UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

An analysis of the tap withdrawal response in male caenorhabditis elegans Mah, Kim Bill 1991

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1991_A8 M23_98.pdf [ 3.95MB ]
Metadata
JSON: 831-1.0100715.json
JSON-LD: 831-1.0100715-ld.json
RDF/XML (Pretty): 831-1.0100715-rdf.xml
RDF/JSON: 831-1.0100715-rdf.json
Turtle: 831-1.0100715-turtle.txt
N-Triples: 831-1.0100715-rdf-ntriples.txt
Original Record: 831-1.0100715-source.json
Full Text
831-1.0100715-fulltext.txt
Citation
831-1.0100715.ris

Full Text

AN ANALYSIS OF THE TAP WITHDRAWAL RESPONSE IN MALE CAENORHABDITIS ELEGANS by KIM BILL MAH B . S c , The U n i v e r s i t y of A l b e r t a , 1988 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE 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 t o the r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA SEPTEMBER 1991 © Kim B i l l Mah, 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 Ps^cV-olo<^. The University of British Columbia Vancouver, Canada D a t e '. s^wu^ r?, mi DE-6 (2/88) i i A b s t r a c t The nematode, Caenorhabditis elegans, d i s p l a y s s e x u a l dimorphism; males a r i s e by t h e process of x-chromosome non-d i s j u n c t i o n . The male i s d i f f e r e n t i a t e d from t h e hermaphrodite by t h e a d d i t i o n o f 14 sensory s t r u c t u r e s i n t h e t a i l , as w e l l as by t h e t o t a l complement of neurons. The male possesses 381 neurons o f which a t l e a s t 87 are male s p e c i f i c , compared w i t h a t o t a l of 302 neurons i n the hermaphrodite. The purpose of t h i s t h e s i s was t o i n v e s t i g a t e whether t h e a d d i t i o n a l s e n s o r y and n e u r a l c i r c u i t r y i n t h e male r e s u l t e d i n a d i f f e r e n c e i n t h e t a p w i t h d r a w a l response i n male C. elegans compared t o t h a t o f the hermaphrodite. F i r s t , t h e b a s a l a c t i v i t y l e v e l o f males was compared t o t h a t o f hermaphrodites. Males e x h i b i t e d s i g n i f i c a n t l y more spontaneous r e v e r s a l s than hermaphrodites', t h e s e r e v e r s a l s were c a t e g o r i z e d as t r u e spontaneous r e v e r s a l s and s e l f - f e e l i n g r e v e r s a l s . The r e v e r s a l magnitude o f t r u e spontaneous r e v e r s a l s d i d not d i f f e r a c r o s s sex, however th e magnitude o f s e l f - f e e l i n g r e v e r s a l s was s i g n i f i c a n t l y g r e a t e r i n males compared t o hermaphrodites. Next, t h e r e f l e x i v e r e v e r s a l response t o t a p was examined. Both males and hermaphrodites e x h i b i t e d a graded magnitude o f response t o an i n c r e a s i n g number of t a p s . M a l e s , however, showed an i n c r e a s i n g i n c i d e n c e of a c c e l e r a t i o n s as t a p number i n c r e a s e d , w h i l e hermaphrodites showed t h e same number of r e v e r s a l s as the number of taps i n c r e a s e d . I n response t o i i i t a p s o f i n c r e a s i n g i n t e n s i t y , both males and hermaphrodites showed an i n c r e a s e i n r e v e r s a l magnitude. Response magnitudes e x h i b i t e d by males were s i g n i f i c a n t l y g r e a t e r t h a n t h o s e o f hermaphrodites. The i n t e r a c t i o n between two a n t a g o n i s t i c r e f l e x e s was t h e n examined. A t a i l - t o u c h f o l l o w e d by a t a p 1 s l a t e r s i g n i f i c a n t l y i n h i b i t e d both the number and s i z e o f t h e r e v e r s a l response i n males and hermaphrodites. There were no s i g n i f i c a n t sex d i f f e r e n c e s a t any t a i l - t o u c h / t a p i n t e r v a l s t e s t e d , a l t h o u g h the hermaphrodites e x h i b i t e d f a c i l i t a t i o n a t t h e 20 and 30 s t a i l - t o u c h / t a p i n t e r v a l . The a d m i n i s t r a t i o n of 25 t a i l - t o u c h e s p r i o r t o the a d m i n i s t r a t i o n of t a i l - t o u c h / t a p s i g n i f i c a n t l y decreased the amount of i n h i b i t i o n produced by t a i l - t o u c h f o l l o w e d by tap 1 s l a t e r i n both males and hermaphrodites. F i n a l l y , males were t e s t e d t o see i f t h e y c o u l d show s i m p l e n o n - a s s o c i a t i v e l e a r n i n g . Both males and hermaphrodites demonstrated h a b i t u a t i o n . The r a t e of h a b i t u a t i o n d i d not d i f f e r between t h e sexes, however, th e a s y m p t o t i c l e v e l o f h a b i t u a t i o n was h i g h e r i n males than i n hermaphrodites. Thus, the a d d i t i o n a l sensory s t r u c t u r e s o f the male d i d not appear t o a f f e c t h a b i t u a t i o n . I t was h y p o t h e s i z e d t h a t the mechanisms g o v e r n i n g t h e r a t e of h a b i t u a t i o n are the same i n both sexes, but t h a t t h e mechanisms governing t h e degree o f h a b i t u a t i o n may be d i f f e r e n t i n the males and hermaphrodites. The r a t e o f spontaneous r e c o v e r y from h a b i t u a t i o n d i d not d i f f e r between i v t h e s e x e s , t h e r e f o r e i t was h y p o t h e s i z e d t h a t t h e mechanisms g o v e r n i n g r e c o v e r y are the same i n males and hermaphrodites. A d d i t i o n a l a n a l y s i s of h a n d l i n g e f f e c t s r e v e a l e d t h a t males showed a s m a l l e r response i f t e s t e d a t 1-2 min r a t h e r t h a n a f t e r 2-24 hr a f t e r t r a n s f e r t o the t e s t p l a t e . Hermaphrodites showed the o p p o s i t e p a t t e r n o f response: l a r g e r responses were observed i f t h e worm was t e s t e d 1-2 min p o s t -t r a n s f e r t h a n i f t e s t e d 2-24 h r p o s t - t r a n s f e r . Thus, t h e s e experiments showed t h a t t h e mechanisms u n d e r l y i n g b e h a v i o u r a l p l a s t i c i t y i n the C. elegans males and hermaphrodites are l i k e l y t o be s i m i l a r , and f u r t h e r t h a t t h e a d d i t i o n a l s e nsory r e c e p t o r s and a s s o c i a t e d n e u r a l c i r c u i t r y do not r e s u l t i n d i f f e r e n c e s i n n o n - a s s o c i a t i v e l e a r n i n g . However, d i f f e r e n c e s i n the responses of males and hermaphrodites were found: spontaneous r e v e r s a l a c t i v i t y i n t h e males was g r e a t e r and h a n d l i n g r e s u l t e d i n d i f f e r e n c e s i n response magnitudes i n males and hermaphrodites. V Table of Contents: A b s t r a c t i i L i s t o f F i g u r e s v i i i Acknowledgements x i I n t r o d u c t i o n 1 G e n e r a l Methods 8 S u b j e c t s 8 M a t e r i a l s 10 Apparatus 10 S c o r i n g and S t a t i s t i c a l A n a l y s i s 11 Experiment I : Spontaneous R e v e r s a l s 11 S u b j e c t s 12 Procedure 12 S c o r i n g and S t a t i s t i c a l A n a l y s i s 13 R e s u l t s 13 D i s c u s s i o n 14 Experiment I I : Response t o Graded Tap D u r a t i o n 19 S u b j e c t s i 20 Procedure 20 S c o r i n g and S t a t i s t i c a l A n a l y s i s 21 R e s u l t s 21 D i s c u s s i o n 27 Experiment I I I : Response t o Graded Tap I n t e n s i t y . . 28 S u b j e c t s 28 Procedure 28 v i S c o r i n g and S t a t i s t i c a l A n a l y s i s 29 R e s u l t s 2 9 D i s c u s s i o n 30 Experiment IV: I n h i b i t i o n 36 S u b j e c t s 37 Procedure 37 S c o r i n g and S t a t i s t i c a l A n a l y s i s 38 R e s u l t s 38 D i s c u s s i o n 39 Experiment V: T a i l - t o u c h H a b i t u a t i o n 42 S u b j e c t s 42 Procedure 42 S c o r i n g and S t a t i s t i c a l A n a l y s i s 43 R e s u l t s 43 D i s c u s s i o n 4 6 Experiment V I : 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 46 S u b j e c t s 47 Procedure 4 8 S c o r i n g and S t a t i s t i c a l A n a l y s i s 48 R e s u l t s 49 D i s c u s s i o n . 55 Experiment V I I : Recovery from H a b i t u a t i o n 56 S u b j e c t s 57 Procedure 57 S c o r i n g and S t a t i s t i c a l A n a l y s i s 58 R e s u l t s . . .'. 58 v i i Discussion 62 General Discussion 62 Conclusions and Directions for Future Research.... 70 References 73 Appendix 1 76 Appendix II 77 Appendix III 78 v i i i L i s t of F i g u r e s : 1. A) The number of spontaneous reversals shown by males and hermaphrodites B) The percent time active of males and hermaphrodites over a 10 min observation period C) The number of true spontaneous reversals shown by males • and hermaphrodites D) The number of s e l f - f e e l i n g reversals shown by males and hermaphrodites 15-16 2. A) The magnitude of spontaneous reversals shown by males and hermaphrodites B) The magnitude of true spontaneous reversals shown by males and hermaphrodites C) The magnitude of s e l f - f e e l i n g reversals shown by males and hermaphrodites 17-18 3. A) The number of males and hermaphrodites showing reversals to different numbers of taps B) The number of males and hermaphrodites showing accelerations to different numbers of taps 22-23 4. The magnitude of reversals shown by males and hermaphrodites to d i f f e r i n g numbers of taps 25-26 5. A) The number of males and hermaphrodites showing reversals to different intensities of tap B) The number of males, and hermaphrodites showing accelerations to different intensities of tap 31-32 6. The magnitude o f r e v e r s a l s shown by males and hermaphrodites t o d i f f e r i n g i n t e n s i t i e s of t a p 33-34 7. A) The number of males arid hermaphrodites showing r e v e r s a l s t o t a p and t a i l - t o u c h f o l l o w e d by a t a p a t v a r y i n g i n t e r v a l s B) The magnitude of response shown by males and hermaphrodites t o t a p and t a i l - t o u c h f o l l o w e d by a t a p a t v a r y i n g i n t e r v a l s 40-41 8. The e f f e c t s o f t a i l - t o u c h h a b i t u a t i o n on i n h i b i t i o n A) The number o f males and hermaphrodites showing r e v e r s a l s t o t a p a l o n e , t o t a i l - t o u c h f o l l o w e d 1 s l a t e r by t a p and t o 25 t a i l - t o u c h e s p r e c e d i n g a t a i l - t o u c h f o l l o w e d 1 s l a t e r by t a p B) The magnitude o f responses shown by males and hermaphrodites t o t a p a l o n e , t o t a i l - t o u c h f o l l o w e d 1 s l a t e r by t a p and t o 25 t a i l - t o u c h e s p r e c e d i n g a t a i l -t o u c h f o l l o w e d 1 s l a t e r by t a p ....44-45 9. The number of r e v e r s a l s shown by males and hermaphrodites 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 and a f t e r shock 50-51 10. A) The magnitude of r e v e r s a l s shown by males 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 and f o l l o w i n g d i s h a b i t u a t i o n B) The magnitude of r e v e r s a l s shown by males 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 and f o l l o w i n g d i s h a b i t u a t i o n 53-54 11. A) The number of r e v e r s a l s shown by males and , hermaphrodites a t the i n i t i a l and f i n a l t a p o f X h a b i t u a t i o n t r a i n i n g and d u r i n g spontaneous r e c o v e r y from h a b i t u a t i o n B) The magnitude of r e v e r s a l s shown by males and hermaphrodites a t the i n i t i a l and f i n a l t a p of h a b i t u a t i o n t r a i n i n g and d u r i n g spontaneous r e c o v e r y from h a b i t u a t i o n 60-61 12. The magnitude of r e v e r s a l s i n response t o a s i n g l e t a p shown by males and hermaphrodites a f t e r 1-2 min p o s t -t r a n s f e r , a t 2 hr and 24 hr p o s t - t r a n s f e r . . . 