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The effects of aging on non-associative learning in the nematode caenorhabditis elegans Beck, Christine Daily O'Brien 1991

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THE EFFECTS OF AGING ON NON-ASSOCIATIVE LEARNING IN THE NEMATODE CAENORHABDITIS ELEGANS by CHRISTINE DAILY O'BRIEN BECK B . S c , The U n i v e r s i t y of A l b e r t a , 1987 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREEE OF MASTER OF ARTS i n THE FACULTY OF GRADUATE STUDIES (Department of Psychology) We accept t h i s t h e s i s as conforming to the req u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA September 1991 © C h r i s t i n e D. O. Beck, 1991 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of * f ^ c A ^ / r w ^ The University of British Columbia Vancouver, Canada Date 3 0 Aug, m < DE-6 (2/88) i i ABSTRACT With the advantages of s i m p l i c i t y and a we11-understood anatomy, development and genome, C.elegans may be an e f f e c t i v e model of the r o l e of the genome i n the e f f e c t s of aging on l e a r n i n g and memory. The purpose of t h i s t h e s i s i s to begin t h i s r e s e a r c h by d e s c r i b i n g the e f f e c t s of aging i n C.elegans on a simple form of l e a r n i n g , h a b i t u a t i o n , i n the tap withdrawal r e f l e x . F i r s t , t h e e f f e c t s of aging on the spontaneous locomotor behavior and simple r e f l e x i v e behavior of C.elegans were examined. Worms were t e s t e d at 4, 7 and 12 days post-hatching. The average l i f e - s p a n of worms r a i s e d i n the c o n d i t i o n s of t h i s l a b o r a t o r y o ( s o l i d medium, 21 C) was 14 to 16 days. The amount of spontaneous a c t i v i t y d i d not change with age, but the nature of t h a t a c t i v i t y d i d change. Worms moved more slowly and both spontaneous and r e f l e x i v e r e v e r s a l s decreased i n magnitude at day 12. Worms at a l l ages e x h i b i t e d graded responses t o taps of d i f f e r e n t i n t e n s i t i e s . The e f f e c t s of aging on h a b i t u a t i o n and d i s h a b i t u a t i o n were then examined. There appeared t o be a d i s s o c i a t i o n of response frequency and magnitude: a l l ages t e s t e d (4, 7 and 12 days post-hatching) showed s i m i l a r changes i n magnitude of r e v e r s a l s due t o h a b i t u a t i o n and d i s h a b i t u a t i o n . However at day 7 the p r o p o r t i o n of worms r e v e r s i n g d i d not decrease d u r i n g h a b i t u a t i o n t r a i n i n g as i t d i d at the other ages (days 4 and 12) t e s t e d . There was a l s o an age-related change i n the recovery from h a b i t u a t i o n ; day 12 worms d i d not recover w i t h i n 30 min of the i i i l a s t h a b i t u a t i o n stimulus, u n l i k e worms t e s t e d a t day 4 and 7 which recovered back t o b a s e l i n e l e v e l s by 30 min. F i n a l l y the e f f e c t s of t a i l - t o u c h h a b i t u a t i o n t r a i n i n g on i n h i b i t i o n of the r e v e r s a l response t o tap was examined at the three t e s t ages. At a l l ages t a i l - t o u c h h a b i t u a t i o n t r a i n i n g decreased the i n h i b i t i o n of r e v e r s a l t o tap by t a i l - t o u c h . C l e a r l y , even day 12 worms are capable of h a b i t u a t i o n independent of f a t i g u e e f f e c t s . The age-related changes seen may be produced by From these experiments i t i s c l e a r that although the behavior of C. elegans does change with age, aged worms are capable of the simple form of l e a r n i n g , h a b i t u a t i o n . Further b e h a v i o r a l t e s t s with normal and mutant worms may help e l u c i d a t e the nature of the ag e - r e l a t e d changes i n l e a r n i n g and memory i n C.elegans and the ge n e t i c mechanisms which u n d e r l i e them. i v Table of Contents A b s t r a c t ,. . . i i L i s t of Figures . . v i Acknowledgements v i i i I n t r o d u c t i o n 1 General Methods 7 Subjects 7 M a t e r i a l s 7 Procedure 8 Experiment 1. Spontaneous locomotor a c t i v i t y . . . .10 Subjects 10 Procedure . . 10 R e s u l t s and D i s c u s s i o n . . 11 Experiment 2. Response to tap and head-touch . . . .18 Subjects 19 Procedure 19 Res u l t s and D i s c u s s i o n 20 Experiment 3. Graded response 26 Subjects 26 Procedure 26 Res u l t s and D i s c u s s i o n . . . . . . . . 28 Response Frequency 29 Response Magnitude . . . . . . . . .29 Experiment 4. Habituation and d i s h a b i t u a t i o n . . . .34 Subjects 35 V Procedure . . . • • .35 Resu l t s and D i s c u s s i o n 35 Response Frequency 36 Response Magnitude 36 Experiment 5. Recovery from h a b i t u a t i o n 44 Subjects 44 Procedure 45 Re s u l t s and D i s c u s s i o n 45 Response Frequency 45 Response Magnitude 50 Experiment 6. I n h i b i t i o n • .50 Subjects 51 Procedure . . . . . . . . . . . .51 Re s u l t s and D i s c u s s i o n 51 Response Frequency 51 Response Magnitude 51 General D i s c u s s i o n . 54 B i b l i o g r a p h y 62 v i L i s t of Figures i 1. Worm length measured at 4, 7 and 12 days post-hatching . . 13 2. The percent time spent a c t i v e measured over a 10 min ob s e r v a t i o n p e r i o d and the spontaneous v e l o c i t y o f movement measured as the di s t a n c e t r a v e l l e d both backward and forward d u r i n g 10 s i n the 10 min observation p e r i o d at each of the three t e s t ages, 4, 7 and 12 days of age 15 3. The number and magnitude of spontaneous r e v e r s a l s d u r i n g the 10 min o b s e r v a t i o n p e r i o d at each of the three t e s t ages, 4, 7 and 12 days post-hatching . 17 4. The number and magnitude of r e v e r s a l s t o tap at 4, 7 and 12 days post-hatching . . . . . . . . . . . 2 2 5. Spontaneous v e l o c i t y , mean magnitude of spontaneous r e v e r s a l s , magnitude of the r e v e r s a l response t o tap comparing worms with d i f f e r e n t times of death at 4 and 7 days post-hatching . . 25 6. The number of worms responding t o taps of d i f f e r e n t i n t e n s i t i e s were measured i n order t o examine graded response t o tap at 4, 7 and 12 days post-hatching 31 7. The magnitude of r e v e r s a l responses t o taps of d i f f e r e n t i n t e n s i t i e s at 4, 7 and 12 days post-hatching . . . . 33 8. The p r o p o r t i o n of worms responding t o repeated s t i m u l i with r e v e r s a l s during h a b i t u a t i o n t r a i n i n g 38 9. The magnitude of r e v e r s a l responses during h a b i t u a t i o n t r a i n i n g V v i i w i t h t r a i n s of taps . 4 0 10. P r o p o r t i o n of worms responding t o s t i m u l i with r e v e r s a l s and the magnitude of r e v e r s a l responses before and a f t e r h a b i t u a t i o n t r a i n i n g and f o l l o w i n g the d i s h a b i t u a t i n g stimulus . . . 4 2 11. P r o p o r t i o n of worms responding with r e v e r s a l s d u r i n g recovery from response decrement from h a b i t u a t i o n t r a i n i n g at 4, 7 and 12 days post-hatching 47 12. The magnitude of r e v e r s a l responses during recovery from h a b i t u a t i o n at 4, 7 and 12 days post-hatching 49 13. The e f f e c t of h a b i t u a t i o n t r a i n i n g with t a i l - t o u c h on i n h i b i t i o n of the r e v e r s a l response t o tap by t a i l - t o u c h . .53 v i i i A c k n o w l e d g e m e n t s F i r s t o f a l l , I w o u l d l i k e t o t h a n k my s u p e r v i s o r C a t h y R a n k i n f o r h e r a d v i c e , good humor and s u p p o r t t h r o u g h o u t t h e work o n t h i s p r o j e c t . T h a n k s a r e due f o r e x c e l l e n t a d v i c e a n d c r i t i c i s m t o Don W i l k i e a n d P e t e r G r a f who s e r v e d o n my t h e s i s c o m m i t t e e . I a l s o w o u l d l i k e t o e x p r e s s my g r a t i t u d e t o o t h e r p e o p l e i n t h e l a b who d i s c u s s e d v a r i o u s a s p e c t s o f t h i s work w i t h me: B r e t t B r o s t e r , Shannon C e r n i u k , B i l l M a h , S t e v e W i c k s a n d T h e r e s a M a r i o n . L a s t b u t n o t l e a s t , I ' d l i k e t o t h a n k Sean f o r h o u r s o f t i r e l e s s b a b y - w r a n g l i n g a n d m o r a l e - b o o s t i n g . 1 I n t r o d u c t i o n T h e l o s s o f l e a r n i n g a n d memory a b i l i t i e s w i t h a d v a n c e d age has b e e n t h e s u b j e c t o f s t u d y i n many s p e c i e s . The o b j e c t i v e o f t h i s r e s e a r c h i s t o u n d e r s t a n d t h e e f f e c t s o f a g i n g i n one s p e c i e s a t b e h a v i o r a l , n e u r a l and g e n e t i c l e v e l s . C h a n g e s i n b e h a v i o r a s an o r g a n i s m ages must r e f l e c t c h a n g e s i n c e l l p h y s i o l o g y . Much r e s e a r c h t o t h i s p o i n t has b e e n c o n c e r n e d w i t h t h e l i n k b e t w e e n c h a n g e s i n s p e c i f i c b e h a v i o r s and t h e c h a n g e s i n n e u r o n a l f u n c t i o n w h i c h may p r o d u c e t h e m . However , a s work i n m o l e c u l a r b i o l o g y has i n d i c a t e d , c h a n g e s i n t h e f u n c t i o n o f c e l l s s u c h as n e u r o n s must i n t u r n r e f l e c t c h a n g e s i n gene e x p r e s s i o n . T h u s a m o d e l o f t h e e f f e c t s o f a g i n g o n l e a r n i n g and memory i n w h i c h as many o f t h e s e l e v e l s may be a p p r o a c h e d a t o n c e i s d e s i r a b l e . T h e c o m p l e x i t y o f t h e mammalian n e r v o u s s y s t e m , c o n t a i n i n g many m i l l i o n s o f n e u r o n s , makes i t d i f f i c u l t t o u n d e r s t a n d t h e e f f e c t s o f a g i n g a t a n e u r o n a l l e v e l . One s o l u t i o n t o t h i s p r o b l e m o f n e r v o u s s y s t e m c o m p l e x i t y i s t o u s e a m o d e l s y s t e m . T h e k e y t o t h i s t y p e o f r e s e a r c h i s i n v e s t i g a t i n g t h e r e l a t i o n s h i p b e t w e e n b e h a v i o r a n d i d e n t i f i e d n e u r o n s i n an o r g a n i s m w i t h a s i m p l e n e r v o u s s y s t e m . A n example o f t h i s a p p r o a c h i s t h e m a r