ages show habituation and dishabituation. The significant dishabituation demonstrates that the response decrement was indeed habituation and not simply the result of sensory adapta-

156 TABLE

1

Experiment

3: the mean magnitude of reversal responsesf5E.M.

during habituation training. Initial

response set at 100%

ISI 1 OS

Age D4 D7

Mean

Mean

Trials

Trials

Dishab

proportion

slope

I-IO

21-30

-hab

0.265

- 8.30

0.026

27.07 2.45 25.01 3.15 24.48 3.35

6.62 1.72 12.25 2.45 9.65 2.50

22.98 7.30 21.48 10.64

71 .I 5 9.98 60.46 a.99 44.48 5.00

41.79

_

6.27 19.57

_

0.329

0.67 ~ 7.61 1.24 - 7.08

0.034

i .oa

0.697

- 3.06

0.035

1.61 - 7.00 1.60 - 5.60 0.93

0.606 0.032

D12

60s

D4 D7

0.599 0.039

D12

0.378 0.045

tion or motor fatigue (Groves and Thompson,

1970).

3.77 I I .a0 2.85

11.47

3.34

_

The only difference across the ages

tested, was in response frequency during habituation. At day 7, worms initially decreased in their response frequency, but then increased again and continued a relatively high response rate throughout habituation training; however, their responses were very small. This pattern of responding was not as pronounced in a later experiment (Experiment 4) on recovery from habituation. Further work on the patterns of response occurrence would help to clarify the significance of these results. Based on the results of the graded response experiment (Experiment 2) we hypothesized that day 12 worms habituate to stimuli delivered at longer interstimulus intervals than do younger animals. Although habituation training at a IO s ISI revealed no age-related difference in response magnitude, the 10 s ISI may have been so short that differences in rate or degree of response decrement were lost. Thus, a 60 s ISI was used to test whether, with longer intervals, older animals might show greater habituation than younger animals.

Results

and Discussion

60 s ISI

Response occurrence As seen in Fig. 3A, B and C, the proportion of worms responding with reversals appeared to decrease more in older worms than in younger ones. Chi-square tests at each age comparing the proportion of worms responding on the initial stimulus with the proportion of worms responding on the final stimulus of the habituation training showed that the proportion training at day 4 habituation training chi-square = 11.25, during habituation habituation training

of worms responding did not change significantly with habituation (chi-square (df I) = 1.8, n.s.) but did decrease significantly with at day 7 and day 12 (day 7: chi-square = 5.0, P < 0.05; day 12: P < 0.05). Furthermore the mean proportion of worms responding training changed significantly with age (see Table I). Throughout day 4 worms responded significantly more frequently with reversals

157

-

3’0

0

TRIALS

DAY

B

7

d

3’0

2.0 TRIALS

DAY

C

12

‘““l!

d

lb

2’0 TRIALS

Fig. 4. During habituation training (IO

s IS1 and 60 s ISI), the mean magnitude of reversal responses

decreased at all ages tested. The mean magnitude5S.E.M.

of reversal responses during habituation

training with trains of taps (IO s ISI and 60 s ISI, 30 stimuli;

n = 20 at each age; day 4 (A),

day 7 (B)

and day 12 (C)). The magnitude of reversal responses was expressed as a percent of each worm’s response to the initial stimulus

than did day 12 worms worms

responding

older

worms

magnitude

The

magnitude

age (Fig.

4A,

of reversal B and 0.

the magnitude

of reversals

t(19)=8.55,

p=O.OOOl;

0.0001).

The

magnitudes

= 16.83,

P = 0.0001).

as much with

Overall,

habituation

the proportion training

of younger

as the proportion

of

responding.

Response with

(F(2,57)

did not decrease

in habituation training.

responses

decreased d7:

rate of habituation,

to the first

during

Habituation

ten stimuli,

habituation

of reversal

significantly

t(19)=12.12, measured

training

magnitude

at each age with

P=O.OOOl;

habituation

d12:

by the regression

did not change with

at a 60 s ISI changed

was present

training

t(19)=10.02,

line slopes

age (see Table

at all ages as (d4: P=

for the reversal

1; f(2,57)

= 1.99,

158

DAY

4

DAY

7

DAY

12

DAY

4

DAY

7

DAY

12

Fig. 5. Habituation and dishabituation were evident in both the proportion of worms responding and the magnitude of reversal responses at all ages tested. Reversal responses before and after habituation training with trains of taps (IO s ISI, 60 stimuli)

and after dishabituation

with electric shock at 4, 7

and 12 days post-hatching (n = 20 at each age). INIT = initial response of the habituation training; HAB = final response of the habituation training; lus. (A) Proportion habituation

of worms

training

response+

S.E.M.

ing stimulus.