66-67 x i Acknowledgements Foremost, I would l i k e t o thank my s u p e r v i s o r , Cathy R a n k i n , f o r her su p p o r t , f o r s h a r i n g her wisdom and e x p e r i e n c e and f o r t e a c h i n g me t h e meaning of t e n a c i t y . Thanks t o t h e members o f my committee: t o Stan Coren f o r h i s k i n d and p a t i e n t s t a t i s t i c a l a d v i c e and t o Rod Wong, who made me r e a l i z e t h a t p h i l o s o p h y and s c i e n c e are not t h a t f a r a p a r t . I thank C h r i s t i n e Beck and N e i l Watson f o r t h e i r t h o u g h t f u l comments and c r i t i c i s m s o f my many ma n u s c r i p t s . Thanks t o M a r i o n Buday and Deanna Ho f o r t h e i r many hours of da t a s c o r i n g . F i n a l l y , I thank my p a r e n t s , Kee and May, f o r b e l i e v i n g i n me. 1 I n t r o d u c t i o n : The relationship between behaviour and i t s physiological underpinnings i s one of the fundamental issues i n biopsychology. A basic tenet of this f i e l d i s that behaviour i s the dire c t result of a process or set of processes occurring at the neural l e v e l . The investigation of the nature of th i s relationship i s dependent upon the level of analysis used. The l e v e l of analysis constrains the generality of experimental findings, and i n turn, i s i t s e l f constrained by the s p e c i f i c type of questions posed and by their resultant answers. Thus, in choosing a model system to investigate the relationship between behaviour and physiology, i t i s c r i t i c a l to define the le v e l of analysis desired, and i n so doing to consider the types of questions for which answers are sought. A central issue with respect to level of analysis involves balancing the variables of neural complexity and behavioural complexity. Neural complexity implies far more than simply a nervous system composed of a large number of neurons. Indeed, an inclusive d e f i n i t i o n of the term must address the a n c i l l a r y concepts of ease of i d e n t i f i c a t i o n of single neurons, neuron to neuron connections, mapping of neural c i r c u i t r y and the lineage of individual neurons. Generally, an increasing number of neurons i s associated with a decrease i n knowledge with respect to the a n c i l l a r y concepts. Behavioural complexity, also, i s descriptive of more than just a large and varied behavioural repertoire. It also refers t o t h e i n t e g r a t i o n of a s e t o f r e l a t i v e l y s i m p l e responses i n t o a complex u n i f i e d b e haviour. Thus, i n o r d e r t o un d e r s t a n d b e h a v i o u r as i t becomes more complex, i t i s n e c e s s a r y t o c o n s i d e r not o n l y the u n i f i e d behaviour but a l s o i t p a r t s . A h i g h degree of b e h a v i o u r a l c o m p l e x i t y i s g e n e r a l l y a s s o c i a t e d w i t h a h i g h degree of n e u r a l c o m p l e x i t y . A l t h o u g h a d i v e r s e b e h a v i o u r a l r e p e r t o i r e may p r o v i d e many o p p o r t u n i t i e s f o r o b s e r v a t i o n and measurement, i t becomes e x t r e m e l y d i f f i c u l t t o i d e n t i f y t h e n e u r a l s u b s t r a t e u n d e r l y i n g t h e b e h a v i o u r . T h i s i s due m a i n l y t o the i n c r e a s e i n the number o f neurons r e q u i r e d t o support t h a t complex be h a v i o u r . However, t h e major advantage o f a d i v e r s e s e t of behaviours i s an i n c r e a s e d a n a l o g y and g e n e r a l i t y t o human behaviour. C o n v e r s e l y , an organism w i t h an e x t r e m e l y b a s i c nervous system may e x h i b i t o n l y a l i m i t e d range of b e h a v i o u r s , and t h i s p l a c e s l i m i t s on the o p t i o n s a v a i l a b l e f o r e x p e r i m e n t a l o b s e r v a t i o n and m a n i p u l a t i o n . A d d i t i o n a l l y , t h e a b i l i t y t o g e n e r a l i z e t o a h i g h e r organism i s l o s t i f t h e model system d i s p l a y s a low degree of n e u r a l s o p h i s t i c a t i o n . D e s p i t e t h e s e drawbacks, t h e s i m p l i c i t y of the nervous system o f such organisms o f f e r s a major advantage i n 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 between p h y s i o l o g y and be h a v i o u r . A s i m p l e systems approach u s i n g Caenorhabditis elegans seems t o s t r i k e a balance between n e u r a l c o m p l e x i t y and b e h a v i o u r a l c o m p l e x i t y . C. elegans i s a s m a l l , m u l t i c e l l u l a r , s o i l d w e l l i n g nematode which i s a n a t o m i c a l l y s i m p l e ; t h e a d u l t hermaphrodite possesses o n l y 959 somatic n u c l e i , of which 302 ar e neurons. The n e u r a l system of C. elegans demonstrates l a r g e l y d e t e rminant development, which has l e d t o t h e mapping of a l l 302 neurons and t h e i r i n t e r c o n n e c t i o n s i n t h e a d u l t hermaphrodite (White, Southgate, Thomson & Brenner, 1986). The l i n e a g e o f each n e u r a l o r somatic c e l l can be t r a c e d t hroughout t h e developmental phases of the nematode ( S u l s t o n , S c h i e r e n b e r g , White & Thomson, 1983), making C. elegans a prime c a n d i d a t e f o r developmental s t u d i e s of b e h a v i o u r / s u b s t r a t e r e l a t i o n s h i p s . Furthermore, t h i s nematode i s t h e s u b j e c t o f i n t e n s i v e g e n e t i c study; over 95% o f i t s genome has been mapped (Coulson, S u l s t o n , Brenner & Karn, 1986), t h e r e b y p r o v i d i n g g r e a t p o t e n t i a l t o l i n k g e n e t i c f u n c t i o n t o n e u r a l f u n c t i o n . As a model system, C. elegans a l s o o f f e r s t h e advantages o f easy c u l t i v a t i o n and low maintenance. C. elegans d i s p l a y s a behaviour which i s e a s i l y a c c e s s i b l e t o o b s e r v a t i o n and t e s t i n g . The nematode swims on i t s s i d e on an agar s u r f a c e by means of c o n t r a c t i o n s o f t h e v e n t r a l and d o r s a l m usculature, l e a d i n g t o t h e p r o p a g a t i o n o f a s i n u s o i d a l wave throughout the l e n g t h of t h e worm r e s u l t i n g i n f o r w a r d p r o p u l s i o n (Wood, 1988). Backward motion, h e r e i n termed a r e v e r s a l , i s a c h i e v e d by changing t h e d i r e c t i o n o f t h e u n d u l a t o r y motions. R e v e r s a l s can o c c u r both s p o n t a n e o u s l y o r i n response t o a s t i m u l u s . Changes i n the d i r e c t i o n o f movement may be induced by t o u c h , v i b r a t i o n a l s t i m u l i , t e mperature changes, i o n s and many c h e m i c a l compounds (Wood, 1988). Thus, t h e presence of forward swimming and subsequent r e v e r s a l t o s t i m u l a t i o n a f f o r d s the o p p o r t u n i t y t o i n v e s t i g a t e a s i m p l e response and the p h y s i o l o g y which c o n t r o l s t h i s r e sponse. Indeed, a model of the n e u r a l c i r c u i t f o r t o u c h s e n s i t i v i t y has been worked out by C h a l f i e e t a l . (1985). The p o s t u l a t e d c i r c u i t c o n t a i n s s i x t o u c h r e c e p t o r s , f i v e p a i r s o f i n t e r n e u r o n s and 69 motor neurons. S t u d i e s o f locomotor behaviour have r e v e a l e d t h a t t h e C. elegans hermaphrodite i s capable of s e v e r a l forms o f non-a s s o c i a t i v e l e a r n i n g , such as s e n s i t i z a t i o 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 (Rankin & C h i b a , 1988; Rankin, Beck & C h i b a , 1990). C. elegans w i l l u s u a l l y show a r e f l e x i v e r e v e r s a l t o a t a p t o t h e P e t r i d i s h which c o n t a i n s the a n i m a l . With t h e a d m i n i s t r a t i o n o f a t r a i n of taps i n the p l a c e o f a s i n g l e t a p , a l a r g e r magnitude r e v e r s a l response i s u s u a l l y seen (Chiba & R a n k i n , 1990). H a b i t u a t i o n o f the r e v e r s a l response o c c u r s as a r e s u l t of a d m i n i s t r a t i o n of a number of taps o f f i x e d i n t e r v a l . Long term r e t e n t i o n (24 hr.) of t h i s h a b i t u a t i o n has been demonstrated (Rankin, e t a l . , 1990). D i s h a b i t u a t i o n o f t h e h a b i t u a t e d r e v e r s a l response i s seen f o l l o w i n g t h e a d m i n i s t r a t i o n o f a n o v e l s t i m u l u s such as a 60 V shock t o t h e agar medium on which an animal i s r e s t i n g . Thus, c o n s i d e r i n g i t s s i m p l e , well-mapped anatomy coupled w i t h a b e h a v i o u r a l r e p e r t o i r e amenable t o e x p e r i m e n t a l m a n i p u l a t i o n , C. elegans p r o v i d e s a p o w e r f u l model system f o r the s t u d y o f t h e r e l a t i o n s h i p between behaviour and i t s n e u r a l u n d e r p i n n i n g s . G e n e t i c and p h y s i o l o g i c a l a n a l y s e s have r e v e a l e d a m u l t i t u d e o f p o s s i b l e mutations i n t h e C. elegans hermaphrodite r e s u l t i n g i n wide r a n g i n g e f f e c t s on the subsequent b e h a v i o u r of t h e nematode ( f o r examples see Brenner, 1974; H o r v i t z , S t e r n b e r g , Greenwald, F i x s e n & E l l i s , 1983). One o f t h e s e m u t a t i o n s r e s u l t s i n t h e male of the s p e c i e s , due t o t h e p r o c e s s o f X-chromosome n o n - d i s j u n c t i o n (Hodgkin, H o r v i t z & Brenner, 1979). In hermaphrodite p o p u l a t i o n s , r e p r o d u c t i o n i s a c c o m p l i s h e d through s e l f - f e r t i l i z a t i o n . Male mutants a r i s e from s e l f - f e r t i l i z a t i o n a t t h e r a t e of o n l y 1 per 1000 animals a t 20 degrees C (Rose & B a i l l i e , 1979) i n the h e r m a p h r o d i t i c p o p u l a t i o n . However, f e r t i l i z a t i o n of t h e oocyte by spermatozoa s u p p l i e d by the male C. elegans r e s u l t s i n a h i g h e r i n c i d e n c e o f male progeny than f e r t i l i z a