i n e m o l l u s c Aplysia californica, i n w h i c h t h e n e u r o p h y s i o l o g i c a l f u n c t i o n o f i d e n t i f i e d n e u r o n s i n c i r c u i t s c o n t r o l l i n g t h e b e h a v i o r s o f g i l l a n d s i p h o n w i t h d r a w a l a r e known. S t u d i e s on a g i n g i n Aplysia h a v e f o u n d l o s s e s i n l e a r n i n g a n d memory. R a t t a n a n d P e r e t z (1981) showed t h a t t h e t h r e s h o l d f o r b e h a v i o r a l r e s p o n s e t o g i l l 2 s t i m u l a t i o n was s i g n i f i c a n t l y higher i n o l d Aplysia than i n young, mature animals. Furthermore they found t h a t o l d Aplysia habituated more r a p i d l y than the younger animals. In a d d i t i o n , these o l d animals d i d not e x h i b i t d i s h a b i t u a t i o n when a neuron (L7) i d e n t i f i e d as one that produces d i s h a b i t u a t i o n i n younger animals was stimulated. Based on these r e s u l t s , Peretz (1989) suggested t h a t i n d i v i d u a l neurons age d i f f e r e n t i a l l y , a f f e c t i n g the behaviors they u n d e r l i e i n d i f f e r e n t ways. Thus he proposed t h a t some b e h a v i o r a l processes/might be expected t o change with age and others not. v The s t u d i e s with Aplysia have advanced our understanding of some of the p h y s i o l o g i c a l processes underlying aging. However, i f we are t o understand how aging i s c o n t r o l l e d at the l e v e l of the genome we must use models' i n which the genetics can be understood as i n as much d e t a i l as p o s s i b l e . Caenorhabditis elegans, a small f r e e - l i v i n g ( n o n - p a r a s i t i c ) nematode has been widely used as a genetic model i n the study of anatomy, development and behavior. Because the neuroanatomy of C. elegans i s simple, c o n t a i n i n g only 302 neurons, the r e l a t i o n s h i p between gene and neuron f u n c t i o n may be narrowly determined. The neuroanatomical map of t h i s nematode has been d e s c r i b e d completely (White, Southgate, Thompson, & Brenner, 1986; G h a l f i e , 1984). Furthermore, the functions of neural c i r c u i t s u n d e r l y i n g c e r t a i n behaviors such as the touch withdrawal c i r c u i t have been demonstrated, e s t a b l i s h i n g the l i n k between behavior and anatomy ( C h a l f i e , S u lston, White, Thompson, Southgate, & Brenner, 1985; 3 C h a l f i e & Au, 1989). C. elegans i s a very simple m u l t i c e l l u l a r organism: at maturation, about 3 days a f t e r hatching, the worm has approximately 1000 somatic c e l l s . The complete developmental l i n e a g e of each somatic c e l l has been mapped us i n g Nomarski microscopy on l i v i n g worms (Sulston, Schierenberg, White, & Thompson, 1983). In a d d i t i o n , the genetics of t h i s nematode are r e l a t i v e l y simple, with only 8 x 10? n u c l e o t i d e p a i r s (approximately h a l f the s i z e of the genome of Drosophila) i n s i x h a p l o i d chromosomes (Sulston & Brenner, 1974; Nigon, 1949). The combined e f f o r t s of many l a b o r a t o r i e s using a v a r i e t y of c l a s s i c a l and molecular genetic techniques have produced a map of over 95% of the C. elegans genome (Coulson, Sulston, Brenner, & Karn, 1986; Hodgkin, Edgley, Riddle, & A l b e r t s o n , 1988). C. elegans e x i s t s p r i m a r i l y as a hermaphrodite, producing both eggs and sperm and.reproducing by s e l f - f e r t i l i z a t i o n . This mode of r e p r o d u c t i o n allows t r u e breeding of mutants. However, mutants with o n l y male r e p r o d u c t i v e systems occur at a low r a t e (Hodgkin, H o r v i t z , & Brenner, 1979; Rose & B a i l l i e , 1979); these males mate with the hermaphrodites and f e r t i l i z e the hermaphrodites' eggs. Thus both homozygous and heterozygous o f f s p r i n g can be produced. Many mutants have been i s o l a t e d . These mutant l i n e s are preserved by f r e e z i n g the l a r v a i n l i q u i d n i t r o g e n . C h a r a c t e r i z e d mutant worms are a v a i l a b l e from a c e n t r a l l i b r a r y of mutants i n the Caenorhabditis Genetics Center at the U n i v e r s i t y of M i s s o u r i , Columbia (Hodgkin, Edgley, Riddle, & A l b e r t s o n , 1988). 4 T h i s b o d y o f b a c k g r o u n d knowledge o n t h e a n a t o m y , d e v e l o p m e n t a n d g e n e t i c s o f C . elegans make i t an e x c e l l e n t c a n d i d a t e m o d e l s y s t e m f o r t h e s t u d y o f b i o l o g i c a l mechani sms u n d e r l y i n g a n y p r o c e s s . H o w e v e r , i f C. elegans i s t o be u s e d as a m o d e l s y s t e m f o r t h e s t u d y o f a g e - r e l a t e d d e f i c i t s i n l e a r n i n g a n d memory, t h e r e must be e v i d e n c e t h a t C. elegans e x p r e s s e s a v a r i e t y o f o b s e r v a b l e b e h a v i o r s a n d l e a r n i n g p r o c e s s e s . L i k e i t s a n a t o m y , t h e b e h a v i o r o f C . elegans i s s i m p l e y e t , v a r i e d e n o u g h t o p r o v i d e a b r o a d r a n g e o f p o s s i b l e a r e a s o f s t u d y . C. elegans l o c o m o t e s b y p r o d u c i n g r h y t h m i c c o o r d i n a t e d c o n t r a c t i o n s o f t h e v e n t r a l a n d d o r s a l m u s c l e s . T h e s e c o n t r a c t i o n s c a u s e u n d u l a t o r y movements down t h e l e n g t h o f t h e b o d y i n t h e d o r s o v e n t r a l p l a n e . When m o v i n g o n a f i r m s u r f a c e s u c h as a g a r , t h e worms l i e o n t h e i r s i d e s . T h e y c a n move t h e i r h e a d s l a t e r a l l y and d o r s o v e n t r a l l y a n d c a n r o t a t e t h e i r b o d i e s o v e r 180 d e g r e e s . The worm r e s p o n d s t o a number o f s t i m u l i i n c l u d i n g t o u c h a n d v i b r a t i o n by c h a n g i n g d i r e c t i o n a n d swimming b a c k w a r d f o r some d i s t a n c e ( S u l s t o n e t a l . , 1975; C h a l f i e & S u l s t o n , 1981; R a n k i n , B e c k , & C h i b a , 1 9 9 0 ) . C. elegans c a n a l s o move up o r down g r a d i e n t s o f c h e m i c a l c o n c e n t r a t i o n , o s m o l a r i t y , a n d t e m p e r a t u r e (Ward , 1973; H e d g e c o c k & R u s s e l , 1 9 7 5 ) . M a t u r e worms o f e a c h s e x have s p e c i f i c s e x u a l b e h a v i o r s : t h e h e r m a p h r o d i t e s l a y eggs ( a l r e a d y f e r t i l i z e d w i t h t h e i r own s p e r m ) , w h e r e a s t h e m a l e s ( w h i c h p r o d u c e o n l y sperm) e n g a g e i n a c o m p l e x s e t o f m a t i n g b e h a v i o r s when t h e y come i n c o n t a c t w i t h a n a d u l t h e r m a p h r o d i t e (Wood, 1 9 8 8 ) . I 5 Sensory-motor behaviors i n C. elegans mediated by the touch withdrawal r e f l e x c i r c u i t are p a r t i c u l a r l y w e l l understood (Sulston e t a l . , 1975; C h a l f i e & Sulston, 1981). When touched on the t a i l w ith a small h a i r , the worm responds by moving forward; to a l i g h t touch across the head, i t moves backward. On the bas i s of e l e c t r o n microscopic r e c o n s t r u c t i o n s and the a n a l y s i s of l a s e r a b l a t i o n s and nervous system mutants, C h a l f i e and co l l e a g u e s (1985) proposed a simple r e f l e x model f o r t a i l touch induced forward locomotion and f o r head touch induced backward locomotion. They determined that the touch c i r c u i t s c o n s i s t of touch receptors connecting t o interneurons which i n tu r n connect t o motorneurons. Recent work on the behavioral p l a s t i c i t y expressed by t h i s organism has confirmed t h a t C. elegans has a r i c h r e p e r t o i r e of l e a r n i n g processes (Rankin & Chiba, 1988; Rankin, Beck, & Chiba, 1990; Kumar, Williams, C u l o t t i , & van der Kooy, 1989). Rankin, Beck and Chiba (1990) showed that the tap withdrawal r e f l e x i n C. elegans e x h i b i t s the major forms of no n - a s s o c i a t i v e l e a r n i n g : h a b i t u a t i o n , d i s h a b i t u a t i o n , and s e n s i t i z a t i o n . The advantage of working with the tap withdrawal r e f l e x i s that the touch withdrawal c i r c u i t , d efined by C h a l f i e and colleagues (1985), has been shown t o be re s p o n s i b l e f o r the tap withdrawal r e f l e x as w e l l (Rankin & C h a l f i e , 1989). With repeated p r e s e n t a t i o n s of a s i n g l e t a c t i l e stimulus such as a s i n g l e tap or t r a i n of taps, the r e v e r s a l response habituates. A f t e r h a b i t u a t i o n t r a i n i n g , the r e v e r s a l response recovers over about 20 t o 30 min t o b a s e l i n e l e v e l s (Rankin & Broster, 1990). The p r e s e n t a t i o n of a novel or noxious stimulus such as a 60 V shock immediately a f t e r the h a b i t u a t i o n of the r e v e r s a l response produces d i s h a b i t u a t i o n or a p a r t i a l recovery t o approximately 50% of the b a s e l i n e response r a t e (Rankin, Beck, & Chiba, 1990; Rankin & B r o s t e r , i n p r e s s ) . In a naive worm, pres e n t a t i o n of a strong stimulus (a t r a i n of taps) produces a higher than b a s e l i n e reponse t o a s i n g l e tap; the response t o tap i s s e n s i t i z e d (Rankin, Beck, & Chiba, 1990). F i n a l l y , memory f o r extensive h a b i t u a t i o n t r a i n i n g l a s t s f o r at l e a s t 24 h, showing that C. elegans i s capable of long-term memory (Rankin, Beck, & Chiba, 1990). Work by van der Kooy and h i s col l e a g u e s (Kumar et a l . , 1989) i n d i c a t e s t h a t C. elegans may be capable o f a s s o c i a t i v e l e a r n i n g i n a taste-approach/avoidance paradigm. However, the neural c i r c u i t u n d e r l y i n g t a s t e - r e l a t e d behaviors has not yet been defined. Thus i t i s c l e a r t h a t C. elegans possesses a range of l e a r n i n g and memory c a p a b i l i t i e s , making i t a promising candidate f o r the i n v e s t i g a t i o n of the b i o l o g i c a l mechanisms u n d e r l y i n g a g e - r e l a t e d d e f i c i t s i n l e a r n i n g and memory. The f i r s t step i n the beh a v i o r