DIS = first response after the dishabituating stimu-

responding to stimuli

and following

with reversals before habituation training,

the dishabituating

trains

the

greater

of greater

age highlights

reversals

susceptibility

and

the

the difference

trained

at a 10

to trials

I-IO

of the reversal

compared

with

trained

reduced: interstimulus magnitude

the

0.05/6

responses

mean

at a 60

s ISI.

of reversals

on

the

during

process

provides

responses

60 s ISI habituation

responses

Likewise,

were with

to of

show

each test B and C;

I-IO

of each

of the

reversal

at each age, the mean to trials The

a picture

were

4A,

to trials

trained used.

at each

training

(Fig.

magnitude

responses us

habituation

both the proportion reversal

worms

mean

t-tests

=

to a greater extent

age.

of each worm

reversal

two-tailed of

the

at a 60 s ISI.

21-30

of the

analysis

with

was

1; F(2,57)

at 10 s and 60 s ISI’s within

of the reversal

trained

the

and older

with

(see Table

Thus

of

training

the younger

to trials

Unpaired This

to the last ten stimuli

to habituate

day 4 worms.

training

compared

magnitude

= 0.0083.

interval

were

of each worm

magnitude

appeared

to habituation

between

s ISI

in response

magnitude

of the effects of habituation

I>. At each age, the mean magnitude

responses

worm

of reversals

at a 60 s ISI than

with

reversal

in habituation training.

than day 7 or day 12 worms

Day 7 and day 12 worms

responding

A comparison

worm

magnitude

of taps administered

evidence

Table

mean

in day 4 worms

P = 0.0001).

worms

after

Mean magnitude of reversal

before habituation training, after habituation training and following the dishabituat-

However,

significantly 11.81,

(6)

Magnitude of reversal responses was expressed as a percent of each worm’s response to the initial stimulus

n.s.).

stimulus.

age. At greater

at a 10 s ISI were 21-30 alpha of the day

of each level effects

4 the

than those

was of

mean during

159

10 s ISI habituation P = 0.0001;

trials

training 21-30

in both

t(38)

early

= 5.41,

and

late training

P = 0.0001).

At day

(trials

I-10

7 the mean

t(38)

= 4.29,

magnitude

of

reversals at a 60 s ISI was greater than at a 10 s ISI in early training but not late training (trials I-IO t(38) = 3.72, P = 0.0006; trials 21-30 t(38) = 1.63, r1.s.). At day 12 the mean magnitude of reversals at a 60 s ISI was greater than at a IO s ISI in early training but not late training (trials I-IO t(38) = 3.32, P= 0.002; trials 21-30 t(38) = 0.57, n.s.>. Thus at each age there was a difference between the mean magnitude of reversals to the first ten stimuli at 10 s and 60 s ISl’s. However, at day 4 the responses to stimuli at a 60 s ISI remained significantly greater than those at a 10 s ISI in late habituation training, while at day 7 and 12 the responses at 10 s and 60 s ISl’s were habituation training.

not significantly

different

in late

In addition we can compare the rate of habituation at 10 s and 60 s ISl’s at each age by comparing the slopes of the regression lines of the first ten stimuli. At day 4 the rate of habituation was significantly greater at a 10 s ISI than at a 60 s ISI (see Table 1; t(38) = -3.007, P = 0.0047). At day 7 and 12 the rate of habituation training was not affected by the duration of the interstimulus interval (see Table 1; t(38) = -0.30, n.s.; t(38) = - 1.04, n.s.>. These comparisons of 10 s and 60 s ISI habituation at each testing age reveal two age-related differences. First, at day 4 the mean magnitude of reversals during 60 s ISI habituation training was significantly greater than that during 10 s ISI training in both early and late trials. However, at day 7 and 12, interstimulus interval only had an effect in early training. This finding confirms that worms at all ages are sensitive to changes in interstimuIus interval. In addition, it suggests that there are age-related differences in the effects of habituation training as stimulus number increases. Second, at day 4 the rate of habituation was slower to a 60 s than a 10 s ISI, but at day 7 and day 12 the rate of habituation was not significantly slower at the longer ISI. This finding suggests an age-related difference in the early part of habituation. Thus, with a longer ISI, older animals appeared to habituate earlier and to a greater degree than younger animals. No such aging effect was seen at the short interstimulus interval.