t i o n by h e r m a p h r o d i t i c sperm, approaching 20-30% of the t o t a l progeny ( p e r s o n a l o b s e r v a t i o n ) . C o p u l a t o r y behaviour i s f r e q u e n t l y i n i t i a t e d when the p o s t e r i o r s ensory r e c e p t o r s o f the male nematode a r e s t i m u l a t e d , u s u a l l y by p h y s i c a l c o n t a c t w i t h another worm, whether i t i s a hermaphrodite o r a male. Upon e n c o u n t e r i n g a hermaphrodite, t h e male swims i n a backwards d i r e c t i o n ( i e : r e v e r s e s ) t o f a c i l i t a t e c o n t a c t between th e c u t i c l e o f t h e hermaphrodite and c o p u l a t o r y bursa found i n t h e t a i l o f t h e male. By r u n n i n g i t s t a i l a l o n g t h e s u r f a c e t o t h e c u t i c l e , t h e male uses t h e sensory s t r u c t u r e s a s s o c i a t e d w i t h t h e t a i l 6 t o l o c a t e t h e v u l v a . Upon f i n d i n g the v u l v a l opening, t h e male f i r s t i n s e r t s t h e c o p u l a t o r y s p i c u l e s t o open t h e v u l v a , and t h e n a t t a c h e s i t s e l f t o the hermaphrodite w i t h t h e s c l e r o t i c hook, which i s f o l l o w e d by e j a c u l a t i o n of t h e spermatozoa. The spermatozoa s u p p l i e d by the male p r e f e r e n t i a l l y d i s p l a c e s t h a t s u p p l i e d by t h e hermaphrodite i n the f e r t i l i z a t i o n p r o c e s s (Ward & C a r r e l , 1979). A n a t o m i c a l l y , the male i s d i f f e r e n t i a t e d from t h e hermaphrodite by i t s l a r g e r complement of somatic n u c l e i : 1031 i n t o t a l , 72 more than the hermaphrodite. Of t h e s e c e l l s , t h e a d u l t male possesses 381 neurons of which a t l e a s t 87 a r e male-s p e c i f i c (Hodgkin, 1988). A d d i t i o n a l l y , t h e nervous system o f t h e male i s much more c o m p l i c a t e d than t h a t o f t h e hermaphrodite (Hodgkin, 1988), and t o date has not been c o m p l e t e l y r e c o n s t r u c t e d . As w i t h t h e hermaphrodite, the l i n e a g e s o f most of t h e c e l l s i n t h e male have been determined ( S u l s t o n & White, 1980). Most o f t h e s e c e l l s a re accounted f o r by t h e f o r m a t i o n o f t h e c o p u l a t o r y apparatus o f the t a i l r e g i o n , i n c l u d i n g t h e vas d e f e r e n s , t h e c l o a c a and a s c l e r o t i c hook, [see Appendices I + I I ] . I n a d d i t i o n t o t h e 2 t a i l s e n s i l l a p o ssessed by t h e hermaphrodite ( S u l s t o n , A l b e r t s o n & Thomson, 1980), t h e male C. elegans can r e c e i v e a d d i t i o n a l sensory i n p u t from t h e t a i l v i a 2 c o p u l a t o r y s p i c u l e s , each o f which i s a s s o c i a t e d w i t h 2 s e n s o r y neurons, a s i n g l e hook s e n s i l l u m and i t s 2 a s s o c i a t e d s e n s o r y neurons,, a p a i r of post c l q a c a l s e n s i l l a i n n e r v a t e d by 2 s e t s o f t h r e e sensory neurons, one p a i r of phasmids ( i e . r e c e p t o r s s p e c i a l i z e d f o r chemoreception), as w e l l as t h r o u g h 18 s e n s o r y r a y s d i s t r i b u t e d b i l a t e r a l l y throughout i t s f a n shaped t a i l and i n n e r v a t e d by 18 p a i r s o f sensory neurons (Wood, 1988) [see Appendices I + I I ] . The arrangement of s e n s o r y c e l l s i n t h e male and hermaphrodite C. elegans i s v e r y s i m i l a r , however f o u r a d d i t i o n a l neuron p r o c e s s e s o f unknown f u n c t i o n are p r e s e n t i n the male (Ward, Thomson, White & Brenner, 1 9 7 5 ) . Thus, t h e r e are major d i f f e r e n c e s between t h e male and hermaphrodite i n terms of sensory r e c e p t o r s , w i t h t h e male h a v i n g 14 a d d i t i o n a l s t r u c t u r e s and t h e i r a s s o c i a t e d s e n s o r y neurons i n the t a i l r e g i o n . The d i f f e r e n c e s i n sensory r e c e p t o r s between males and hermaphrodites may r e s u l t i n d i f f e r e n c e s i n response t o head and t a i l t o u c h o r i n d i f f e r e n c e s i n b e h a v i o u r a l p l a s t i c i t y . F o r i n s t a n c e , a b e h a v i o u r a l a n a l y s i s l e a d Rankin e t a l . ( 1990 ) t o h y p o t h e s i z e t h a t a t a p a c t i v a t e d the t o u c h r e c e p t o r s i n both t h e head and t a i l of t h e nematode. The a d d i t i o n a l s e n s o r y r e c e p t o r s i n t h e C. elegans male might render t h e a n i m a l more o r l e s s r e s p o n s i v e t o t a p s t i m u l a t i o n , o r might l e a d t o a d i f f e r e n t b e h a v i o u r a l response than t h a t d i s p l a y e d by t h e hermaphrodite. These a d d i t i o n a l sensory s t r u c t u r e s may p l a y a r o l e i n response t o touch and may t h e r e f o r e n e c e s s i t a t e an a n a l y s i s o f t h e touch w i t h d r a w a l c i r c u i t f o r t h e male. The f o l l o w i n g s e r i e s of experiments was designed i n o r d e r t o determine whether the a d d i t i o n a l c i r c u i t r y p r e s e n t i n male C. 8 elegans has any e f f e c t on responses t o v i b r a t i o n a l s t i m u l a t i o n as compared t o t h a t of the hermaphrodite. S i n c e a d e t a i l e d g e n e t i c a n a l y s i s o f t h e male has been c a r r i e d out (Hodgkin, 1974), the p o t e n t i a l e x i s t s t o c o r r e l a t e b e h a v i o r w i t h g e n e t i c s i n male C.elegans. I f t h e r e a r e any b e h a v i o u r a l d i f f e r e n c e s between males and hermaphrodites, t h i s g e n e t i c a n a l y s i s may be u s e f u l i n i n v e s t i g a t i o n s o f t h e m o l e c u l a r mechanisms u n d e r l y i n g the b e h a v i o u r a l d i f f e r e n c e s . I n t h e f i r s t experiments, t o determine whether b a s a l a c t i v i t y l e v e l s d i f f e r e d , the spontaneous r e v e r s a l a c t i v i t y o f males was r e c o r d e d and compared t o t h a t of hermaphrodites. I n th e next experiments, the re s p o n s i v e n e s s o f r e f l e x i v e r e v e r s a l s t o d i f f e r e n t d u r a t i o n s and i n t e n s i t i e s of v i b r a t o r y s t i m u l a t i o n was e s t a b l i s h e d i n both sexes. To determine whether t h e i n t e r a c t i o n o f a n a t a g o n i s t i c r e f l e x e s f o l l o w e d t h e same r u l e s i n males as i n hermaphrodites (Rankin, 1991), an a n a l y s i s o f th e i n h i b i t i o n o f r e v e r s a l t o t a p by t a i l - t o u c h was performed. F i n a l l y , b e h a v i o u r a l p l a s t i c i t y was i n v e s t i g a t e d by comparing males and hermaphrodites on measures o f 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 spontaneous r e c o v e r y from h a b i t u a t i o n . G e n e r a l Methods S u b j e c t s I n a l l experiments, C. elegans hermaphrodites s e r v e d as c o n t r o l s w h i l e male C. elegans ( s t r a i n N2 B r i s t o l ) s e r v e d as t h e e x p e r i m e n t a l s u b j e c t s . A t o t a l of 220 hermaphrodites and 220 males were used. Male/hermaphrodite b r e e d i n g c o l o n i e s were s e t up, a l l o w i n g f o r c r o s s - f e r t i l i z a t i o n t o o c c u r . T h i s method ensured an adequate s u p p l y of male C. elegans f o r r e s e a r c h purposes. These b r e e d i n g c o l o n i e s were s y n c h r o n i z e d t o ensure t h a t t e s t a n imals were th e same age. S e x u a l l y mature males (n=10) were p l a c e d on a P e t r i d i s h w i t h 4 t o 5 hermaphrodites a t t h e l a t e L4 t o young a d u l t s tage f o r a 24 hour p e r i o d . T h i s procedure a l l o w e d f o r maximal exposure o f oocytes t o male sperm d u r i n g t h e time f e r t i l i z a t i o n n o r m a l l y occurs i n t h e hermaphrodite, and has been shown t o y i e l d t h e g r e a t e s t number o f c r o s s progeny (Hodgkin, 1983). The animals were s u b s e q u e n t l y t r a n s f e r r e d t o another d i s h and a l l o w e d t o l a y eggs, and t h e n removed a p p r o x i m a t e l y 2 t o 3 hours l a t e r . The r e s u l t i n g male progeny from t h e eggs l a i d on t h i s p l a t e were t e s t e d 4 days l a t e r . W i t h each s u c c e s s i v e g e n e r a t i o n , the genome of C. elegans d i s p l a y s a s m a l l amount of g e n e t i c d r i f t . Over t h e c o u r s e o f t h e e x p e r i m e n t s , many g e n e r a t i o n s have passed, i n c r e a s i n g t h e l i k e l i h o o d of d r i f t w i t h each mating of males and hermaphrodites. To ensure t h a t t h i s g e n e t i c d r i f t was not r e s p o n s i b l e f o r any observed d i f f e r e n c e s i n t h e b e h a v i o u r o f th e male as compared t o t h a t of the hermaphrodite, a group o f h e r m a p h r o d i t i c nematodes were i n c l u d e d i n each experiment as a c o n t r o l . Thus, s y n c h r o n i z e d c o l o n i e s of hermaphrodites were 1 0 a l s o s e t up. S y n c h r o n i z a t i o n of t h e c o l o n i e s ensured t h a t t e s t a n i m a l s had reached t h e a d u l t stage of development. G r a v i d hermaphrodites were p l a c e d on a p e t r i d i s h and l e f t t o l a y eggs f o r a p e r i o d of 2-3 hours. Progeny were t e s t e d i n t h e a d u l t s t a g e a p p r o x i m a t e l y 96 hours a f t e r t h e eggs were l a i d ( B y e r l y , Cassada & R u s s e l l , 1976). M a t e r i a l s A l l a n imals were m a i n t a i n e d a t 20 degrees C e l s i u s on 5 cm P e t r i p l a t e s f i l l e d w i t h Nematode Growth Medium (Brenner, 1974) s t r e a k e d w i t h Escherichia coli on the s u r f a c e o f t h e agar t o p r o v i d e a source o f food f o r C. elegans. I n d i v i d u a l a n i m a l s were t r a n s f e r r e d t o unstreaked ( i e . no food p r e s e n t ) agar f i l l e d p l a t e s f o r t e s t i n g . I n a l l c a s e s , f o l l o w i n g t r a n s f e r , a n i m a l s were a l l o w e d a minimum of 1 t o 2 minutes t o accommodate t o t h e t e s t p l a t e p r i o r t o e x p e r i m e n t a l m a n i p u l a t i o n . Apparatus T e s t p l a t e s were h e l d i n p l a c e on the microscope s t a g e by a h o l d e r a t t a c h e d t o a m i c r o m a n i p u l a t o r and s t a n d . Taps were t r a n s m i t t e d t o t h e s i d e of the t e s t p l a t e by a m e c h a n i c a l t a p p e r a t t a c h e d t o an e l e c t r o m a g n e t i c r e l a y s w i t c h . Shocks were a d m i n i s t e r e d w i t h a spanning e l e c t r o d e c o n s i s t i n g o f a p a i r o f w i r e s which were p l a c e d i n the agar a p p r o x i m a t e l y 1 mm on e i t h e r s i d e o f t h e a n i m a l . Both the t a p and shock parameters 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 [see Appendix I I I ] . 