a l a n a l y s i s of aging i n C. elegans i s t o determine the normal e f f e c t s of aging on the simple n o n - a s s o c i a t i v e forms of l e a r n i n g already d e s c r i b e d i n C. elegans. Thus, the purpose of the experiments de s c r i b e d here was t o d e f i n e the e f f e c t s of age f i r s t , on b a s e l i n e a c t i v i t y l e v e l s , second, on the tap withdrawal r e f l e x and f i n a l l y , on two forms of no n - a s s o c i a t i v e l e a r n i n g , h a b i t u a t i o n and d i s h a b i t u a t i o n , expressed by the tap withdrawal r e f l e x . In studying h a b i t u a t i o n 7 and d i s h a b i t u a t i o n , I attempted t o i s o l a t e the a g e - r e l a t e d changes by approaching the question of the e f f e c t s of h a b i t u a t i o n t r a i n i n g from s e v e r a l d i f f e r e n t p e r s p e c t i v e s . F i r s t , I examined the dynamics of h a b i t u a t i o n i t s e l f , and the appearance and extent of d i s h a b i t u a t i o n . Next, I examined how recovery from h a b i t u a t i o n changes duri n g aging. F i n a l l y , I examined how aging a f f e c t s the way t h a t h a b i t u a t i o n t r a i n i n g i n t e r a c t s with response competition or i n h i b i t i o n . General Methods Subjects Mature hermaphroditic C. elegans ( s t r a i n N2 from B r i s t o l , England) were used throughout these s t u d i e s . When grown i n l i q u i d c u l t u r e at 20° C, the l i f e - c y c l e of C. elegans i s approximately 21 days (Woods, 1988); however, i n the c o n d i t i o n s maintained d u r i n g these experiments ( s o l i d medium, 20° C) the average l i f e s p a n was 14 t o 16 days. Tests of behavior were performed at 4, 7 and 12 days post-hatching. At 4 days post-hatching worms are at the peak of egg-laying, at 7 days egg-laying i s complete, and by 12 days post-hatching worms are w e l l i n t o the p o s t - r e p r o d u c t i v e p e r i o d . M a t e r i a l s Worms were maintained on Nematode Growth Medium (NGM) a g a r - f i l l e d P e t r i p l a t e s (5 cm diameter) and fed Escherichia coli ( s t r a i n OP50) which was streaked or spotted on the s u r f a c e of the agar. Subjects were maintained and t e s t e d with the methods and apparatus used i n previous b e h a v i o r a l s t u d i e s (Rankin, Beck, & 8 Chiba, 1990). Behavioral observations were made through a stereomicroscope with attached vi d e o r e c o r d i n g equipment. Mechanical and e l e c t r i c a l s t i m u l a t i o n were c o n t r o l l e d by a Grass S88 s t i m u l a t o r . V i b r a t i o n a l s t i m u l i were produced by a mechanical tapper c o n t r o l l e d by an electromagnetic r e l a y ; the mechanical tapper tapped the side of the P e t r i p l a t e h o l d i n g the worm. E l e c t r i c a l shocks were produced using a spanning e l e c t r o d e ; the two wires were placed on e i t h e r s i d e of the animal on the su r f a c e of the agar approximately 2 mm apart. Each shock stimulus c o n s i s t e d of a t r a i n of shocks (each shock was 10 ms i n duration) d e l i v e r e d over 600 ms at a frequency of 10.Hz. Procedure The hermaphroditic C. elegans t h a t were used i n these s t u d i e s were hatched synchronously. To ensure synchrony, mature eg g - l a y i n g worms were s e l e c t e d from the general p o p u l a t i o n and were p l a c e d on an agar p l a t e streaked with E. coli. These worms were permitted t o l a y eggs f o r 3 to 4 h and were then removed from the p l a t e . The eggs hatched i n 9 t o 11 h. At 3 days post-hatching (72 to 84 hours) the maturing worms were placed i n d i v i d u a l l y on numbered agar p l a t e s spotted with E. coli. In experiments where i n d i v i d u a l worms were followed throughout p o s t - r e p r o d u c t i v e development, the worms were t e s t e d a t three chosen ages: 4, 7 and 12 days. Tests were performed on p l a t e s without food. A f t e r each t e s t , the worms were i n d i v i d u a l l y p l a c e d on new p l a t e s with f r e s h E. coli to ensure a c o n s i s t e n t food source and t o prevent confusing the subjects with t h e i r o f f s p r i n g . 9 In experiments where worms were t e s t e d at only one of the t e s t ages, worms were p l a t e d i n d i v i d u a l l y and r a i s e d t o t h a t t e s t age. Worms were r e p l a t e d approximately every 2 days t o maintain i s o l a t i o n . B e h a v i o r a l responses were scored i n s e v e r a l ways depending on the experiment. The r e v e r s a l response (swimming backward) t o v i b r a t i o n a l s t i m u l i (taps) was the c h i e f dependent measure i n these experiments. In order to have been considered a r e v e r s a l t o tap, the response must have occurred w i t h i n 1 s of the stimulus. Any v i s i b l e backward movement (>. .02 mm) was considered a r e v e r s a l and was scored. Both the frequency and the magnitude of these r e v e r s a l s were scored. The magnitude of the r e v e r s a l responses were q u a n t i f i e d by t r a c i n g the r e v e r s a l paths on t o acetate sheets. These t r a c i n g s were then d i g i t i z e d u s ing a B i t Pad Plus d i g i t i z i n g t a b l e t , and the d i g i t i z e d tracings.were measured using MacMeasure software on a Macintosh SE personal computer. S t a t i s t i c a l a n a l y s i s of p r o p o r t i o n data was performed w i t h a Cochran Q t e s t when the design i n v o l v e d repeated measures; otherwise p r o p o r t i o n data were analyzed with chi-squared t e s t s . Magnitude data were analyzed using ANOVAs with F i s h e r ' s post-hoc comparisons when s t a t i s t i c a l s i g n i f i c a n c e was achieved. The l e v e l of s i g n i f i c a n c e was set at alpha = .05 unless the performance of m u l t i p l e t e s t s made i t necessary to adjust the alpha l e v e l downward. 10 Experiment I. Spontaneous locomotor a c t i v i t y In order t o determine whether there were changes i n the b a s e l i n e a c t i v i t y during aging that might a f f e c t the expression of p l a s t i c i t y by the tap withdrawal r e f l e x , the l e v e l of spontaneous locomotor a c t i v i t y of the worms during l a t e (post-reproductive) development was measured. The measures of spontaneous locomotor a c t i v i t y were 1) time spent a c t i v e (any forward or backward swimming movement was considered a c t i v i t y ) over a 10 min o b s e r v a t i o n p e r i o d 2) the v e l o c i t y of spontaneous swimming and 3) the number and magnitude of spontaneous r e v e r s a l s w i t h i n the 10-min observa t i o n p e r i o d . Subjects The same 21 hermaphroditic worms were t e s t e d f o r spontaneous a c t i v i t y three times, once at each of the t e s t ages: 4, 7 and 12 days a f t e r hatching. As the average l i f e - s p a n of the s u b j e c t s was roughly 14 to 16 days under these c o n d i t i o n s and many worms d i e before t h i s age, 45 worms had t o be t e s t e d at day 4 (35 a t day 7) t o o b t a i n data f o r 21 animals on day 12. These data allowed an i n v e s t i g a t i o n of behavioral d i f f e r e n c e s between worms t h a t d i e d young (between day 4 and day 7 or between day 7 and day 12) and those t h a t s u r v i v e d 12 days. Procedure Spontaneous locomotor a c t i v i t y was observed over a 10 min p e r i o d ; the time a c t i v e was determined by measuring the amount of time the worm spent swimming (any v i s i b l e forward or backward movement). The v e l o c i t y of movement, measured as the d i s t a n c e 11 t r a v e l l e d over a s p e c i f i c 10-s i n t e r v a l (from 5 min 0 s t o 5 min 10 s i n each observation period) was a l s o c a l c u l a t e d . The number of spontaneous r e v e r s a l s (swimming backward i n the absence of an obvious e x t e r n a l stimulus) was scored over the same p e r i o d . The magnitude of these spontaneous r e v e r s a l s was determined by video stop-frame a n a l y s i s and computer-aided d i g i t i z i n g of r e v e r s a l l e n g t h . R e s u l t s and D i s c u s s i o n A measure of worm length at each t e s t age was made by t r a c i n g the worms' magnified image (50 X) from the video screen (see F i g u r e 1). A repeated-measures ANOVA with post-hoc comparisons showed t h a t the length of the worms s i g n i f i c a n t l y i n c r e a s e d between day 4 and day 7 but not between day 7 and 12 (F(2,40) = 12.062, E = .0001). Because of t h i s age-dependent change i n s i z e , the r e v e r s a l magnitudes were standardized by d i v i d i n g them by the worm's own length at that age so that responses by worms at d i f f e r e n t ages c o u l d be d i r e c t l y compared (Chiba & Rankin, 1990). The spontaneous v e l o c i t y and the mean magnitude of spontaneous r e v e r s a l s were found t o change with age. However, time a c t i v e and the number of spontaneous r e v e r s a l s d i d not change with age (see Figures 2A and B). The time spent a c t i v e , measured over a 10-min p e r i o d f o r each worm at each t e s t age, was analyzed with a repeated-measures ANOVA; no s i g n i f i c a n t d i f f e r e n c e was found among the t e s t ages (F(2,40) = 2.675, £) = .0812). However, spontaneous v e l o c i t y , or the speed with which the. worms moved, d i d change with age. Spontaneous v e l o c i t y was measured as the t o t a l d i s t a n c e 12 F i g . 1. Worm length +/- SEM (n = 21) magnified 5Ox and measured from a video screen at 4 days, 7 days and 12 days post-hatching. WORM LENGTH (mm) 3 U l _ l _ U l _l 14 F i g . 