Experiment

4: Recovery from habituation

Recovery from habituation may be memory. The stronger the memory, Thus if the processes underlying the recovery from habituation might also

thought of as a measure of the strength of short-term the slower the recovery from habituation might be. formation of short-term memories change with age, change with age.

Subjects Twenty

worms

were tested at each of the test ages 4, 7 and 12 days post-hatching.

Procedure Habituation was established by delivering 60 trains of taps at a 10 s ISI; at the end of habituation training, single trains of taps at 30 s, 10 min, 20 min and 30 min were

160 B

00

DAY 4

DAY 12

I HAB

INIT

30s

20M

10M

30M

DAY

D

DAY 7

C

12

150-j

150,

01

T

0’ 30s

HAB

Fig. 6. (A) The

10M

proportion

20M

of worms

, HAB

30M

responding

10M

30s

to trains of taps with

reversals

habituation training and subsequent recovery tests at all ages tested. Proportion

20M

changed during

of worms responding

with reversals during recovery following habituation training (10 s ISI, 60 stimuli;

n = 20 at each age)

shown. Tests of recovery were given 30 s, 10 min, 20 min, and 30 min post-habituation. response to the first habituation stimulus; The magnitude of reversal responses

30M

lNlT=

HAB = the response to the last habituation stimulus.

the (B)

at the end of habituation training and during recovery from

habituation at day 4. Magnitude of reversal responses was expressed as a percent of each worm’s reversal

response

to the initial

stimulus

in habituation

training.

(C) The

magnitude of reversal

responses at the end of habituation training and during recovery from habituation at day 7. (D) The magnitude of reversal

responses

at the

end

of

habituation

delivered

demonstrate

Results

training

and

during

recovery

from

to test for recovery

4, it produced

4-day-old

habituation at day 12.

from habituation. A 10 s ISI was used because, in Experiment the same degree of habituation at all ages. This procedure has been used to recovery from habituation 20 to 30 min after the last habituating stimulus in

worms

(Rankin

and Broster,

1990,

1992).

and Discussion

In the analysis of the recovery of the magnitude of the reversal response after habituation training, reversal magnitude was standardized as percent initial response as in the previous experiment. Only the frequency and magnitude data from the 30 min recovery tests were statistically analyzed because the variance in the intermediate tests (at 30 s, 10 min and 20 min) was too great to be usefully included. The magnitude data from all tests are depicted in Fig. 6B, C and D.

161

Response occurrence The occurrence of reversals, scored as the proportion of worms responding to each stimulus with a reversal, showed that both habituation and recovery from habituation were evident at 4, 7 and 12 days of age (see Fig. 6A). Cochran Q tests at each age comparing the initial, habituated and recovered (30 min post-habituation) response occurrence confirmed that at each age there were significant changes with the treatments (day 4: Q(2) = 17.231,

P < 0.001;

day

7:

Q(2) = 13.0,

P < 0.01;

day

12:

Q(2) = 19.0,

P < 0.001 I Response magnitude A different pattern is seen in the analysis of the magnitude of reversal responses. As shown in Fig. 66 and C at 4 days and 7 days post-hatching worms showed significant recovery of response magnitude over habituated levels by 30 min following habituation training; in contrast, worms tested at 12 days post-hatching (Fig. 6D) did not show significant recovery over habituated levels by 30 min post-habituation (day 4: F(2,38) = 23.56, P< .OOOOl; day 7: F(2,38)= 16.95, P< 0.0001; day 12: F(2,38)= 3.43, P < 0.0001). The slower

recovery

from habituation

in day 12 worms

may reflect a change

in the way

short-term memory for habituation is expressed in older worms. It is not clear whether this longer time to recover is related to the greater sensitivity to habituation to long ISIS seen in the day 12 worms. Further investigations into this issue are needed before conclusions can be drawn.