11 A l l o b s e r v a t i o n s were made through a W i l d M3Z st e r e o m i c r o s c o p e w i t h b r i g h t - f i e l d i l l u m i n a t i o n and r e c o r d e d on v i d e o t a p e u s i n g an a t t a c h e d Panasonic D5000 h i g h - r e s o l u t i o n v i d e o camera and Panasonic v i d e o c a s s e t t e r e c o r d e r . S c o r i n g and S t a t i s t i c a l A n a l y s i s U s i n g s t o p frame v i d e o a n a l y s i s , t h e r e v e r s a l b e h a v i o u r o f each a n i m a l was t r a c e d from v i d e o t a p e s onto a c e t a t e s h e e t s . A z e r o ( o r blan k ) v a l u e was a s s i g n e d t o a response i n which an an i m a l showed no r e v e r s a l . Measurements o f the magnitude o f th e t r a c i n g (and hence r e v e r s a l ) were rendered u s i n g a B i t Pad P l u s d i g i t i z i n g t a b l e t u s i n g MacMeasure s o f t w a r e on an Apple M a c i n t o s h SE computer. Data i n v o l v i n g p r o p o r t i o n s o f animals showing r e v e r s a l s were a n a l y z e d u s i n g an o v e r a l l ANOVA w i t h dichotomous d a t a (Winer, Brown & M i c h e l s , 1991). Data i n v o l v i n g magnitude i n f o r m a t i o n were a n a l y z e d u s i n g o v e r a l l ANOVAs w i t h a p p r o p r i a t e f o l l o w - u p t e s t s as wa r r a n t e d . Trend a n a l y s i s was used t o a n a l y z e magnitude d a t a o b t a i n e d i n response t o graded s t i m u l i . R e g r e s s i o n a n a l y s i s was employed t o t e s t r a t e measures ( i e . r a t e of h a b i t u a t i o n o r r a t e o f r e c o v e r y from h a b i t u a t i o n ) . Repeated measures d a t a were ;» a n a l y z e d w i t h a repe a t e d measures ANOVA. Nominal a l p h a was s e t a t 0.05 f o r a l l experiments. Experiment I: Spontaneous r e v e r s a l s A spontaneous r e v e r s a l i s d e f i n e d as backward motion i n th e absence of any o b s e r v a b l e e x t e r n a l s t i m u l u s . C h i b a and 1 2 Rankin (1989) have demonstrated t h a t a d u l t C. elegans hermaphrodites show a mean frequency of 2.5 spontaneous r e v e r s a l s per minute i n the absence of any o b s e r v a b l e s t i m u l i . I t has been h y p o t h e s i z e d t h a t the presence and i n t e g r a t i o n o f new n e u r o n a l c i r c u i t r y may a f f e c t the spontaneous b e h a v i o u r o f t h e a n i m a l (Chiba & Rankin, 1989; Rankin, S t o p f e r , Marcus & Carew, 1987). The n e u r a l c i r c u i t r y i n male C. elegans, w i t h t h e a d d i t i o n o f 14 p o s t e r i o r sensory s t r u c t u r e s and 79 male s p e c i f i c neurons, i s d i s s i m i l a r from t h a t o f t h e hermaphrodite. T h i s experiment was designed t o e x p l o r e whether t h i s d i f f e r e n c e was r e f l e c t e d i n the frequency and magnitude of spontaneous r e v e r s a l a c t i v i t y i n t h e male a d u l t as compared t o t h e hermaphrodite. S u b j e c t s : Twenty male and twenty h e r m a p h r o d i t i c C. elegans were used i n t h i s experiment. A l l animals were a t l e a s t f o u r days but not' e x c e e d i n g f i v e days o l d . P r o c e d u r e : Immediately f o l l o w i n g t r a n s f e r t o a t e s t p l a t e , animals were a l l o w e d t o accommodate t o t h e i r new environment f o r one minute. A t e n minute p e r i o d of spontaneous a c t i v i t y was t h e n r e c o r d e d . T e s t s o f males and hermaphrodites were a l t e r n a t e d t o c o n t r o l f o r any a g i n g e f f e c t s d u r i n g t h e c o u r s e o f t h e experiment. 1 3 S c o r i n g and S t a t i s t i c a l A n a l y s e s ; V i d e o t a p e s were s c o r e d f o r t o t a l number and magnitude of r e v e r s a l s d u r i n g the t e n minute o b s e r v a t i o n p e r i o d . The t o t a l number and average magnitude of the r e v e r s a l s were a n a l y z e d u s i n g o v e r a l l ANOVAs. R e s u l t s ; R e v e r s a l Frequency: The frequency o f spontaneous r e v e r s a l s d u r i n g t h e 10 minute t e s t p e r i o d was s i g n i f i c a n t l y h i g h e r i n male C. elegans t h a n i n hermaphrodites [ F i g u r e 1A, F(1,38)=14.37, rKO.OOl]. T h i s e f f e c t was not due a d i f f e r e n c e i n a c t i v i t y l e v e l s s i n c e t h e r e were no s i g n i f i c a n t d i f f e r e n c e s between males and hermaphrodites i n time a c t i v e as measured by t o t a l t i me a c t i v e (time spent swimming forward and backward), [ F i g u r e IB, t(19)=1.209, p=0.1207], A c l o s e r e x a m i n a t i o n of the v i d e o t a p e d b e h a v i o u r showed t h a t t h e l a r g e number of spontaneous r e v e r s a l s i n males c o n s i s t e d o f two types of r e v e r s a l s , 1) s e l f - f e e l i n g r e v e r s a l s and 2) t r u e spontaneous r e v e r s a l s r e s e m b l i n g t h o s e shown by hermaphrodites. A s e l f - f e e l i n g r e v e r s a l was d e f i n e d as a r e v e r s a l i n which the t a i l of the animal made c o n t a c t w i t h i t s own body. The s e l f - f e e l i n g r e v e r s a l accounted f o r a p p r o x i m a t e l y 19% of t h e t o t a l number of r e v e r s a l s observed i n males w h i l e a c c o u n t i n g f o r l e s s than 3% o f r e v e r s a l s shown by hermaphrodites. I n comparing the frequency o f t r u e and spontaneous r e v e r s a l s , males showed a s i g n i f i c a n t l y g r e a t e r 1 4 number o f both t r u e spontaneous r e v e r s a l s [ F i g u r e IC, F(1,38)=8.35, p<0.01] and s e l f - f e e l i n g r e v e r s a l s [ F i g u r e ID, F(1,38)=13.69, p<0.001] than d i d hermaphrodites. R e v e r s a l Magnitude: The average s i z e of males a t t e s t (mean length=49.35, SEM=0.73) was s m a l l e r than t h a t of hermaphrodites (mean length=59.30, SEM=1.20). To ensure t h a t d i f f e r e n c e s i n r e v e r s a l magnitude were not due t o an e f f e c t o f worm l e n g t h , t h e magnitude of each r e v e r s a l was s t a n d a r d i z e d by d i v i d i n g t h e r e v e r s a l l e n g t h by the l e n g t h of t h a t p a r t i c u l a r worm (Chiba & Ra n k i n , 1990; Beck, 1991). When t r u e spontaneous r e v e r s a l s and s e l f - f e e l i n g r e v e r s a l s were grouped t o g e t h e r , the average l e n g t h o f spontaneous r e v e r s a l s was s i g n i f i c a n t l y s m a l l e r i n males than i n hermaphrodites [ F i g u r e 2A, F(1,38)=5.11, p<0.05]. When t h e magnitude of o n l y t r u e spontaneous r e v e r s a l s was a n a l y z e d , t h e r e were no s i g n i f i c a n t d i f f e r e n c e s between males and hermaphrodites [ F i g u r e 2B, F(l,38)=0.34, p=0.5639]. However, t h e magnitude of s e l f - f e e l i n g r e v e r s a l s was s i g n i f i c a n t l y l a r g e r i n males than i n hermaphrodites [ F i g u r e 2C, F(1,38)=8.27, p<0.01]. Moreover, t h e magnitude o f t h e s e l f -f e e l i n g r e v e r s a l i n males was s i g n i f i c a n t l y s m a l l e r t h a n t h e t r u e spontaneous r e v e r s a l shown by t h e males [t(19)=2.054, p=0.027, o n e - t a i l e d t e s t ] . D i s c u s s i o n : The frequency of both t r u e and s e l f - f e e l i n g spontaneous 1 5 F i g u r e 1A. Mean number of r e v e r s a l s + SEM shown by males (n=2 0) and hermaphrodites (n=20) over a t e n minute o b s e r v a t i o n p e r i o d . Males showed s i g n i f i c a n t l y more r e v e r s a l s than hermaphrodites. F i g u r e IB. Mean time a c t i v e + SEM o f males (n=20) and hermaphrodites (n=20) expressed as a percentage o f t o t a l o b s e r v a t i o n time (10 min). Males and hermaphrodites d i d not d i f f e r i n a c t i v i t y l e v e l s d u r i n g the t e n minute o b s e r v a t i o n p e r i o d . F i g u r e IC. Mean number of t r u e spontaneous r e v e r s a l s + SEM ( e x c l u d i n g s e l f - f e e l i n g r e v e r s a l s ; see t e x t ) shown by males (n=20) and hermaphrodites (n=20) over a t e n minute o b s e r v a t i o n p e r i o d . Males showed s i g n i f i c a n t l y more t r u e spontaneous r e v e r s a l s t h a n hermaphrodites. F i g u r e ID. Mean number of s e l f - f e e l i n g r e v e r s a l s + SEM shown by males (n=20) and hermaphrodites (n=20) over a t e n minute o b s e r v a t i o n p e r i o d . Males showed s i g n i f i c a n t l y more s e l f -f e e l i n g r e v e r s a l s than hermaphrodites. NUMBER OF REVERSALS O x m 3J 1 7 F i g u r e 2A. Mean magnitude of spontaneous r e v e r s a l s + SEM shown by males (n=20) and hermaphrodites (n=20). T h i s was o b t a i n e d by t a k i n g t h e mean magnitude o f a l l t h e spontaneous r e v e r s a l shown by each worm and d i v i d i n g t h a t mean magnitude by t h e l e n g t h o f t h a t p a r t i c u l a r worm. Males showed a s i g n i f i c a n t l y s m a l l e r average r e v e r s a l magnitude than hermaphrodites. F i g u r e 2B. Mean magnitude of t r u e spontaneous r e v e r s a l s + SEM shown by males (n=20) and hermaphrodites (n=20). C a l c u l a t i o n o f mean magnitude was the same as i n F i g u r e 2A, w i t h t h e e x c e p t i o n t h a t o n l y t r u e spontaneous r e v e r s a l s were c o n s i d e r e d . There were no d i f f e r e n c e s i n r e v e r s a l magnitude between males and hermaphrodites. F i g u r e 2C. Mean magnitude of s e l f - f e e l i n g r e v e r s a l s + SEM shown by males (n=20) and hermaphrodites (n=20). C a l c u l a t i o n o f mean magnitude was t h e same as in,.Figure 2A, w i t h t h e e x c e p t i o n t h a t o n l y s e l f - f e e l i n g r e v e r s a l s were c o n s i d e r e d . The magnitude o f s e l f - f e e l i n g r e v e r s a l s was s i g n i f i c a n t l y l a r g e r i n males than i n hermaphrodites. CO 1 9 r e v e r s a l s was h i g h e r i n males than i n hermaphrodites. Upon c l o s e i n s p e c t i o n , i t appears t h a t t h e s e l f - f e e l i n g r e v e r s a l c o n s t i t u t e s a unique c l a s s , q f r e v e r s a l a c t i v i t y . I n t h e male, when t h e t a i l c o n t a c t s i t s own body, the e n t i r e f a n - l i k e t a i l s t r u c t u r e i s p l a c e d f i r m l y and rubbed v i g o r o u s l y on i t s own c u t i c l e d u r i n g t h e r e v e r s a l ; when the t a i l o f the hermaphrodite c o n t a c t s i t s own body, i t w i l l o n l y b r i e f l y brush i t s c u t i c l e w i t h i t s t a i l . Thus, i t appears t h a t the s e l f - f e e l i n g r e v e r s a l o f t h e male i s an a c t i v e attempt t o mate w h i l e t h a t of t h e hermaphrodite i s more p e r f u n c t o r y i n n a t u r e . The d i s t i n c t n a t u r e of