2A. The percent time spent a c t i v e +/- SEM measured over a 10 min o b s e r v a t i o n p e r i o d at each of the three t e s t ages, 4, 7 and 12 days of age (n = 21). B. The v e l o c i t y of spontaneous movement (iran/s) +/- SEM was measured as the d i s t a n c e t r a v e l l e d both backward and forward d u r i n g 10 s i n the 10 min observation p e r i o d at each of the three t e s t ages, 4, 7 and 12 days post-hatching (n = 21). DISTANCE (mm) PERCENT TIME ACTIVE / TIME (s) oo 10 *» o» oo 16 Fig. 3A. The number of spontaneous reversals (swimming backward in the absence of any obvious external stimuli) during the 10 min observation period at each of the three test ages, 4, 7 and 12 days post-hatching. B. Mean spontaneous reversal magnitude was calculated for each worm at each age by taking the mean of the magnitude of a l l the spontaneous reversals that worm exhibited during the 10 min observation period and dividing i t by the worm's body length. REVERSAL MAGNITUDE /WORM LENGTH m o o o to o o O) o oo NUMBER OF REVERSALS > to o u o o 18 t r a v e l l e d i n a s p e c i f i c 10 s peri o d (from 5 min 0 s t o 5 min 10 s) duri n g the 10 min observation p e r i o d . V e l o c i t y i s expressed as d i s t a n c e t r a v e l l e d (mm) / time ( s ) . I t was found t h a t a t day 12 worms swam s i g n i f i c a n t l y more slowly than at day 4 or 7 (F(2,40) = 23.158; rj = .0001) . Spontaneous r e v e r s a l s were analyzed both i n terms of the number of spontaneous r e v e r s a l s each worm expressed and the mean magnitude of the r e v e r s a l s each worm expressed (see F i g u r e s 3A and B). The number of spontaneous r e v e r s a l s during the 10 min o b s e r v a t i o n p e r i o d d i d not change with age (F(2,40) = 1.634, rj = .2037). However, the mean magnitude of spontaneous r e v e r s a l s at day 12 were s i g n i f i c a n t l y smaller than at day 4 or 7 (F(2,40) = 16.407, E = .0001). Thus there was a decrease i n the v e l o c i t y of spontaneous movement and i n the magnitude of spontaneous r e v e r s a l s with age. The p o s s i b i l i t y t h a t these changes i n spontaneous movement are a l s o r e f l e c t e d i n the r e v e r s a l response to v i b r a t i o n a l s t i m u l a t i o n w i l l be examined i n the next experiment. Experiment 2. Response to tap and head touch. In a d d i t i o n t o the spontaneous r e v e r s a l s d e s c r i b e d above, worms a l s o show r e f l e x i v e r e v e r s a l s i n response to a v a r i e t y of s t i m u l i . In previous experiments we have shown that i n 4 day o l d a d u l t s , a tap t o the d i s h e l i c i t s a withdrawal response of swimming backward c a l l e d a r e v e r s a l (Rankin, Beck, & Chiba, 1990). In t h i s experiment, a g e - r e l a t e d changes i n the tap withdrawal r e f l e x were 19 examined. The pr o p o r t i o n and magnitude of the r e v e r s a l responses t o tap were measured; i n a d d i t i o n the frequencies of other types of response t o tap ( a c c e l e r a t i o n s and pauses) were scored. F i n a l l y each worm's response t o a l i g h t touch t o the head with a h a i r was t e s t e d . In t h i s context, the head-touch t e s t was administered a f t e r the tap t e s t t o determine whether the worm was capable of a normal r e v e r s a l , as a l l healthy worms reverse t o head-touch ( C h a l f i e e t a l . , 1985). Thus, i f a worm d i d not respond t o a head-touch with a r e v e r s a l , i t was assumed t o be i n c a p a c i t a t e d and was not included i n the r e s u l t s . Although the magnitude of the r e v e r s a l t o head-touch might a l s o change with age i t was not scored because the strength of the head-touch c o u l d not be c o n t r o l l e d because the stimulus was hand-delivered w i t h a f i n e h a i r . Subjects At 4, 7 and 12 days post-hatching, f o l l o w i n g the r e c o r d i n g of spontaneous a c t i v i t y , worms were t e s t e d f o r t h e i r response t o tap and head-touch. Procedure The s i n g l e tap and the head-touch were administered immediately f o l l o w i n g the observation p e r i o d f o r spontaneous a c t i v i t y w i t h a 3 min i n t e r v a l between tap and head touch. Head-touch was administered with a f i n e h a i r t o the head (the r e g i o n o f the pharynx) of the worm. Enough pressure was exerted t o bend the h a i r as i t touched the subject's head; pressure from the h a i r d i d not damage the worms. R e s u l t s and D i s c u s s i o n Both the frequency and the magnitude of the r e v e r s a l response to tap were scored (see Figures 4A and B). When the frequency of r e v e r s a l response was analyzed with a Cochran Q t e s t , no change with age was evident (2(2) = 2.8, rj > .05). However, the magnitude of r e v e r s a l responses to tap, standardized by each worm's le n g t h , was s i g n i f i c a n t l y smaller at day 12 than at day 4 (there was no s i g n i f i c a n t d i f f e r e n c e between day 7 and e i t h e r day 4 or 12; F(2,38) = 9.923, p> = .0003). The o b s e r v a t i o n that r e v e r s a l s t o tap were s m a l l e r at day 12 than at day 4 i s s i m i l a r t o the f i n d i n g i n Experiment 1 t h a t spontaneous r e v e r s a l s were smaller at day 12 than a t day 4. In both cases the magnitude of the r e v e r s a l s changed while the frequency of r e v e r s a l s d i d not. The decrease i n v e l o c i t y of spontaneous movement, i n magnitude of spontaneous and r e f l e x i v e r e v e r s a l at day 12 may have been r e l a t e d t o these worms' approaching deaths. A number of worms d i e d d u r i n g these experiments (22% d i e d between day 4 and 7; 31% d i e d between day 7 and 12). By comparing the behavior of worms which d i e d e a r l i e r t o those t h a t s u r v i v e d t o the day 12 t e s t , we may t e s t t h i s hypothesis. To determine whether d e t e r i o r a t i o n r e l a t e d t o an i n d i v i d u a l ' s l i f e - s p a n a f f e c t e d the spontaneous and r e f l e x i v e a c t i v i t y of the worms, the behavior at day 4 of worms that s u r v i v e d t o 12 days was compared with the behavior of those that d i e d between day 4 and 7 and with the behavior of those that died between day 7 and 12. 21 F i g . 4A. The number of worms responding t o tap with a r e v e r s a l at 4, 7 and 12 days post-hatching (n = 21). B. The magnitude of r e v e r s a l s t o tap ( i n c l u d i n g only those worms t h a t responded with a r e v e r s a l ) d i v i d e d by each worm's body le n g t h . MAGNITUDE REVERSAL /WORM LENGTH m o o o (Jl o U l _l NUMBER OF WORMS REVERSING 23 In a d d i t i o n , the behavior at day 7 of worms th a t s u r v i v e d t o 12 days was compared with the behavior of those t h a t d i e d between day 7 and 12. Only those behaviors that changed with age (the spontaneous v e l o c i t y of movement, see Figure 5A; the mean magnitude of spontaneous r e v e r s a l s , see Figure 5B; and the magnitude of the r e v e r s a l response to tap, see F i g u r e 5C) were examined. At day 7, worms that d i e d before day 12 moved more slo w l y than worms t h a t s u r v i v e d t o day 12 (day 7: t(32) = -3.399, rj = .0019). There was no apparent r e l a t i o n s h i p between spontaneous v e l o c i t y and l i f e - s p a n at the day 4 t e s t (day 4: F(2,42) = 1.269, r> = .2917). N e i t h e r the mean magnitude of spontaneous r e v e r s a l s (day 4: F(2,42) = .355, £ = .703; day 7: t(33) = 1.656, rj = .1072), nor the magnitude of r e v e r s a l response to tap was r e l a t e d t o time of death at e i t h e r day 4 or day 7 (day 4: F(2,42) = .355, p. = .703; day 7: t(33) = 1.656, p. = .1072). Whether the change i n spontaneous v e l o c i t y occurred because of approaching death i s not c l e a r ; however t h i s p o s s i b i l i t y must be considered. One p o s s i b l e explanation f o r the decrease i n response magnitude i n 12 day o l d worms i s that there might be a change i n sensory a b i l i t y w ith age; a lower s e n s i t i v i t y i n o l d e r worms c o u l d produce a decrease i n the s i z e of the responses to t a c t i l e s t i m u l a t i o n . An examination of the r e v e r s a l r e f l e x i n response t o a s e r i e s of taps graded i n i n t e n s i t y (beginning with a very weak stimulus) at each of the three t e s t ages might help t o c l a r i f y t h i s i s s u e . 24 F i g . 5. The performance of worms that s u r v i v e d u n t i l day 12 ( > D12; n = 21) compared with the performance of worms t h a t d i e d between day 4 and day 7 (4D - 7D; n = 10) and day 7 and day 12 (7D - 12D; n = 14). D4 TEST = performance at day 4; D7 TEST = performance at day 7. A. Spontaneous v e l o c i t y +/- SEM shown comparing worms with d i f f e r e n t l i f e - s p a n s . B. Mean magnitude of spontaneous r e v e r s a l s +/- SEM ( d i v i d e d by worm length) shown comparing worms with d i f f e r e n t l i f e - s p a n s . C. Magnitude of r e v e r s a l response t o tap +/- SEM ( d i v i d e d by worm length) shown comparing worms with d i f f e r e n t l i f e - s p a n s . 0.35-1 ^ 0.30-B 4D-7D 7D-1 2D L I F E - S P A N >12D 4D T E S T 7D T E S T 1.0-1 0 . 0 4D - 7D 7D - 12D L I F E - S P A N > 12D D4 T E S T D7 T E S T UJ Q 2 I -< z 5 UJ 1 . 5 n 1.0-< DZ UJ > UJ DZ DZ O 0.5-0 . 