General

Discussion

Examination of the spontaneous and reflexive behavior of the worms indicated that there was a decrease in some measures of motor activity with age. The magnitude of spontaneous reversals and the reversal response to tap diminished with age. The mean magnitude of spontaneous reversals and the magnitude of response to single taps was related to life span. In future studies it may be useful to probe the relationship in C. elegans between an individual worm’s life span, their physiological state, the quality of their activity and the effects of that activity on behavioral plasticity. To test the sensitivity of the aged worms to changes in stimulus intensity, above-threshold stimuli of different intensities (weak, moderate and strong) were administered at a 10 min ISI in either ascending (weak, moderate, strong) or descending (strong, moderate, weak) orders of intensity. At day 4, 7 and 12 worms showed graded responses to stimuli of graded intensities. However, at day 12 there was an interaction between the order of presentation and the effect of stimulus intensity. In the ascending order of presentation, the responses to moderate and strong stimuli were of the same magnitude as the small responses seen to the weak stimulus. This may have been due to habituation to the tap stimulus. This finding was unexpected as Rankin and Broster (1992) found no evidence of response decrement when they administered four taps of equal intensities at a 10 min ISI to day 4 worms. Exposure to the environmental conditions of the graded response’experiment alone did not affect reversal magnitude of day 12 worms. In addition, since 12-day-old worms showed an average of 34.857 + 2.145 spontaneous reversals during a IO-min observation period, it is unlikely that three evoked reversals would lead to exhaustion. Thus, the unresponsiveness observed in day 12 worms in the graded response experiment when

162

stimuli

were administered in order of ascending intensity may have indeed been caused by

habituation to tap. This finding supports the hypothesis that processes underlying tion in C. elegans change with age.

habitua-

Habituation at a 10 s ISI and dishabituation to shock were present in worms at all ages and did not appear to change greatly during post-reproductive development. Although there were no apparent differences in the rate or amount of habituation to a 10 s ISI between age groups, habituation training using a longer ISI (60 s) revealed that older worms showed greater habituation at long interstimulus intervals than day 4 worms as reflected by both the proportion of worms responding and the magnitude of reversal response. These findings are similar to those of Rattan and Peretz (1981) who found that older Aplysia habituated more quickly than young, mature Aplysia. However, unlike Rattan and Peretz’s observations in Aplysia, we observed dishabituation at all ages of C. elegans tested. Worms tested at 4 and 7 days showed recovery from habituation (10 s ISI) of both the frequency and response magnitude 30 min after habituation training, but worms tested at 12 days did not. This deficit in recovery suggests a persistence of habituation in the older worms. This persistence is unlikely to be explained in full by a factor such as more rapid physical exhaustion since day 12 worms with the same habituation training showed dishabituation (facilitation of the habituated response) immediately after a mild electric shock. Perhaps this persistence reflects a change in short-term memory for habituation training in the older worms. Further studies on the dynamics of habituation and recovery from habituation and on long-term memory in aged worms may help the understanding of mechanisms underlying the age-related changes in habituation. In summary, older worms show smaller spontaneous reversals and smaller reversals to tap than younger worms. In addition older worms show greater habituation to stimuli delivered at long interstimulus intervals and recover from habituation more slowly than do younger worms. These changes appear to be independent of fatigue. The studies described here provide groundwork for further research on the effects of aging on learning and memory in C. elegans. Future research should explore the behavioral, neural and genetic mechanisms that underlie changes in learning and memory with age. First, behavioral experiments on the parameters of habituation can further define the effects of aging on non-associative learning. Second, laser ablation studies and work with mutants missing particular neurons will aid in understanding the roles of individual neurons in the aging nervous system. Finally mutants with specific changes in the aging process may be used to examine the genetic mechanisms underlying changes in learning and memory with age. For example, Johnson (1987) isolated a single gene mutation that produces a 60-I 10% increase in life span; attempts are underway to clone this gene. By integrating the findings of these various lines of research a better understanding of the effects of aging on behavioral plasticity in C. elegans

may be achieved.

Acknowledgements We thank Cathy Chiba for her editorial input on an earlier version of this manuscript, and Brett Broster, Shannon Cerniuk, Bill Mah, Theresa Marion and Steve Wicks for useful discussions. This work was supported by a BCHCRF grant to C.H.R.

163

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