t h e s e l f -f e e l i n g r e v e r s a l of t h e male i s f u r t h e r e v i n c e d by t h e f i n d i n g t h a t t h e average l e n g t h of t h e s e l f - f e e l i n g r e v e r s a l e x h i b i t e d by t h e male i s l a r g e r than t h a t shown by t h e hermaphrodite as w e l l s m a l l e r t h a n t r u e spontaneous r e v e r s a l s shown by t h e male. I n c o n t r a s t t o s e l f - f e e l i n g r e v e r s a l s , t r u e spontaneous r e v e r s a l s o c c u r r e d w i t h the same magnitude i n b o t h s e x e s , a l b e i t w i t h g r e a t e r frequency i n the males. G i v e n t h a t the males demonstrate a spontaneous a c t i v i t y p a t t e r n d i s t i n c t from t h a t of hermaphrodites, Experiment I I was d e s i g n e d t o determine whether these d i f f e r e n c e s might o c c u r i n response t o mechanosensory s t i m u l a t i o n . Experiment I I : Response to Graded Tap D u r a t i o n A s i n g l e t a p t o a d i s h c o n t a i n i n g an a d u l t hermaphrodite C. elegans has been shown t o cause the animal t o r e v e r s e (Chiba & R a n k i n , 1990). I n c r e a s i n g the number o f ta p s a d m i n i s t e r e d t o 20 a d u l t hermaphrodites has been shown t o i n c r e a s e t h e magnitude o f t h e r e v e r s a l s as w e l l as t o decrease t h e p r o p o r t i o n o f a n i m a l s showing th e r e v e r s a l response (Chiba & R a n k i n , 1990). S i n c e t h e male possesses 14 more sensory s t r u c t u r e s t h a n t h e hermaphrodite, th e p o s s i b i l i t y e x i s t s t h a t the male i s more s e n s i t i v e t o a v i b r a t o r y s t i m u l u s . T h i s experiment was d e s i g n e d t o t e s t whether male 'C.elegans would show a r e v e r s a l i n response t o a t a p , and i f a f f i r m a t i v e , t o t e s t t h e e f f e c t o f i n c r e a s i n g t h e s t i m u l u s magnitude by a d m i n i s t e r i n g a t r a i n o f 3 and 6 t a p s . The p r o p o r t i o n o f males r e s p o n d i n g , as w e l l as t h e magnitude of t h e r e v e r s a l , was measured i n a l l c o n d i t i o n s . A l l responses made by the males were compared t o t h o s e o f hermaphrodites t o determine whether t h e r e were d i f f e r e n c e s i n t h e i r r e s p e c t i v e responses t o t a p s . S u b j e c t s ; S i x t y males and s i x t y hermaphrodites were used w i t h ages of a t l e a s t f o u r but not exceeding f i v e days of age. P r o c e d u r e ; Animals were t e s t e d under t h r e e c o n d i t i o n s ; a s i n g l e t a p , a t r a i n o f t h r e e taps and a t r a i n o f s i x t a p s . The d i f f e r e n t numbers of, t a p s were produced by v a r y i n g t h e t r a i n d u r a t i o n s e t t i n g on the S88 s t i m u l a t o r . At 8.5 p u l s e s per second, a d u r a t i o n o f 100 ms r e s u l t e d i n a s i n g l e t a p , a d u r a t i o n o f 300 ms r e s u l t e d i n a t r a i n of t h r e e t a p s , w h i l e a d u r a t i o n o f 600 ms produced a t r a i n o f t h r e e t a p s . S i n g l e t a p s were a d m i n i s t e r e d t o 20 males and 20 hermaphrodites, a t r a i n o f 21 t h r e e t a p s was g i v e n i n d i v i d u a l l y t o a second group o f 20 males and 20 hermaphrodites, and a t r a i n of s i x ta p s was g i v e n t o a t h i r d group of 20 males and 20 hermaphrodites. S c o r i n g and S t a t i s t i c a l A n a l y s e s : Measurements i n c l u d e d t h e p r o p o r t i o n o f animals r e v e r s i n g t o t h e d i f f e r i n g t r a i n s of taps and the magnitude o f t h e r e v e r s a l responses. P r o p o r t i o n a l d a t a were a n a l y z e d w i t h a t w o - f a c t o r ANOVA u s i n g dichotomous d a t a (Winer, Brown & M i c h e l s , 1991). W i t h i n and between group comparisons on p r o p o r t i o n d a t a were a n a l y z e d by the method o f L e a s t Square Means. Magnitude d a t a were t e s t e d u s i n g an o v e r a l l t w o - f a c t o r ANOVA as w e l l as by the, method of L e a s t Square Means. Responses where the animal showed no r e v e r s a l were r e p l a c e d w i t h t h e group mean f o r the s t a t i s t i c a l a n a l y s i s (Winer, 1971). R e s u l t s : R e v e r s a l Frequency: As t h e number of taps i n c r e a s e d , t h e frequency o f r e v e r s a l s i n hermaphrodites d i d not change. In c o n t r a s t , males showed fewer r e v e r s a l responses as the number of taps i n c r e a s e d ( F i g u r e 3A); t h e number of worms r e v e r s i n g t o t h r e e and s i x t a p s was s i g n i f i c a n t l y l e s s than t o a s i n g l e t a p (p=0.0201 and p=0.0006, r e s p e c t i v e l y ) . I n f a c t , males showed an i n c r e a s i n g tendency t o respond w i t h a g r e a t e r number of a c c e l e r a t i o n s as t a p number i n c r e a s e d ( F i g u r e 3B). These o b s e r v a t i o n s were c o n f i r m e d by a t w o - f a c t o r ANOVA which r e v e a l e d s i g n i f i c a n t main e f f e c t s f o r F i g u r e 3A. The number of males (n=20) and hermaphrodites (n=20) showing a r e v e r s a l t o 1, 3 o r 6 t a p s . Males showed fewer r e v e r s a l s as t h e number of taps a d m i n i s t e r e d i n c r e a s e d . The number o f r e v e r s a l s shown by hermaphrodites d i d not change s i g n i f i c a n t l y w i t h an i n c r e a s e i n t a p number. F i g u r e 3B. The number of males (n=20) and hermaphrodites (n=20) showing an a c c e l e r a t i o n t o 1, 3 o r 6 t a p s . Males showed an i n c r e a s i n g number of a c c e l e r a t i o n s as the number o f t a p s a d m i n i s t e r e d i n c r e a s e d . The number of a c c e l e r a t i o n s shown by hermaphrodites d i d not change s i g n i f i c a n t l y as t a p number i n c r e a s e d . number of t a p s [F(2,114)=3.141, p=0.047], sex [F(1,114)=8.702, p=0.0039] and a s i g n i f i c a n t i n t e r a c t i o n between sex and number o f t a p s [F(2,114)=3.450, p=0.0351]. R e v e r s a l Magnitude: As t h e number of taps i n c r e a s e d , t h e r e was an o v e r a l l i n c r e a s e i n r e v e r s a l magnitude [F(2,114)=9.324, p=0.0002]. T h i s r e l a t i o n s h i p o c c u r r e d i n both males [ p o s i t i v e l i n e a r t r e n d , F(1,114)=6.461, p<0.025] and i n hermaphrodites [ p o s i t i v e l i n e a r t r e n d , F( 1,114)=11.575, p<0.001]. ( F i g u r e 4 ) . However, males and hermaphrodites d i d not respond w i t h t h e same p a t t e r n o f r e v e r s a l magnitudes t o the d i f f e r e n t number of t a p s , as r e v e a l e d by t h e s i g n i f i c a n t i n t e r a c t i o n between sex and number of t a p s [F(2,114)=4.837, p=0.0096]. Indeed, males responded w i t h n e a r l y e q u i v a l e n t r e v e r s a l magnitudes t o one and t h r e e t a p s , but t h e y responded w i t h s u b s t a n t i a l l y l a r g e r responses t o s i x t a p s . I n c o n t r a s t , hermaphrodites responded e q u a l l y t o t h r e e and s i x t a p s , w h i l e showing a s u b s t a n t i a l l y s m a l l e r response t o a s i n g l e t a p . O v e r a l l , t h e average r e v e r s a l magnitude o f t h e hermaphrodites was l a r g e r than t h a t of t h e males [F(l,114)=35.094, p<0.0001]. At i n d i v i d u a l t a p c o n d i t i o n s , t h i s d i f f e r e n c e approached s i g n i f i c a n c e o n l y a t t h e t h r e e t a p c o n d i t i o n ( s i m p l e e f f e c t s , F( 1,114)=3.498, p<0.08), w i t h t h e s i n g l e and s i x t a p c o n d i t i o n showing no s i g n i f i c a n t e f f e c t ( s i m p l e e f f e c t s , F( 1,114)=0.0973, p>0.25 and F(1,114)=0.6786, p>0.25, r e s p e c t i v e l y ) . F i g u r e 4. Mean r e v e r s a l magnitude + SEM of males (n=20) and hermaphrodites (n=20) i n response t o 1, 3 o r 6 t a p s . R e v e r s a l magnitude f o r each animal was s t a n d a r d i z e d by d i v i d i n g t h e r e v e r s a l observed by t h e l e n g t h of t h e animal showing t h a t r e v e r s a l . Both males and hermaphrodites showed a s i g n i f i c a n t i n c r e a s e i n response magnitude as t a p number i n c r e a s e d . Males responded w i t h a p p r o x i m a t e l y equal magnitude t o 1 and 3 t a p s . Hermaphrodites responded w i t h the same approximate magnitude t o 3 and 6 t a p s . 26 M A L E HERM 27 D i s c u s s i o n The hermaphrodites responded w i t h approximately-e q u i v a l e n t r e v e r s a l magnitudes t o t h e t r a i n s o f t h r e e and s i x t a p s , w h i l e t h e males tended t o respond t o t h e s i n g l e t a p and t r a i n o f t h r e e t a p s e q u a l l y . S u p e r f i c i a l l y , t h i s might suggest t h a t t h e t h r e s h o l d f o r d i s c r i m i n a t i o n of d i f f e r e n c e s i n number of t a p s i s h i g h e r i n males than i n hermaphrodites. However, t h i s h y p o t h e s i s i s not supported by the observed i n c i d e n c e o f r e s p o n d i n g t o t h e t h r e e t a p c o n d i t i o n s . Indeed, t h e t o t a l number o f responses d i s p l a y e d by t h e male and hermaphrodite was not s i g n i f i c a n t l y d i f f e r e n t , however, males showed more a c c e l e r a t i o n s than hermaphrodites a t h i g h e r t a p d u r a t i o n . R a n k i n and her c o l l e a g u e s (1990) h y p o t h e s i z e d t h a t t a p a c t i v a t e s mechanosensory r e c e p t o r s i n both t h e head and t a i l of t h e a n i m a l . I f t h i s i s the case, t h e 14 a d d i t i o n a l s e n s o r y r e c e p t o r s i n t h e t a i l of the male may r e s u l t i n an a s y m m e t r i c a l s e n s i t i v i t y a t t h e p o s t e r i o r end of the a n i m a l , w i t h t h e t a i l b e i n g more s e n s i t i v e t o s t i m u l a t i o n than t h e head. I n response t o an i n c r e a s i n g number of t a p s , t h e h y p o t h e s i z e d a s y m m e t r i c a l r e s p o n s i v e n e s s c o u l d r e s u l t i n a p o s i t i v e b i a s toward i n p u t from t h e p o s t e r i o r of t h e a n i m a l , and thus l e a d t o an i n c r e a s i n g number of a c c e l e r a t i o n s . The h y p o t h e s i s of asymmetrical s e n s i t i v i t y can a l s o be used t o e x p l a i n the f i n d i n g t h a t males demonstrate s m a l l e r r e v e r s a l magnitudes than hermaphrodites. The i n c r e a s e d s e n s i t i v i t y i n t h e t a i l may produce a competing s i g n a l which i s 2 8 not s t r o n g enough t o c o m p l e t e l y overcome t h e r e v e r s a l r e sponse, i n s t e a d , i t might o n l y produce a s i g n a l s t r o n g enough t o reduce t h e magnitude o f the r e v e r s a l s which do o c c u r . Thus, assuming t h a t t h e