0 4D • 7D 7D - 12D L I F E - S P A N > 12D D4 T E S T D7 T E S T 26 Experiment 3. Graded response. In t h i s experiment, the e f f e c t s of aging on the responses t o taps of d i f f e r e n t i n t e n s i t i e s were examined. The tap i n t e n s i t i e s chosen were ones which, during p i l o t s t u d i e s , induced r e v e r s a l s of d i f f e r e n t magnitudes i n 4 day o l d worms. Subjects Twenty naive worms were t e s t e d at each age f o r a t o t a l of 60 worms. Procedure Worms were placed i n d i v i d u a l l y on t e s t p l a t e s with a smal l amount of E.coli ( s t r a i n OP50) 24 h before t e s t i n g . Tap i n t e n s i t y was a l t e r e d by changing the voltage from the Grass S-88 s t i m u l a t o r t o the electromagnetic r e l a y which c o n t r o l l e d the tapper. The o b j e c t i v e i n s e l e c t i n g the s t i m u l i i n t e n s i t i e s was t o choose ones t h a t c o n s i s t e n t l y evoked r e v e r s a l s , yet covered a range of i n t e n s i t i e s from the strong tap used i n other experiments i n t h i s t h e s i s t o much weaker taps that might evoke sma l l e r r e v e r s a l responses. The lowest i n t e n s i t y voltage (38 V) produced a b a r e l y perceptable tap, while the highest i n t e n s i t y v o l t a g e (60 V) produced a strong tap and was the i n t e n s i t y used i n the other experiments i n t h i s t h e s i s . An intermediate v o l t a g e of 40 V produced a tap of intermediate i n t e n s i t y . The taps were administered i n d i v i d u a l l y t o worms at 10 min i n t e r v a l s . In s t u d i e s of recovery from h a b i t u a t i o n , Rankin and B r o s t e r ( i n press) showed t h a t taps administered at 10 min i n t e r v a l s d i d not produce s i g n i f i c a n t response decrement i n worms t h a t were 4 days 27 o l d . Of the 20 worms t e s t e d at each age (4, 7 and 12 days po s t - h a t c h i n g ) , 10 worms re c e i v e d the s t i m u l i i n ascending order of i n t e n s i t y (38, 40 and 60 V) and 10 worms r e c e i v e d the s t i m u l i i n descending order of i n t e n s i t y (60, 40 and 38 V ) . I n t e n s i t y of s t i m u l a t i o n , not order of prese n t a t i o n , was the manipulation of i n t e r e s t here; t h e r e f o r e i f p o s s i b l e i t would be app r o p r i a t e t o pool the data across order of pr e s e n t a t i o n w i t h i n each age. However, i f the order of pre s e n t a t i o n (ascending or descending) had an e f f e c t on the responses, that would i n d i c a t e t h a t the data from the ascending and descending orders of p r e s e n t a t i o n should be cons i d e r e d s e p a r a t e l y . At each age, the r e v e r s a l responses were compared with a two-factor (Order x I n t e n s i t y ) repeated-measures ANOVA t o t e s t f o r an e f f e c t of the order of p r e s e n t a t i o n or an i n t e r a c t i o n between the order of p r e s e n t a t i o n and stimulus i n t e n s i t y . At the ages at which there was no e f f e c t of the order of p r e s e n t a t i o n and no i n t e r a c t i o n between order and i n t e n s i t y , the data from the ascending and descending orders of p r e s e n t a t i o n were pooled. The magnitude of the r e v e r s a l s at each age f o r d i f f e r e n t i n t e n s i t i e s were analyzed with a repeated-measures ANOVA. Only those r e s p o n s e s i t h a t were r e v e r s a l s were i n c l u d e d i n the a n a l y s i s of magnitude because the o b j e c t i v e of t h i s experiment was s p e f i c a l l y t o r e v e a l the e f f e c t of i n t e n s i t y on the magnitude of r e v e r s a l response. Non-reversals (pauses, a c c e l e r a t i o n s , no change i n behavior or no response: approximately 5% of a l l responses) were replaced with the mean of the group f o r the 28 purposes of the s t a t i s t i c a l a n a l y s i s . At each t e s t age, 4, 7 and 12 days post-hatching, the numbers of r e v e r s a l s t o taps of d i f f e r e n t i n t e n s i t i e s was analyzed with a Cochran Q t e s t . R e s u l t s and D i s c u s s i o n To determine whether the data from the groups which r e c e i v e d the s t i m u l i i n ascending and descending orders c o u l d be pooled together, the response magnitudes from worms t h a t r e c e i v e d the s t i m u l i i n ascending order were compared with the response magnitudes from those worms that r e c e i v e d the s t i m u l i i n descending order across the three stimulus i n t e n s i t i e s at each t e s t age (4, 7 and 12 days post-hatching). To c o n t r o l experiment-wise e r r o r r a t e , the alpha l e v e l (.05) was d i v i d e d by the number of t e s t s t h at might be performed on the data from each age (3 t e s t s : one i n i t i a l comparison of the orders of s t i m u l a t i o n , and p o s s i b l y two follow-up t e s t s , one f o r each o r d e r ) ; .05 / 3 = .016. At day 4 and day 7, there was an e f f e c t of stimulus i n t e n s i t y , but no e f f e c t of stimulus order nor any i n t e r a c t i o n between stimulus order and i n t e n s i t y (day 4: Order: F(l,18) = 1.855, rj < .19; I n t e n s i t y : F(2,36) = 5.725, p. = .0069; Order x I n t e n s i t y : F(2,36) = .65, rj = .5283; day 7: Order: F(l,18) = .922, p. = .3497; I n t e n s i t y : F(2,36) = 5.511, p_ = .0082; Order x I n t e n s i t y : F(2,36) = .367, p. = .6955). However, at 12 days the order of s t i m u l a t i o n a f f e c t e d the response of the worms t o s t i m u l i of d i f f e r e n t i n t e n s i t i e s i n a d d i t i o n t o a s i g n i f i c a n t e f f e c t of i n t e n s i f y (day 12: Order: F(l,18) = 7.152, p_ = .0155; I n t e n s i t y : F(2,36) = 11.601, p_ = .0001; Order x I n t e n s i t y : 29 .0004). Therefore, while data from ascending and descending orders of p r e s e n t a t i o n from day 4 and 7 worms were pooled w i t h i n each age, the data from day 12 were not. At day 12, the data from ascending and descending orders of p r e s e n t a t i o n were analyzed s e p a r a t e l y . Response Frequency. As seen i n Figures 6A and B, at days 4 and 7 no change was evident i n number of worms responding w i t h d i f f e r e n t stimulus i n t e n s i t i e s (day 7: 0.(2) =2.4; JJ > .05). At day 12, the data from worms th a t r e c e i v e d the s t i m u l i i n ascending and descending orders were considered s e p a r a t e l y (see F i g u r e 6C). No change i n the number of worms responding was seen i n e i t h e r group (ascending: 0.(2) = 1.2, JD > .05; descending: 0.(2) = 2.0, p_ > .05). Since these s t i m u l i were s e l e c t e d because they a l l c o n s i s t e n t l y produced r e v e r s a l responses, i t i s not s u r p r i s i n g t h a t no change i n frequency of response with i n t e n s i t y was observed. I t i s p o s s i b l e that with the a d d i t i o n of lower i n t e n s i t y s t i m u l i nearer t o the sensory t h r e s h o l d , more of a decrease i n the frequency of r e v e r s a l responses would be seen at a l l ages. Response Magnitude. At day 4 (see Figure 7A), worms responded w i t h s i g n i f i c a n t l y smaller r e v e r s a l s t o a 38 V tap than t o e i t h e r a 40 or 60 V tap (F(2,38) = 5.833, rj = .0062). At day 7 (see Fig u r e 7B), worms responded with s i g n i f i c a n t l y l a r g e r r e v e r s a l s t o a 60 V tap than e i t h e r a 40 or 38, V tap (F(2,38) = 7.791, r> = .0015). At day 12, the data from worms th a t r e c e i v e d the s t i m u l i i n ascending and descending orders were considered s e p a r a t e l y . F i g . 6. The number of worms that responded t o taps of d i f f e r e n t i n t e n s i t i e s with r e v e r s a l s at 4, 7 and 12 days post-hatching (n = 20 at each age). At days 4 and 7, the data from worms t h a t r e c e i v e d the s t i m u l i i n d i f f e r e n t orders were pooled, while at day 12 the data from worms that r e c e i v e d the s t i m u l i i n ascending order (ASCENDING; n = 10) and the data from worms t h a t r e c e i v e d the s t i m u l i i n descending order (DESCENDING; n = 10) are shown s e p a r a t e l y . NUMBER OF WORMS REVERSING O NUMBER OF WORMS REVERSING 03 NUMBER OF WORMS REVERSING > 32 F i g . 7. The magnitude of r e v e r s a l responses +/- SEM t o taps of d i f f e r e n t i n t e n s i t i e s at 4, 7 and 12 days post-hatching (n = 20 at each age). The magnitude of r e v e r s a l s was d i v i d e d by each worm's body l e n g t h . Only those responses t h a t were r e v e r s a l s were i n c l u d e d i n the a n a l y s i s . At days 4 and 7, the data from worms t h a t r e c e i v e d the s t i m u l i i n d i f f e r e n t orders were,pooled, while at day 12 the data from worms that r e c e i v e d the s t i m u l i i n ascending order (ASCENDING; n = 10) and the data from worms th a t r e c e i v e d the s t i m u l i i n descending order (DESCENDING; n = 10) are shown s e p a r a t e l y . "* o > o m z o o m g z z co co 34 Day 12 worms (see Figure 7C) that r e c e i v e d s t i m u l a t i o n i n ascending order (38, 40 and 60 V) d i d not show a graded response t o s t i m u l i of d i f f e r e n t i n t e n s i t i e s (F(2,18) = 1.77, p_ = .1986). However, day 12 worms that r e c e i v e d the s t i m u l i i n descending order (60, 40 and 38 V) d i d respond with s i g n i f i c a n t l y l a r g e r r e v e r s a l s t o the 60 V tap than t o e i t h e r the 38 or 40 V taps (F(2,18) = 17.713, p. = .0004). This f i n d i n g suggests t h a t day 12 worms, l i k e day 4 and 7 worms, are capable of responding t o s t i m u l i of d i f f e r e n t i n t e n s i t i e s with graded responses, but t h a t the e f f e c t of repeated s t i m u l a t i o n may mask t h e . e f f e c t of stimulus i n t e n s i t y even when the s t i m u l i are administered at 10-min i n t e r v a l s . This response decrement was evident o n l y i n the ascending order of s t i m u l a t i o n , p o s s i b l y because the response magnitude t o the l a s t stimulus of the descending order (38 V tap) was alre a d y so small that any response decrement was l o s t i n a f l o o r e f f e c t . I t may be th a t o l d e r worms are more s u s c e p t i b l e t o h a b i t u a t i o n t r a i n i n g than younger worms. I f so, day 12 worms might be expected t o e x h i b i t a f a s t e r r a t e of response decrement du r i n g h a b i t u a t i o n t r a i n i n g . Experiment 4. Habituation and d i s h a b i t u a t i o n . The e f f e c t s of aging on h a b i t u a t i o n and d i s h a b i t u a t i o n i n C. elegans were examined at each of the three t e s t ages, 4 days, 7 days and 12 days post-hatching. 