males a r e as s e n s i t i v e o r more s e n s i t i v e t o t a p as herma p h r o d i t e s , t h e males s h o u l d demonstrate s m a l l e r r e v e r s a l responses than hermaphrodites. To f u r t h e r e x p l o r e the p o s s i b i l i t y o f i n c r e a s e d s e n s i t i v i t y t o t a p i n the male, Experiment I I I was d e s i g n e d t o t e s t t h e response o f males and hermaphrodites t o a s i n g l e t a p of d i f f e r i n g i n t e n s i t y . Experiment I I I : Response to Graded Tap I n t e n s i t y As was suggested i n Experiment I I , a s y m m e t r i c a l s e n s i t i v i t y i n males may have a f f e c t e d t h e i r p a t t e r n o f responses t o t a p . By v a r y i n g the i n t e n s i t y o f a s i n g l e t a p , t h i s experiment was designed t o compare t h e s e n s i t i v i t y o f males and hermaphrodites t o weak and s t r o n g t a p s . S u b j e c t s : S i x t y males and s i x t y hermaphrodites were used w i t h ages o f a t l e a s t f o u r but not exceeding f i v e days o f age. Pro c e d u r e : I n t h i s s e r i e s o f experiments, worms were p l a c e d i n d i v i d u a l l y on un s t r e a k e d t e s t p l a t e s a p p r o x i m a t e l y two hours p r i o r t o t e s t i n g . Tap i n t e n s i t y was v a r i e d by a l t e r i n g t h e i n t e n s i t y o f v o l t a g e p r e s e n t e d t o t h e e l e c t r o m a g n e t i c r e l a y c o n t r o l l i n g t h e t a p p e r . A s i n g l e t a p w i t h an i n t e n s i t y o f 34 V was a d m i n i s t e r e d t o 20 males and 20 hermaphrodites. A s i n g l e t a p o f 40 V i n t e n s i t y was g i v e n i n d i v i d u a l l y t o a second group 20 males and 20 hermaphrodites. A s i n g l e t a p of 60 V i n t e n s i t y was g i v e n t o a t h i r d group of 20 males and 20 hermaphrodites. The 34 V i n t e n s i t y produced a b a r e l y p e r c e p t i b l e ( t o t h e e x p e r i m e n t e r ) t h r e s h o l d t a p ; the 40 V i n t e n s i t y t a p was n o t i c e a b l y s t r o n g e r , w h i l e t h e 60 V i n t e n s i t y produced t h e s t r o n g e s t t a p . S c o r i n g and S t a t i s t i c a l A n a l y s e s ; Measurements i n c l u d e d t h e p r o p o r t i o n of animals r e v e r s i n g t o t h e d i f f e r i n g i n t e n s i t i e s of t a p and t h e magnitude o f t h e r e v e r s a l b e h a v i o u r . P r o p o r t i o n a l d a t a were a n a l y z e d u s i n g a t w o - f a c t o r AVOVA. Between groups and w i t h i n group d i f f e r e n c e s were a n a l y z e d w i t h the L e a s t Square Means method. W i t h i n group magnitude d a t a were a n a l y z e d u s i n g an o v e r a l l t w o - f a c t o r ANOVA. Responses where the animal showed no r e v e r s a l were r e p l a c e d w i t h t h e group mean f o r the s t a t i s t i c a l a n a l y s i s (Winer, 1971). R e s u l t s ; R e v e r s a l Frequency: As t a p i n t e n s i t y i n c r e a s e d , t h e number of r e v e r s a l s shown by both males and hermaphrodites i n c r e a s e d [ F i g u r e 5A, F{2,114)=7.171, p=0.0012]. A l l worms showed s i g n i f i c a n t l y fewer r e v e r s a l s t o 34 V i n t e n s i t y t a p s than t o 40 o r 60 V t a p s (p<0.05). Furthermore, t h e r e was no d i f f e r e n c e between males and hermaphrodites i n the number o r p a t t e r n of r e v e r s a l s e x h i b i t e d over a l l t a p i n t e n s i t i e s [sex: F{1,114)=0.665, 30 p=0.416; sex by t a p i n t e n s i t y i n t e r a c t i o n : F(2,114)=0.222, p=0.8014]. There were v e r y few a c c e l e r a t i o n s i n response t o t h e t a p , and t h e r e were no d i f f e r e n c e s i n a c c e l e r a t i o n f r e q u e n c y between t h e males and hermaphrodites ( F i g u r e 5B). R e v e r s a l Magnitude: Animals of both sexes responded t o i n c r e a s e s i n t a p i n t e n s i t y w i t h i n c r e a s i n g r e v e r s a l magnitudes [ F i g u r e 6, F(2,114)=4.957, p=0.0086]. At a l l t a p i n t e n s i t i e s , response magnitudes were g r e a t e r i n males than i n hermaphrodites, [F(1,114)=11.983, p=0.0008]. The r e l a t i o n s h i p between t a p i n t e n s i t y and i n c r e a s i n g r e v e r s a l magnitude was c h a r a c t e r i z e d by a p a r a l l e l l i n e a r r e l a t i o n s h i p i n both males and hermaphrodites, as i n d i c a t e d by the n o n - s i g n i f i c a n t i n t e r a c t i o n between gender and t a p i n t e n s i t y [F( 1,114)=0.201, p=0.8180]. However, th e l i n e a r t r e n d was s i g n i f i c a n t o n l y i n t h e hermaphrodites [males: F( 1,114)=3.090, p>0.05; hermaphrodites: F(l,114)=6.904, p<0.01]. D i s c u s s i o n : As t h e i n t e n s i t y of t h e taps i n c r e a s e d , t h e f r e q u e n c y of r e v e r s a l s i n males and hermaphrodites d i d not d i f f e r ; however, i n response t o i n c r e a s i n g t a p i n t e n s i t y , b oth males and hermaphrodites showed an i n c r e a s e i n r e v e r s a l magnitude. Males e x h i b i t e d l a r g e r r e v e r s a l magnitudes t h a n hermaphrodites a t a l l t a p i n t e n s i t i e s . T h i s was an unexpected r e s u l t , s i n c e hermaphrodites showed l a r g e r r e v e r s a l magnitudes 31 F i g u r e 5A. The number of males (n=20) and hermaphrodites (n=20) showing a r e v e r s a l t o taps of 34, 40 and 60 V i n t e n s i t y . S i g n i f i c a n t l y fewer males and' hermaphrodites r e v e r s e d t o a t a p of 34 V i n t e n s i t y , whereas most animals r e v e r s e d t o t a p s o f 40 and 60 V. There were no sex d i f f e r e n c e s a t any t a p i n t e n s i t y . F i g u r e 5B. The number of males (n=20) and hermaphrodites (n=20) showing an a c c e l e r a t i o n t o t a p s o f 34, 40 and 60 V i n t e n s i t y . There were no s i g n i f i c a n t d i f f e r e n c e s i n t h e number o f a c c e l e r a t i o n s shown by males a t any t a p i n t e n s i t y . Hermaphrodites showed no a c c e l e r a t i o n s a t any t a p i n t e n s i t y . 32 _ i < U l > HI DC CC UJ m 34 V 40 V 60 V M A L E HERM B <n z o I-< oc U l - I U l o u < 20 -i 15 -1 0 -34 V 40 V 60 V DC U l m 5 -HERM 33 F i g u r e 6. Mean r e v e r s a l magnitude + SEM of males (n=20) and hermaphrodites (n=20) i n response t o taps o f 34, 40 and 60 V i n t e n s i t y . R e v e r s a l magnitude f o r each animal was s t a n d a r d i z e d by d i v i d i n g t h e r e v e r s a l observed by t h e l e n g t h o f t h e a n i m a l showing t h a t r e v e r s a l . Both males and hermaphrodites showed l a r g e r r e v e r s a l s as t a p i n t e n s i t y i n c r e a s e d . Response magnitudes were s i g n i f i c a n t l y h i g h e e r i n males than i n hermaphrodites a t each t a p i n t e n s i t y . 34 M A L E HERM 3 5 t h a n t h e males i n the graded d u r a t i o n experiment. T h i s d i f f e r e n c e might r e f l e c t d i f f e r e n c e s i n h a n d l i n g c o n d i t i o n s between t h e two experiments. I n the graded d u r a t i o n experiment, animals were t e s t e d o n l y 1-2 minutes a f t e r b e i n g p l a c e d on t h e t e s t p l a t e . In c o n t r a s t , i n t h e graded i n t e n s i t y e x periment, animals were l e f t on t e s t p l a t e s f o r 2 hours p r i o r t o t e s t i n g . The l o n g e r d u r a t i o n may have a l l o w e d a n i m a l s t o r e c o v e r from t h e e f f e c t s of h a n d l i n g . The hermaphrodites showed a s i g n i f i c a n t l y l a r g e r response i f t e s t e d i m m e d i a t e l y a f t e r h a n d l i n g . Thus, the l a r g e magnitude responses o f hermaphrodites i n the graded d u r a t i o n experiment might have been due t o s e n s i t i z a t i o n as a r e s u l t of h a n d l i n g . I f t h e s e f i n d i n g s were r e p l i c a t e d when h a n d l i n g c o n d i t i o n s were th e same, an a l t e r n a t i v e h y p o t h e s i s might e x p l a i n t h e d i f f e r e n c e s shown by males and hermaphrodites i n t h e graded d u r a t i o n and graded i n t e n s i t y e x p e r i m e n t s . The h y p o t h e s i s assumes a q u a l i t a t i v e d i f f e r e n c e between th e number of t a p s and i n t e n s i t y of t a p s , as opposed t o a q u a n t i t a t i v e d i f f e r e n c e . Consequently, i n f o r m a t i o n about t h e number of t a p s might be p r o c e s s e d d i f f e r e n t l y than i n f o r m a t i o n about i n t e n s i t y of a s i n g l e t a p , l e a d i n g t o a d i f f e r e n t response p a t t e r n f o r number and i n t e n s i t y o f t a p s . Such a h y p o t h e s i s i s t e s t a b l e by c o v a r y i n g t h e t a p i n t e n s i t y and number. Another way t o assess t h e r e l a t i o n s h i p between i n p u t from t h e head and t a i l of t h e male worm might be t o p l a c e i n p u t from t h e head and t a i l r e c e p t o r s i n d i r e c t c o m p e t i t i o n . T h i s was t e s t e d i n t h e next experiment i n which response c o m p e t i t i o n between t a i l - t o u c h and t a p was examined. Experiment IV: I n h i b i t i o n A t a p t o a d i s h c o n t a i n i n g C. elegans w i l l u s u a l l y r e s u l t i n a r e v e r s a l by the animal i f i t i s swimming i n a f o r w a r d d i r e c t i o n o r i s m o t i o n l e s s p r i o r t o d e l i v e r y o f a t a p . C o n v e r s e l y , a t o u c h t o the t a i l of t h e animal w i l l r e s u l t i n f o r w a r d motion. When a t a p i s g i v e n i n c l o s e t e m p o r a l p r o x i m i t y t o a t a i l t o u c h , c o m p e t i t i o n between t h e r e v e r s a l response and t h e forward swimming response w i l l o c c u r . I t has been shown t h a t a t a i l touch p r e c e d i n g a t a p w i l l l e a d t o an i n h i b i t i o n o f r e v e r s i n g o r a decrement i n t h e r e v e r s a l response observed i n hermaphrodites, however, a t a p a d m i n i s t e r e d p r i o r t o a t a i l t ouch does not i n h i b i t t h e response t o t o u c h (Rankin, 1991). I n an experiment t e s t i n g the d u r a t i o n o f t h i s t a i l t o u c h i n d u c e d i n h i b i t i o n of r e v e r s a l t o t a p , Rankin (1991) showed t h a t i n hermaphrodites the i n h i b i t o r y e f f e c t o f t a i l t o u c h l a s t e d l e s s than 10 s, and that 20 s a f t e r t a i l t o u c h t h e r e appeared t o be a f a c i l i t a t i o n of t h e a m p l i t u d e o f t h e r e v e r s a l t o t a p . The sensory apparatus of the t a i l of t h e male C. elegans c o n t a i n s 14 more sensory s t r u c t u r e s than t h e hermaphrodite. T h i s may r e s u l t i n a response b i a s r e s u l t i n g from an i n c r e a s e d amount of i n f o r m a t i o n r e c e i v e d by the p o s t e r i o r end o f t h e male. T h i s experiment was designed t o t e s t whether males were c a p a b l e o f showing the i n h i b i t i o n response and i f so, t o t e s t t h e h y p o t h e s i s o f response b i a s by comparing th e d u r a t i o n and r e l a t i v e a m p l i t u d e of the i n h i b i t i o n response observed i n males w i t h t h a t observed i n hermaphrodites. S u b j e c t s : A t o t a l of 40 s u b j e c t s were used, c o n s i s t i n g o f 20 males and 20 hermaphrodites. P r o c e d u r e : Animals were p l a c e d i n d i v i d u a l l y on t h e t e s t p l a t e 24 hours i n advance of t e s t i n g . A s m a l l amount o f E. coli was p l a c e d on t h e t e s t p l a t e w i t h the worm t o ensure th e s u b j e c t was not f o o d d e p r i v e d . Each animal underwent seven d i f f e r e n t s t i m u l u s c o n d i t i o n s , s e p a r a t e d by i n t e r v a l s o f a p p r o x i m a t e l y 15-20 min. The c o n d i t i o n s were: 1) a s i n g l e t a p , 2) a t a i l - t o u c h f o l l o w e d 1 s l a t e r by a t a p , 3) a t a i l - t o u c h f o l l o w e d 5 s l a t e r by a t a p , 4) a t a i l - t o u c h f o l l o w e d 10 s l a t e r by a t a p , 5) a t a i l -t o u c h f o l l o w e d 20 s l a t e r by a t a p , 6) a t a i l - t o u c h f o l l o w e d 30 s l a t e r by a t a p , 7) a s i n g l e t a p a l o n e . C o n d i t i o n 1 e s t a b l i s h e d a b a s e l i n e l e v e l of response t o t a p a l o n e . C o n d i t i o n s 2-6 t e s t e d f o r the e x i s t e n c e of t h e i n h i b i t i o n e f f e c t and e x p l o r e d t h e temporal parameters o f t h e e f f e c t i t s e l f . C o n d i t i o n 7 was i n t e n d e d t o t e s t whether th e response t o t a p a l o n e showed any evidence of h a b i t u a t i o n due t o t h e a d m i n i s t r a t i o n o f a l l t h e s t i m u l i of c o n d i t i o n s 2-6. 38 T a i l - t o u c h was a d m i n i s t e r e d by hand by s t r o k i n g t h e t a i l o f t h e a n i m a l w i t h an e y e l a s h mounted t o t h e t i p o f a 30 gauge ne e d l e . S c o r i n g and S t a t i s t i c a l A n a l y s e s ; The frequency of r e v e r s a l s t o t a p was examined a c r o s s sexes t o determine between-sex d i f f e r e n c e s . W i t h i n - s e x r e v e r s a l f r e q u e n c y was measured a c r o s s i n c r e a s i n g t a i l - t o u c h t a p i n t e r v a l s t o determine the e f f e c t s o f t h i s i n t e r v a l on i n h i b i t i o n o f r e v e r s a l s . A d d i t i o n a l l y , t h e magnitude o f t h e t a p response was measured i n each sex and a n a l y z e d w i t h a one f a c t o r r e p e a t e d measures ANOVA, and m u l t i p l e comparisons were c a r r i e d out w i t h F i s h e r ' s PLSD. R e s u l t s ; R e v e r s a l Frequency: A l l a n imals r e v e r s e d t o the i n i t i a l t a p . Both males and hermaphrodites showed a l a r g e decrease i n r e v e r s a l responses t o t a p a t t h e one second t a i l - t o u c h / t a p i n t e r v a l (p<0.01 i n both c a s e s ) . A t a l l o t h e r i n t e r v a l s , t h e r e were no s i g n i f i c a n t d i f f e r e n c e s i n t h e number of animals r e v e r s i n g . A d d i t i o n a l l y , males and hermaphrodites showed no d i f f e r e n c e s i n t h e number of r e v e r s a l s shown a t any of the t a p i n t e r v a l s ( F i g u r e 7A) . R e v e r s a l Magnitude: To d i r e c t l y compare i n h i b i t i o n between males and hermaphrodites, r e v e r s a l magnitude was s t a n d a r d i z e d by e x p r e s s i n g each worm's response as a percentage o f i t s i n i t i a l r esponse t o t a p , which was s e t a t 100%. I n males, the 1 and 5 s t a i l - t o u c h / t a p i n t e r v a l r e s u l t e d i n s i g n i f i c a n t l y s m a l l e r r e v e r s a l magnitudes compared t o the i n i t i a l response t o t a p alone [F(6,114)=13.814, rxO.OOl, F i s h e r ' s PLSD: p<0.01 and p<0.05 f r e s p e c t i v e l y ) . There were no o t h e r d i f f e r e n c e s a t any o t h e r i n t e r v a l s ( F i g u r e 7B). I n hermaphrodites, s m a l l e r r e v e r s a l magnitudes were observ e d a t both t h e 1 and 5 s t a i l - t o u c h / t a p i n t e r v a l [F(6,114)=15.844, p<0.001, F i s h e r ' s PLSD: p<0.01 i n b o t h cases) when compared w i t h the i n i t i a l response. Moreover, t h e responses o f hermaphrodites a t the 20 and 30 s i n t e r v a l and a t t h e l a s t t a p were s i g n i f i c a n t l y l a r g e r than t h e i r i n i t i a l r esponses (p<0.01, F i g u r e 7B). Between groups a n a l y s e s r e v e a l e d no sex d i f f e r e n c e s i n r e v e r s a l magnitude a t any of the t a i l - t o u c h t a p i n t e r v a l s [F(l,38)=0.929, p=0.3413]. D i s c u s s i o n : In males and hermaphrodites, a t a i l - t o u c h a d m i n i s t e r e d p r i o r t o a t a p r e s u l t e d i n an i n h i b i t i o n o f both t h e f r e q u e n c y of r e v e r s a l s and the magnitude of r e v e r s a l s a t s h o r t t o u c h - t a p i n t e r v a l s . S i n c e t h e r e was no s i g n i f i c a n t d i f f e r e n c e i n i n h i b i t i o n between t h e sexes, i t i s u n l i k e l y t h a t t h e r e e x i s t s a response b i a s i n t h e male as a r e s u l t of the i n c r e a s e d s e n s o r y i n p u t made a v a i l a b l e by the p o s t e r i o r sensory r e c e p t o r s o f t h e male. I n t e r e s t i n g l y , t h e males d i d not appear t o show f a c i l i t a t i o n a t 20 and 30 s i n t e r v a l t a i l - t o u c h c o n d i t i o n s as hermaphrodites 40 F i g u r e 7A. Number of males (n=20) and hermaphrodites (n=20) showing r e v e r s a l s t o t a p and touch-tap i n t e r v a l s o f 1, 5, 10, 20 and 30 s. Both males and hermaphrodites show s i g n i f i c a n t i n h i b i t i o n o f r e v e r s a l a c t i v i t y t o t a p a t t h e 1 s t a i l - t o u c h / t a p i n t e r v a l . F i g u r e 7B. Mean magnitude of r e v e r s a l s + SEM o f males (n=20) and hermaphrodites (n=20) t o t a p and touch-tap i n t e r v a l s o f 1, 5, 10, 20 and 30 s. R e v e r s a l magnitude i s expressed as a percentage o f each a n i m a l s r e v e r s a l t o the i n i t i a l t a p (which was s e t a t 100%). Males and hermaphrodites showed s i g n i f i c a n t i n h i b i t i o n o f r e v e r s a l magnitude t o t a p a t t h e 1 and 5 s t a i l - t o u c h / t a p i n t e r v a l . Hermaphrodites showed s i g n i f i c a n t f a c i l i t a t i o n o f r e v e r s a l magnitude t o t a p a t t h e 20 and 30 s t a i l - t o u c h / t a p i n t e r v a l and a t t h e f i n a l t a p . Males d i d not show s i g n i f i c a n t f a c i l i t a t i o n . 1 '4, % 1 V/-41 • MALE ffl HERM TAP 1S 5S 10S 20S 30S TAP TAIL-TOUCH/TAP INTERVAL 200 T T X MALE HERM TAP "IS 5S 10S 20S 30S TAP TAIL-TOUCH/TAP INTERVAL d i d . However, t h e r e was a t r e n d toward l a r g e r responses ( i e . f a c i l i t a t i o n ) i n males, and t h e d i f f e r e n c e i n r e v e r s a l magnitude between males and hermaphrodites was not s i g n i f i c a n t l y d i f f e r e n t a t t h e s e t a i l - t o u c h / t a p i n t e r v a l s . Experiment V: T a i l - t o u c h H a b i t u a t i o n The n a t u r e of t h e i n t e r a c t i o n between two a n t a g o n i s t i c r e f l e x e s can be changed by e x p e r i e n c e , d e m o n s t r a t i n g t h e presence of p l a s t i c i t y i n r e f l e x i n t e r a c t i o n . H a b i t u a t i o n t o t a i l - t o u c h by t h e a d m i n i s t r a t i o n of 25 t a i l - t o u c h e s p r i o r t o a d m i n i s t r a t i o n o f t h e t a i l - t o u c h / t a p has been shown t o decrease t h e amount o f i n h i b i t i o n produced by t a i l - t o u c h ( Rankin, 1991). T h i s experiment i n v e s t i g a t e d i f the responses of males d i f f e r e d from hermaphrodites i n h a b i t u a t i o n t o t a i l - t o u c h due t o t h e presence of t h e a d d i t i o n a l sensory r e c e p t o r s i n t h e male. S u b j e c t s ; A t o t a l o f 40 s u b j e c t s were used, c o n s i s t i n g o f 20 males and 20 hermaphrodites. P r o c e d u r e : Each a n i m a l r e c e i v e d t h r e e d i f f e r e n t s t i m u l u s c o n d i t i o n s , s e p a r a t e d by i n t e r v a l s of a p p r o x i m a t e l y 15-20 min. The c o n d i t i o n s were: 1) a s i n g l e t a p , 2) a t a i l - t o u c h f o l l o w e d 1 s l a t e r by a t a p , 3) 25 t a i l - t o u c h e s a t a 2 s i n t e r s t i m u l u s i n t e r v a l f o l l o w e d 1 s l a t e r by a t a p . C o n d i t i o n 1 e s t a b l i s h e d a b a s e l i n e l e v e l of response t o t a p a l o n e . C o n d i t i o n 2 t e s t e d f o r t h e e x i s t e n c e of t h e i n h i b i t i o n e f f e c t . C o n d i t i o n 3 t e s t e d whether h a b i t u a t i o n t o t a i l touch decreased t h e i n h i b i t i o n o f r e v e r s a l t o t a p . S c o r i n g and S t a t i s t i c a l A n a l y s e s ; Both t h e i n c i d e n c e and magnitude o f r e v e r s a l s i n males and hermaphrodites was scored and a n a l y z e d w i t h t - t e s t s . R e s u l t s ; R e v e r s a l Frequency: A l l worms r e v e r s e d t o t h e i n i t i a l t a p . I n hermaphrodites, t a i l - t o u c h induced i n h i b i t i o n a t a 1 s i n t e r v a l was complete; no animals r e v e r s e d t o t a p i n t h i s c o n d i t i o n . When 25 t a i l - t o u c h e s preceded the 1 s t a i l - t o u c h / t a p , 16 worms r e v e r s e d t o t a p ( F i g u r e 8A). In males, a t a i l - t o u c h 1 s p r i o r t o t a p reduced t h e number o f r e v e r s a l s seen; o n l y 2 animals r e v e r s e d t o t a p . When the 1 s t a i l - t o u c h t a p was preceded by 25 t a i l - t o u c h e s , a l l 20 males showed a r e v e r s a l . An u n p a i r e d t - t e s t i n d i c a t e d t h a t t h e number o f males r e v e r s i n g i n the t a i l - t o u c h h a b i t u a t i o n c o n d i t i o n was s i g n i f i c a n t l y l a r g e r than t h e number o f hermaphrodites r e v e r s i n g i n t h e same c o n d i t i o n [t(38)=2.179, p=0.0178]. R e v e r s a l Magnitude: To compare t a i l - t o u c h h a b i t u a t i o n i n males and hermaphrodites, r e v e r s a l magnitude was s t a n d a r d i z e d by e x p r e s s i n g each worm's response as a percentage o f i t s i n i t i a l r esponse t o t a p , which i t s e l f was s e t a t 100%. F i g u r e 8A. Number of males and hermaphrodites (n=20 f o r each sex) showing r e v e r s a l s t o t a p a l o n e ; t a p preceded by 1 s by 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 (25 t a i l - t o u c h e s , i n t e r s t i m u l u s i n t e r v a l = 2 s) f o l l o w e d 2 s l a t e r by a t a i l - t o u c h / t a p ( i n t e r s t i m u l u s i n t e r v a l between t a i l - t o u c h and t a p = l s ) . Both males and hermaphrodites showed s i g n i f i c a n t i n h i b i t i o n o f r e v e r s a l s when t h e t a i l - t o u c h / t a p i n t e r v a l was 1 s (TT/TAP I S ) . A f t e r h a b i t u a t i o n t r a i n i n g w i t h 25 t a i l - t o u c h e s , both sexes showed a s i g n i f i c a n t i n c r e a s e i n the number of r e v e r s a l s shown t o t a p f o l l o w i n g t a i l - t o u c h (TTHAB25) over t a i l - t o u c h / t a p a l o n e . F i g u r e 8B. Magnitude of r e v e r s a l s + SEM shown by males and hermaphrodites (n=20 f o r each sex) t o t a p a l o n e ; t a p preceded by 1 s by 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 (25 t a i l -t o u c h e s , i n t e r s t i m u l u s i n t e r v a l = 2 s) f o l l o w e d 2 s l a t e r by a t a i l - t o u c h / t a p ( i n t e r s t i m u l u s i n t e r v a l between t a i l - t o u c h and t a p = l s ) . R e v e r s a l magnitude was s i g n i f i c a n t l y i n h i b i t e d by a t a i l - t o u c h p r e c e d i n g t a p . The amount o f i n h i b i t i o n was s i g n i f i c a n t l y decreased by the a d m i n i s t r a t i o n o f 25 t a i l -t ouches p r i o r t o t a i l - t o u c h t a p a d m i n i s t r a t i o n . 