35 Subjects 20 naive worms were used at each of the three t e s t ages (4, 7 and 12 days) f o r a t o t a l of 60 worms. Procedure T r a i n s of taps (1 t r a i n = 6 taps at a frequency of 10 Hz) were the s t i m u l i used i n both the h a b i t u a t i o n / d i s h a b i t u a t i o n experiment and the recovery from h a b i t u a t i o n experiment because t h i s stimulus was found t o produce l a r g e r responses than the s i n g l e tap (Chiba & Rankin, 1990) and thus a f f o r d e d a l a r g e response range i n which changes i n p l a s t i c i t y might be observed. In the present study, at day 4 the mean r e v e r s a l response to tap was 1.275 ± .141 ( i SEM) w h ile the mean response to a t r a i n of taps was 2.005 t .157. T r a i n s of taps have been used i n our other s t u d i e s of n o n - a s s o c i a t i v e l e a r n i n g (Rankin, Beck, & Chiba, 1990; Rankin & B r o s t e r , i n p r e s s ) . In t h i s experiment, 60 t r a i n s of taps were administered a t a 10 s i n t e r s t i m u l u s i n t e r v a l ( I S I ) . Ten seconds a f t e r the l a s t h a b i t u a t i n g stimulus, a 60 V t r a i n of shocks (a stimulus t h a t produces d i s h a b i t u a t i o n i n 4 day o l d s ; Rankin, Beck, & Chiba, 1990) was administered. Within 20 s a f t e r the d i s h a b i t u a t i n g stimulus, 12 more t r a i n s of taps was administered at a 10 s ISI t o t e s t f o r d i s h a b i t u a t i o n . R e s u l t s and D i s c u s s i o n The a n a l y s i s of the frequency and the magnitude of the r e v e r s a l response during h a b i t u a t i o n and d i s h a b i t u a t i o n showed d i f f e r e n t p a t t e r n s of r e s u l t s across ages. 36 Response frequency. The frequency of r e v e r s a l s , scored as the p r o p o r t i o n of worms responding t o each stimulus with a r e v e r s a l , showed t h a t both h a b i t u a t i o n and d i s h a b i t u a t i o n were evident at 4, 7 and 12 days of age (see Figure 8 and 10A) . A c c e l e r a t i o n s (approximately 3% of the responses) were omitted from the a n a l y s i s and t r e a t e d as missing data. Cochran Q t e s t s at each age (4, 7 and 12 days of age) confirmed that at each age there were s i g n i f i c a n t changes across the i n i t i a l response, h a b i t u a t i o n ( l a s t response of the h a b i t u a t i o n t r a i n i n g ) and d i s h a b i t u a t i o n ( f i r s t response a f t e r the d i s h a b i t u a t i n g stimulus) (day 4: 0.(2) = 10.64, p_ < .02; day 7: 0.(2) = 7.625, p_ < .05; day 12: 0.(2) = 21.375, p_ < .001). However, as seen i n Figure 8, the p a t t e r n of responding d u r i n g h a b i t u a t i o n t r a i n i n g d i d appear d i f f e r e n t at day 7. At 4 and 12 days of age, the number of worms responding decreased e a r l y i n the h a b i t u a t i o n t r a i n i n g and remained low throughout. At 7 days of age, the worms appeared t o stop responding e a r l y i n h a b i t u a t i o n t r a i n i n g then begin responding again as the h a b i t u a t i o n t r a i n i n g continued. This f i n d i n g was r e f l e c t e d i n a change i n the frequency of response averaged across a l l 60 h a b i t u a t i o n t r i a l s with age; the mean frequency of response was s i g n i f i c a n t l y greater at day 7 than at day 4 or day 12 (day 4: X = .265 t .026 ; day 7: .606 t .032; day 12: .329 ± .034; F(2,57) = 33.667, p. = .0001) . Response magnitude. In order t o compare response magnitude across ages the r e v e r s a l magnitudes were standardized by expr e s s i n g each response as a percent of the i n i t i a l r e v e r s a l 37 F i g . 8. The p r o p o r t i o n of worms responding to s t i m u l i with r e v e r s a l s during h a b i t u a t i o n t r a i n i n g t o t r a i n s of taps (10 s ISI, 60 stim; n = 20 at each age). PROPORTION WORMS REVERSING PROPORTION WORMS REVERSING DO PROPORTION WORMS REVERSING o o o ro _J_ o i o CD _ 1 _ o 00 _ 1 _ o _ l o o ro i o o 05 _ l _ 00 o _ l o o o ro o o CT> o 00 o _ l 39 F i g . 9. The magnitude of r e v e r s a l responses +/- SEM d u r i n g h a b i t u a t i o n t r a i n i n g with t r a i n s of taps (10 s ISI, 60 s t i m u l i ) . The magnitude of r e v e r s a l responses was expressed as a percent of each worm's response t o the i n i t i a l stimulus i n h a b i t u a t i o n t r a i n i n g . A DAY 4 1 20 n B Z Hi - (/> I— z Z O UJ CL o co CC £ in cc a 1 20 -i 1 00 80 -60 -40 -20 1 20 -i 100 Hf 1 0 20 30 TRIALS DAY 7 40 i 20 — r ~ 30 40 TRIALS DAY 12 50 60 50 60 t 80H UJ GL o w DC JJi U J DC CL 60 -40 -20 -41 F i g . 10. Reversal responses before and a f t e r h a b i t u a t i o n t r a i n i n g with t r a i n s of taps (10 s ISI, 60 s t i m u l i ) and a f t e r d i s h a b i t u a t i o n with e l e c t r i c shock at 4, 7 and 12 days post-hatching (n = 20 at each age). INIT = i n i t i a l response of the h a b i t u a t i o n t r a i n i n g ; HAB = f i n a l response o f the h a b i t u a t i o n t r a i n i n g ; DIS = f i r s t response a f t e r the d i s h a b i t u a t i n g stimulus. A. P r o p o r t i o n of worms responding t o s t i m u l i with r e v e r s a l s before and a f t e r h a b i t u a t i o n t r a i n i n g and f o l l o w i n g the d i s h a b i t u a t i n g s t i m u l u s . B. Magnitude of the r e v e r s a l response +/- SEM before and a f t e r h a b i t u a t i o n t r a i n i n g and f o l l o w i n g the d i s h a b i t u a t i n g s t i m u l u s . Magnitude of r e v e r s a l responses was expressed as a percent of each worm's r e v e r s a l response t o the i n i t i a l stimulus i n h a b i t u a t i o n t r a i n i n g . PROPORTION WORMS REVERSING > 43 response (which was set at 100%) f o r each worm (see Figures 9A, B and C ). A c c e l e r a t i o n responses were not i n c l u d e d i n the a n a l y s i s because they represent d i s c r e t e motor responses t h a t cannot be compared t o r e v e r s a l s ; however, the absence of a response or a pause were i n c l u d e d as scores of zero (Rankin, Beck, & Chiba, 1990). M i s s i n g data points (approximately 3% of the responses) i n repeated measures analyses were replaced by the group and c o n d i t i o n mean (Glass & Hopkins, 1984). Worms at a l l ages showed s i g n i f i c a n t h a b i t u a t i o n (see Fi g u r e 9A, B and C) and d i s h a b i t u a t i o n (see Figure 10B) i n the magnitude of r e v e r s a l response. Repeated measures ANOVA's were performed on each of the three ages. To c o n t r o l family-wise e r r o r r a t e , the alpha l e v e l was reduced from .05 to .05/3 = .016 (Glass and Hopkins, 1984). As seen i n Figure 10B, at a l l ages the i n i t i a l response was s i g n i f i c a n t l y greater than the habituated response and the d i s h a b i t u a t e d response, and the d i s h a b i t u a t e d response was s i g n i f i c a n t l y g r e a t e r than the habituated response (4 days: F(2,38) = 134.358, p_ < .0001; 7 days: F(2,38) = 280.389, rj < .0001; 12 days: F(2,38) = 786.631, E < .0001). I t was not p o s s i b l e t o compare the degree of ha b i t u a t i o n or d i s h a b i t u a t i o n across the ages t e s t e d because the habituated response of worms at a l l ages were small enough that the c h a r a c t e r i s t i c s of any change measured ag a i n s t t h a t low l e v e l of responding may have been l o s t i n a f l o o r e f f e c t . The r a t e of response decrement during h a b i t u a t i o n t r a i n i n g was analyzed by c a l c u l a t i n g the slope of the r e g r e s s i o n l i n e s f o r 44 r e v e r s a l responses t o the f i r s t f i v e s t i m u l i i n the h a b i t u a t i o n t r a i n i n g f o r each worm and then t a k i n g the mean of these slopes f o r each age (day 4 X = -22.125 t 2.375; day 7 X = -25.512 t 1.695; day 12 X = -19.721 £ 2.317). A comparison of the slopes showed t h a t there was no s i g n i f i c a n t change i n the r a t e of h a b i t u a t i o n as a f u n c t i o n of age (F(2,56) = 1.786, rj = .177). While the f i n d i n g s from t h i s experiment seemed t o i n d i c a t e t h a t t h e r e was no change with age i n the r e v e r s a l response magnitude i n h a b i t u a t i o n and d i s h a b i t u a t i o n , there was a change with age i n response vfrequency during h a b i t u a t i o n . A d i s s o c i a t i o n of the response magnitude and response frequency was apparent at day 7, where worms continued t o respond t o s t i m u l i d u r i n g h a b i t u a t i o n t r a i n i n g but responded with small r e v e r s a l s . T h i s d i s s o c i a t i o n may r e f l e c t some change i n the underlying memory mechanisms at th a t age. Experiment 5. Recovery from h a b i t u a t i o n Recovery from h a b i t u a t i o n may be thought of a form of short-term memory; the stronger the memory, the slower the recovery from h a b i t u a t i o n might be. Thus, i f short-term memory changes with age, the r a t e of recovery from h a b i t u a t i o n might a l s o change with age. Subjects Twenty worms were t e s t e d at each of the t e s t ages 4, 7 and 12 days post-hatching f o r a t o t a l of 60 worms. 45 Subjects Twenty worms were t e s t e d at each of the t e s t ages 4, 7 and 12 days post-hatching f o r a t o t a l of 60 worms. Procedure H a b i t u a t i o n was e s t a b l i s h e d by d e l i v e r i n g 60 t r a i n s of taps a t a 10 s ISI; at the end of ha b i t u a t i o n t r a i n i n g , s i n g l e t r a i n s o f taps at 30 s, 10 min, 20 min and 30 min were d e l i v e r e d t o t e s t f o r recovery from h a b i t u a t i o n . This procedure has been used t o demontrate recovery from h a b i t u a t i o n 20 t o 30 min a f t e r the l a s t h a b i t u a t i n g stimulus i n 4 day o l d worms (Rankin & B r o s t e r , 1990, i n p r e s s ) . R e s u l t s and D i s c u s s i o n In the a n a l y s i s of the recovery of the magnitude of the r e v e r s a l response a f t e r h a b i t u a t i o n t r a i n i n g , r e v e r s a l magnitude was stan d a r d i z e d as percent i n i t i a l response as i n the previous experiment. Only the frequency and magnitude data from the 30 min recovery t e s t were s t a t i s t i c a l l y analyzed because the v a r i a n c e i n the intermediate t e s t s (at 30 s, 10 min and 20 min) was too great to be u s e f u l l y i n c l u d e d . The data from a l l t e s t s are d e p i c t e d i n Fig u r e s 11 and 12A, B and C. Response frequency. The frequency of r e v e r s a l s , scored as the p r o p o r t i o n of worms responding t o each stimulus with a r e v e r s a l , showed t h a t both h a b i t u a t i o n and recovery from h a b i t u a t i o n were evident a t 4, 7 and 12 days of age (see Figure 11). Cochran Q t e s t s a t each age comparing the i n i t i a l , h abituated and recovered (30 min post-habituation)'response frequency confirmed t h a t a t 46 F i g . 11. P r o p o r t i o n of worms responding with r e v e r s a l s d u r i n g recovery from response decrement from h a b i t u a t i o n t r a i n i n g (10 s IS I , 60 s t i m u l i ; n = 20 at each age). Tests of recovery were given 30 s, 10 min, 20 min, and 30 min p o s t - h a b i t u a t i o n . INIT = the response t o the f i r s t h a b i t u a t i o n stimulus; HAB = the response t o the l a s t h a b i t u a t i o n stimulus. 47 — i i r—p 1 : i r— INIT HAB 30S 10M 20M 30M 48 F i g . 12. The magnitude of r e v e r s a l responses +/- SEM d u r i n g recovery from h a b i t u a t i o n (n = 20 at each age). The data are expressed as percent i n i t i a l response which f o r each worm was set at 100%. The s o l i d h o r i z o n t a l l i n e at 100% represents t h i s i n i t i a l response during h a b i t u a t i o n t r a i n i n g . HAB = the r e v e r s a l response t o the l a s t h a b i t u a t i o n stimulus i n the t r a i n i n g . Recovery was t e s t e d at 30 s, 10 min, 20 min and 30 min p o s t - h a b i t u a t i o n . 50 each age there were s i g n i f i c a n t changes with the treatments (day 4: 2(2) = 17.231, rj < .001; day 7: 0,(2) = 13.0, rj < .01; day 12: 0.(2) = 19.0, p_ < .001) . Response magnitude. A d i f f e r e n t p a t t e r n i s seen i n the a n a l y s i s of the magnitude of r e v e r s a l responses (see Figures 12A, B and C). A l l ages showed s i g n i f i c a n t h a b i t u a t i o n (day 4: F(2,38) = 23.563, E < .0001; day 7: F(2,38) = 16.953, £ < .0001; day 12: F(2,38) = 3.426, E < .0001). By 30 minutes a f t e r h a b i t u a t i o n t r a i n i n g , 4 day and 7 day worms showed s i g n i f i c a n t recovery of response magnitude over habituated l e v e l s . However, worms t e s t e d a t 12 days post-hatching d i d not show recovery over habituated l e v e l s . The e f f e c t s of recovery on the response magnitude were f u r t h e r examined by comparing the d i f f e r e n c e between habituated response l e v e l s and 30 min po s t - h a b i t u a t i o n response l e v e l s across ages. Experiment 6. I n h i b i t i o n As a f i n a l t e s t of the e f f e c t s of aging on behavior, the i n h i b i t i o n of one a n t a g o n i s t i c r e f l e x by another as d e s c r i b e d by Rankin ( i n press) was examined. F i r s t , the response t o tap alone was t e s t e d . Second, the i n h i b i t i o n of the r e v e r s a l withdrawal i n response t o tap by t a i l - t o u c h was examined. T h i r d , the i n h i b i t i n g stimulus, t a i l - t o u c h , was habituated and the i n t e r a c t i o n between the r e f l e x e s was examined again. The e f f e c t of h a b i t u a t i o n t r a i n i n g on response competition was then compared at the three t e s t ages. 51 Subjects Twenty worms at each age (4, 7 and 12 days) were t e s t e d f o r a t o t a l of 60 worms. Procedure There were three treatments i n t h i s procedure. A l l animals r e c e i v e d a tap alone, a tap preceded w i t h i n 1 s by a t a i l - t o u c h , and t a i l - t o u c h h a b i t u a t i o n t r a i n i n g (2 s ISI; 50 s t i m u l i ) immediately followed by a tap preceded w i t h i n 1 s by a t a i l - t o u c h . The treatments were given i n the same order f o r a l l worms so that the h a b i t u a t i o n t o t a i l - t o u c h d i d not i n t e r f e r e with the other responses. There was a 20 to 30 min i n t e r v a l between a l l t e s t s . In order t o determine the e f f e c t s of the t a i l - t o u c h on the r e v e r s a l t o tap,'worms that d i d not respond t o the s i n g l e tap with a r e v e r s a l were e l i m i n a t e d from the experiment (approximately 10% of worms t e s t e d ) . The r e v e r s a l responses t o the tap were t r a c e d and d i g i t i z e d f o r a l l groups. R e s u l t s and Di s c u s s i o n Response frequency. At each age there was a marked i n h i b i t i o n of the frequency of the r e v e r s a l response t o tap when tap was preceded by t a i l - t o u c h and a decrease i n the i n h i b i t i o n of the frequency of response from t h a t l e v e l when the t a i l - t o u c h was preceded by h a b i t u a t i o n t r a i n i n g with t a i l - t o u c h (see F i g u r e 13A). Response magnitude. The magnitude of r e v e r s a l responses were sta n d a r d i z e d by expressing them as a percent of each worm's r e v e r s a l response t o the s i n g l e tap which was s e t a t 100%. In measuring the e f f e c t s of t a i l - t o u c h and t a i l - t o u c h h a b i t u a t i o n on 52 F i g . 13. The e f f e c t of h a b i t u a t i o n t r a i n i n g on i n h i b i t i o n of the r e v e r s a l response t o tap by t a i l - t o u c h (n = 20 at each age). A. The number of worms r e v e r s i n g t o tap alone (TAP), t a i l - t o u c h f o l l o wed w i t h i n 1 s by tap (TT), and t a i l - t o u c h h a b i t u a t i o n t r a i n i n g ( 2 s ISI, 50 s t i m u l i ) followed w i t h i n 2s by t a i l - t o u c h / t a p . B. The magnitude of r e v e r s a l s +/- SEM to tap alone (TAP), t a i l - t o u c h followed w i t h i n 1 s by tap (TT), and t a i l - t o u c h h a b i t u a t i o n t r a i n i n g (2 s ISI, 50 s t i m u l i ) followed w i t h i n 2 s by t a i l - t o u c h / t a p . The magnitude of the r e v e r s a l s t h a t occurred were expressed as a percent of each worm's response t o the tap alone which was s e t at 100%. PERCENT MAGNITUDE REVERSALS o o o > > > -< •< < - l -si * » ro NUMBER WORMS REVERSING > 1 1 1 O D D > > > -< -< < - i ->l *» ro 54 the magnitude of r e v e r s a l s (see Figure 13B), o n l y animals t h a t reversed under these c o n d i t i o n s were i n c l u d e d i n the a n a l y s i s . Repeated measures ANOVAs at each age showed t h a t the r e v e r s a l magnitude was s i g n i f i c a n t l y lower i n the t a i l - t o u c h / t a p c o n d i t i o n than the tap alone c o n d i t i o n and s i g n i f i c a n t l y higher i n the h a b i t u a t e d t a i l - t o u c h / t a p c o n d i t i o n than i n the t a i l - t o u c h / t a p c o n d i t i o n (day 4: F(2,38) = 48.679, £ = .0001; day 7: F(2,38) = 11.027, p_ = .0002;,day 12: F(2,38) = 19.618, p_ = .0001). C l e a r l y , t a i l - t o u c h i n h i b i t e d both the frequency of r e v e r s a l response and the magnitude of the r e v e r s a l response. H a b i t u a t i o n t r a i n i n g with the t a i l - t o u c h diminished the amount of i n h i b i t i o n ; thus t h e r e was an increase i n both the frequency and the magnitude of the r e v e r s a l response to tap. The observation that worms showed r e v e r s a l s to tap f o l l o w i n g t a i l - t o u c h h a b i t u a t i o n suggests t h a t the h a b i t u a t i o n d i d not simply produce f a t i g u e . From the r e s u l t s of t h i s experiment, one can conclude t h a t even aged worms are capable of h a b i t u a t i o n independent of f a t i g u e e f f e c t s . General Dis c u s s i o n These experiments have demonstrated t h a t i n C. elegans the e f f e c t s of aging may be seen i n spontaneous and r e f l e x i v e behaviors, and more importantly i n changes i n l e a r n i n g and memory. V e l o c i t y of spontaneous movement diminished with age, as d i d the magnitude of spontaneous r e v e r s a l s and the r e v e r s a l response to tap. In a d d i t i o n spontaneous v e l o c i t y of locomotion t e s t e d a t 7 55 days of age was lower i n worms that d i e d before the day 12 t e s t than worms t h a t s u r v i v e d u n t i l day 12. However, the mean magnitude of spontaneous r e v e r s a l s and the magnitude of response t o s i n g l e taps was not r e l a t e d t o time of death. C l e a r l y aging d i d have an e f f e c t on the spontaneous and r e f l e x i v e behaviors. These changes must be kept i n mind when c o n s i d e r i n g the e f f e c t s of aging i n b e h a v i o r a l p l a s t i c i t y . To t e s t the s e n s i t i v i t y t o t a c t i l e s t i m u l i of the aged worms, s t i m u l i of d i f f e r e n t i n t e n s i t i e s were administered. Worms a t a l l ages t e s t e d showed graded responses t o taps of d i f f e r e n t i n t e n s i t i e s . I n t e r e s t i n g l y , the response magnitude of day 12 worms appeared t o decrease during the a d m i n i s t r a t i o n of the three s t i m u l i given at a 10 min ISI. Rankin and B r o s t e r ( i n press) found no evidence of h a b i t u a t i o n when they administered taps at a 10 min ISI t o day 4 worms. I t may be that o l d e r worms are p a r t i c u l a r l y v u l n e r a b l e t o h a b i t u a t i o n . H a b i t u a t i o n t o repeated s t i m u l a t i o n at a 10 s ISI and d i s h a b i t u a t i o n t o 60 V shock were present i n worms at a l l ages and d i d not appear t o change g r e a t l y during p o s t - r e p r o d u c t i v e development. However, at day 7 there appeared t o be a d i s s o c i a t i o n of the p r o b a b i l i t y of response and the magnitude of response. At day 4 and day 12 these measures appeared t o f o l l o w the same p a t t e r n , however i n day 7 worms the p r o b a b i l i t y of response stayed high while response magnitude diminished. I t i s not c l e a r why t h i s change with age i n response frequency d u r i n g h a b i t u a t i o n t r a i n i n g occurred; however a c l o s e r examination of the 56 behavior of worms between the ages t e s t e d (4, 7 and 12 days post-hatching) might provide f u r t h e r information on t h i s phenomenon. The r a t e of h a b i t u a t i o n t o s t i m u l a t i o n at a 10 s ISI d i d not appear t o change with age. This f i n d i n g does not support the suggestion a r i s i n g from Experiment 4 (Graded response) t h a t day 12 worms may be p a r t i c u l a r l y v u l n e r a b l e t o h a b i t u a t i o n . However i t may be t h a t the i n t e r s t i m u l u s i n t e r v a l used i n the h a b i t u a t i o n t r a i n i n g (10 s ISI) was short enough that worms of a l l ages habi t u a t e d q u i c k l y , masking any d i f f e r e n c e i n r a t e of h a b i t u a t i o n . -During the l a t e r part of h a b i t u a t i o n t r a i n i n g ( t r i a l s 50 through 60), the r e v e r s a l magnitude t o v i b r a t i o n a l s t i m u l i when e x h i b i t e d appears t o be grea t e r than the r e v e r s a l magnitude t o the low i n t e n s i t y tap (38 V) whether the low i n t e n s i t y tap was given f i r s t or l a s t (day 12 h a b i t u a t i o n of r e v e r s a l magnitude from t r i a l s 50 t o 60: X = .339 - .073; day 12 r e v e r s a l magnitude t o 38 V tap X = .096 £ .036); thus i t may be that l i t t l e f u r t h e r h a b i t u a t i o n of r e v e r s a l magnitude could be e x h i b i t e d . An examination o f the e f f e c t s of aging on the rate of h a b i t u a t i o n t o longer ISI's might be help c l a r i f y t h i s question. Worms t e s t e d at 4 and 7 days of age showed recovery from h a b i t u a t i o n of both the frequency and response magnitude 30 min p o s t - h a b i t u a t i o n t r a i n i n g , but worms t e s t e d at 12 days d i d not. Thi s d e f i c i t i n recovery might r e f l e c t a p e r s i s t e n c e of h a b i t u a t i o n i n the o l d e r worms. This p e r s i s t e n c e i s u n l i k e l y t o be e x p l a i n e d by a f a c t o r such as more r a p i d exhaustion s i n c e day 57 12 worms with the same ha b i t u a t i o n t r a i n i n g showed d i s h a b i t u a t i o n ( f a c i l i t a t i o n of the habituated response) immediately a f t e r a m i l d e l e c t r i c shock. In a d d i t i o n , h a b i t u a t i o n of t a i l - t o u c h p r i o r t o t e s t s of i n h i b i t i o n helped e s t a b l i s h that h a b i t u a t i o n can be d i s t i n g u i s h e d from simple f a t i g u e e f f e c t s i n worms of a l l ages. Because h a b i t u a t i o n t r a i n i n g t o t a i l - t o u c h i n c r e a s e d the r e v e r s a l response t o tap by r e l e a s i n g i t from t a i l - t o u c h i n h i b i t i o n at each t e s t age, c l e a r l y even aged worms are capable of h a b i t u a t i o n independent of f a t i g u e or response diminishment r e l a t e d t o age. Furthe r s t u d i e s on the dynamics of h a b i t u a t i o n and recovery from h a b i t u a t i o n and on long-term memory i n aged worms may help the understanding of mechanisms underlying the a g e - r e l a t e d changes i n h a b i t u a t i o n . , An assumption un d e r l y i n g the use of simple-system models of l e a r n i n g and memory i s that these simple forms of l e a r n i n g , h a b i t u a t i o n and d i s h a b i t u a t i o n , seen i n so many s p e c i e s , must share some common b i o l o g i c a l mechanisms. I f the u n d e r l y i n g mechanisms are conserved across species, i t may be p o s s i b l e t o make v i a b l e p r e d i c t i o n s about the patterns of l e a r n i n g i n one species based on the patterns of l e a r n i n g i n another. Work i n other species on the e f f e c t s of aging on h a b i t u a t i o n has not been extensive and f i n d i n g s have been mixed. E i s e n s t e i n and colleagues (1990) found t h a t i n human males the ga l v a n i c s k i n response hab i t u a t e d more q u i c k l y i n younger subjects ( e a r l y 20's) than i n o l d e r ones ( l a t e 20's). They r e l a t e d t h i s d e f i c i t with the l o s s of dopamine receptors i n the caudate nucleus observed i n PET 58 s t u d i e s between age 20 and 30. F r a l e y and Springer (1981) found t h a t middle-aged and o l d e r mice (12 to 24 mo) d i d not r e t a i n h a b i t u a t i o n t r a i n i n g f o r as long as 2 month o l d mice. Parsons, Fagan and Spear (1973) found no change i n short-term r e t e n t i o n of h a b i t u a t i o n t r a i n i n g i n o l d age i n r a t s . As d i s c u s s e d e a r l i e r , Rattan and Peretz (1987) found more r a p i d h a b i t u a t i o n i n o l d e r Aplysia and an absence of d i s h a b i t u a t i o n . The r e s u l t s from the present experiments o f f e r l i t t l e support f o r any g e n e r a l i t i e s t h a t might be drawn from the above s t u d i e s . I t i s p o s s i b l e t h a t aged C. elegans habituate more r a p i d l y than younger a d u l t s ; however t h i s suggestion has not y e t been confirmed. Aged C. elegans showed d i s h a b i t u a t i o n a f t e r e l e c t r i c shock; t h i s f i n d i n g does not support the a s s e r t i o n of Rattan and Peretz (1987) t h a t d i s h a b i t u a t i o n i s s p e c i f i c a l l y d i s a b l e d i n o l d e r organisms (although a recent personal communication from Peretz ( A p r i l , 1991) i n d i c a t e d that b e h a v i o r a l experiments on Aplysia have shown t h a t d i s h a b i t u a t i o n may be e x h i b i t e d by o l d e r Aplysia; the d e f i c i t seen formerly appears t o have been dependent on the s p e c i f i c h a b i t u a t i o n t r a i n i n g procedure used). From Experiment 6 (Recovery from/habituation) there i s evidence of a longer r e t e n t i o n of h a b i t u a t i o n which seems t o c o n t r a d i c t the f i n d i n g s of F r a l e y and Springer (1981) t h a t middle-aged and aged mice showed s h o r t e r r e t e n t i o n of h a b i t u a t i o n t r a i n i n g than young mature a d u l t s . C l e a r l y there i s as yet no s i n g l e d e s c r i p t i o n of the e f f e c t s of aging on h a b i t u a t i o n . One of the d i f f i c u l t i e s w i t h comparing these studies i s that methodologies used are 59 d i v e r s e and f u l l parametric examinations of the l e a r n i n g phenomena s t u d i e d have not been done. Further development of a model f o r the e f f e c t s of aging on simple forms of l e a r n i n g such as h a b i t u a t i o n i n C. elegans may help to c l a r i f y the r o l e of aging e f f e c t s i n other s p e c i e s . The s t u d i e s d e s c r i b e d here provide a groundwork f o r f u r t h e r r e s e a r c h on the e f f e c t s of aging on l e a r n i n g and memory i n C. elegans. There are s e v e r a l d i r e c t i o n s i n which t h i s r e s e a r c h c o u l d continue. F i r s t , the mechanisms u n d e r l y i n g the a g e - r e l a t e d changes observed i n h a b i t u a t i o n and recovery from h a b i t u a t i o n might be i n v e s t i g a t e d with s t u d i e s f o c u s s i n g on the parameters of those changes. Second, known mutant s t r a i n s with e f f e c t s on c e r t a i n types of l e a r n i n g or responses might be examined. For example the use of a s t r a i n of worms which i s incapable of locomotion yet s t i l l feeds may a i d i n the development of a l t e r n a t i v e response measures that may be used i n s t u d i e s of l e a r n i n g such as pharyngeal bulb pumping. F i n a l l y g e n e t i s t s i n t e r e s t e d i n i n v e s t i g a t i n g the b i o l o g i c a l mechanisms of aging have begun work on the genetic c o n t r o l s of aging mechanisms i n C. elegans. Johnson and h i s colleagues (Johnson, Friedman, F o l t z , F i t z p a t r i c k , & Shoemaker,1989) chose t o focus on s e a r c h i n g f o r a mutant with an extended l i f e span, with the l o g i c t h a t there are many reasons why a mutant may have a shortened l i f e span, but a mutation producing an extended l i f e span i s more l i k e l y t o be d i r e c t l y r e l a t e d t o the mechanisms underlying aging. Johnson and h i s c o l l e a g u e s (1989) i s o l a t e d and mapped a s i n g l e gene mutation 60 t h a t produces a 60-110% increase i n l i f e span; attempts are underway t o clone t h i s gene. In t h i s s t r a i n of worms, the extended l i f e span i s not due to the extension of any e a r l y developmental stage, but to a lengthening of the p o s t - r e p r o d u c t i v e l i f e span. A s e r i e s of recombinant mutation experiments f o c u s s i n g on genes t h a t modify development have shown t h a t e a r l y development, reproduction and l i f e span are a l l under independent g e n e t i c c o n t r o l (Johnson, 1987). However, i n a l l s t r a i n s , decreased motor a c t i v i t y c o r r e l a t e s with l i f e span; longer l i v e d s t r a i n s undergo slower motor a c t i v i t y decay than wild-type s t r a i n s . Thus i t may be that aging and motor a c t i v i t y decay share a common genetic mechanism. F i n a l l y m i l d food d e p r i v a t i o n causes an i n c r e a s e i n the l i f e s p a n with a decrease i n f e r t i l i t y . T h i s e f f e c t i s seen i n both wild-type and mutant s t r a i n s of C. elegans. Thus, the e f f e c t of food d e p r i v a t i o n on l i f e - s p a n seems t o be independent of the extended l i f e span produced by the aging mutation (Johnson, 1987). The work of Johnson and h i s colleagues not o n l y gives us candidate aging mechanisms, but a l s o a means of t e s t i n g the proposed mechanisms b e h a v i o r a l l y . By comparing aging i n normal and mutant worms i t may be p o s s i b l e t o d e f i n e some of the mechanisms underlying l e a r n i n g and memory d e f i c i t s r e s u l t i n g from the aging process. C l e a r l y a model of aging e f f e c t s on l e a r n i n g and memory i n C. elegans provides a r i c h set of p o s s i b l e research d i r e c t i o n s . By e s t a b l i s h i n g i n t h i s set of experiments the pa t t e r n s of change wit h aging i n these simple forms of l e a r n i n g , h a b i t u a t i o n and d i s h a b i t u a t i o n , we may begin t o develop a simple-systems model of the aging e f f e c t s on l e a r n i n g and memory. Using C. elegans, such a model may provide us with a unique opportunity t o i n v e s t i g a t e the g e n e t i c c o n t r o l of changes i n l e a r n i n g and memory i n senescence. 62 Bi b l i o g r a p h y C h a l f i e , M. (1984). Neuronal development i n Caenorhabditis elegans. Trends i n Neuroscience. 1_, 197-202. C h a l f i e , M., & Au, M. (1989). Genetic c o n t r o l of d i f f e r e n t i a t i o n of the Caenorhabditis elegans touch receptor neurons. Science, 243. 1027-1033. C h a l f i e , M., & Sulston, J . E. (1981). (Developmental g e n e t i c s of the mechanosensory neurons of Caenorhabditis elegans. Developmental Biology, 82. 358-370. 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