20 n 15H 1 0 MALE HERM T A P TT/TAP 1 S TT/HAB25 B 1 20 1 00 80 60 40 20 0 MALE HERM T A P TT/TAP 1 S TT/HAB25 Both males and hermaphrodites e x h i b i t e d a l a r g e i n h i b i t i o n o f r e v e r s a l magnitude i n the 1 s t a i l - t o u c h / t a p c o n d i t i o n . I n o b o t h s e x e s , t h e a d m i n i s t r a t i o n of 25 t a i l - t o u c h e s p r i o r t o a t a i l -t o u c h / t a p a t a 1 s i n t e r v a l s i g n i f i c a n t l y i n c r e a s e d t h e r e v e r s a l magnitude [ F i g u r e 8B, males: t(16)=-4.018, p=0.0005; hermaphrodites: t(17)=-3.473, p=0.0015]. A l t h o u g h males e x h i b i t e d a l a r g e r response than hermaphrodites t o t a p a f t e r t h e t a i l - t o u c h h a b i t u a t i o n , t h i s d i f f e r e n c e was not s i g n i f i c a n t [t(36)=0.249, p=0.249]. D i s c u s s i o n : The a d m i n i s t r a t i o n of 25 t a i l - t o u c h e s p r i o r t o a t a i l -t o u c h / t a p w i t h a 1 s i n t e r v a l r e s u l t e d i n a decrease o f i n h i b i t i o n i n both males and hermaphrodites. The absence o f any sex d i f f e r e n c e s suggests t h a t 1) the c i r c u i t s i n f l u e n c i n g t h e decrement i n t a i l - t o u c h i n h i b i t i o n due t o h a b i t u a t i o n a re not a f f e c t e d by t h e presence of the a d d i t i o n a l s e n s o r y r e c e p t o r s i n t h e t a i l of the male and 2) h a b i t u a t i o n can be demonstrated by male C. elegans. The a b i l i t y o f male C. elegans t o show h a b i t u a t i o n was more f u l l y e x p l o r e d i n Experiment V I . Experiment VI: 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 H a b i t u a t i o n can be d e f i n e d as a decreased response t o r e p e a t e d s t i m u l a t i o n . D i s h a b i t u a t i o n i s t h e f a c i l i t a t i o n o f th e h a b i t u a t e d response due t o the p r e s e n t a t i o n o f a n o v e l o r noxious s t i m u l u s (Groves & Thompson, 1970). The a b i l i t y of an a n i m a l t o show d i s h a b i t u a t i o n has i m p o r t a n t i m p l i c a t i o n s f o r h a b i t u a t i o n ; i t demonstrates t h a t h a b i t u a t i o n i s not due t o s e n s o r y a d a p t a t i o n o r muscular f a t i g u e (Groves & Thompson, 1970). 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 o f t h e r e v e r s a l response have been demonstrated i n C. elegans hermaphrodites (Rankin e t a l . , 1990; Rankin & B r o s t e r , 1991). Rankin and her c o l l e a g u e s (1990) have shown t h a t t h e a d m i n i s t r a t i o n o f a s e r i e s o f 40 t r a i n s of taps w i t h an i n t e r s t i m u l u s i n t e r v a l o f 10 s produced s i g n i f i c a n t h a b i t u a t i o n , w h i l e t h e a d m i n i s t r a t i o n o f a 60 V shock immediately f o l l o w i n g the t a p s r e s u l t e d i n a s i g n i f i c a n t d i s h a b i t u a t i o n o r a mean i n c r e a s e t o a p p r o x i m a t e l y 50% o f t h e i n i t i a l response. To d a t e , i n our l a b o r a t o r y , h a b i t u a t i o n i n C. elegans has been ac c o m p l i s h e d by t h e a d m i n i s t r a t i o n of a r e p e a t e d s e r i e s of t r a i n s o f t a p s . The c u r r e n t experiment t e s t e d whether h a b i t u a t i o n c o u l d be induced by s i n g l e t a p s i n s t e a d o f t r a i n s . F u r t h e r , i t was designed t o t e s t whether male C. elegans were c a p a b l e o f 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 and t o compare t h e i r response p a t t e r n s t o those of hermaphrodites. S u b j e c t s : Twenty male and twenty h e r m a p h r o d i t i c C. elegans were used i n t h i s experiment. A l l animals were a t l e a s t f o u r days but not e x c e e d i n g f i v e days o l d . 4 8 P r o c e d u r e ; S i x t y s i n g l e taps w i t h an i n t e r s t i m u l u s i n t e r v a l o f 10 s were d e l i v e r e d f o l l o w e d by the a d m i n i s t r a t i o n o f a 60 V shock w i t h i n 10 s o f t h e l a s t t a p . W i t h i n 10-20 s o f t h e shock, a f u r t h e r s e r i e s o f 10 tap s w i t h an i n t e r s t i m u l u s i n t e r v a l of 10 seconds was a d m i n i s t e r e d . S c o r i n g and S t a t i s t i c a l A n a l y s i s Only animals which showed a r e v e r s a l t o t h e i n i t i a l t a p were s c o r e d . A d d i t i o n a l l y , a response on which animals showed no r e v e r s a l was a s s i g n e d a v a l u e of z e r o , w h i l e a response i n which an animal showed an a c c e l e r a t i o n was r e p l a c e d w i t h t h e group mean. A l t h o u g h replacement of m i s s i n g d a t a w i t h t h e group mean r e s u l t s i n a decrease i n the observed v a r i a n c e , t h i s replacement d i d not have a l a r g e impact on s t a t i s t i c a l a n a l y s e s s i n c e fewer than 1% o f da t a p o i n t s were m i s s i n g . These c r i t e r i a a l l o w e d f o r c o n s t r u c t i o n o f an h a b i t u a t i o n c u r v e and f o r a comparison o f t h e l e v e l of d i s h a b i t u a t i o n o f a h a b i t u a t e d response. The responses t o the f i r s t seven s t i m u l i were a n a l y z e d w i t h a repe a t e d measure ANOVA ( p r o p o r t i o n a l data) o r a r e g r e s s i o n a n a l y s i s (magnitude data) t o determine whether h a b i t u a t i o n was p r e s e n t and i f so, t o determine i t s r a t e . The f i n a l 10 s t i m u l i p r i o r t o the a d m i n i s t r a t i o n o f t h e d i s h a b i t u a t i n g s t i m u l u s (shock) were a n a l y z e d w i t h a r e p e a t e d measures ANOVA ( p r o p o r t i o n a l data) t o compare t h e f i n a l l e v e l s o f h a b i t u a t i o n reached between the sexes. F i n a l l y , t h e presence o f d i s h a b i t u a t i o n was t e s t e d by comparing t h e average of the l a s t 10 stimuli to the response to the tap immediately following the shock administration. Results: Reversal Frequency: The number of males and hermaphrodites reversing to tap decreased s i g n i f i c a n t l y (ie. habituated) over the course of the f i r s t seven taps [F(6,228)=5.565, p=0.0001]. Although males and hermaphrodites showed a similar pattern of decrement i n ( reversal frequency, males responded with a greater frequency of reversals than hermaphrodites over the 7 taps [Figure 9, main effe c t for sex: F(1,38)=4.163, p=0.0483, interaction of tap by sex: F(6,228)=1.351, p=0.2354, NS]. During the la s t 10 stimulus presentations preceding the shock, the number of reversals i n both sexes had s t a b i l i z e d , with males showing a s i g n i f i c a n t l y higher frequency of reversals than hermaphrodites [non-sig. main effect of tap: F(9,342)=0.723, p=0.6789, non-sig. interaction of tap by sex: F(9,342)=0.723, p=0.6789, s i g . main effect of sex: F(l,38)=22.537, p=0.0001]. To determine i f the shock increased the number of reversals shown over habituated levels, the the mean of the l a s t 10 responses was compared with a paired t - t e s t to the response immediately following the shock. The l a s t 10 responses were used to provide a better estimate'of pre-shock responding than would be obtained by a single point. In terms of reversal frequency, males did not display dishabituation, 50 F i g u r e 9. Number of r e v e r s a l s shown by males (n=20) and hermaphrodites (n=20) a t each s t i m u l u s p r e s e n t a t i o n 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 and a f t e r a d m i n i s t r a t i o n o f d i s h a b i t u a t i n g s t i m u l u s (60 V s h o c k ) . The shock a d m i n i s t r a t i o n i s r e p r e s e n t e d by t h e v e r t i c a l l i n e a p pearing a t immediately a f t e r s t i m u l u s # 60. Males and hermaphrodites showed h a b i t u a t i o n , a l t h o u g h t h e number of males r e v e r s i n g t o t a p was s i g n i f i c a n t l y h i g h e r t h a n hermaphrodites a t almost every t a p . Hermaphrodites showed s i g n i f i c a n t d i s h a b i t u a t i o n f o l l o w i n g shock a d m i n i s t r a t i o n whereas males d i d not. 52 j u s t m i s s i n g s i g n i f i c a n c e [t(19)=1.612, p-0.0617], however, hermaphrodites d i d show s i g n i f i c a n t d i s h a b i t u a t i o n [t(19)=2.398, p=0.0134]. R e v e r s a l Magnitude: To compare 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 males and hermaphrodites, r e v e r s a l magnitude was s t a n d a r d i z e d by e x p r e s s i n g each worm's responses as a percentage o f i t s i n i t i a l r esponse t o t a p , which i t s e l f was s e t a t 100%. The r a t e o f response decrement was determined by computing t h e mean o f the s l o p e s o f r e g r e s s i o n l i n e s o b t a i n e d f o r i n d i v i d u a l animals f o r the f i r s t 7 s t i m u l u s p r e s e n t a t i o n s . Both males and hermaphrodites showed h a b i t u a t i o n , as i n d i c a t e d by n e g a t i v e s l o p e s which were s i g n i f i c a n t l y d i f f e r e n t from z e r o [ F i g u r e 10A and 10B, males: mean slope=-13.29, SEM=2.841, t(19)=5.574, p<0.0001; hermaphrodites: mean slope=-9.489, SEM=1.702, t(19)=5.574, p<0.0001]. There were no s i g n i f i c a n t d i f f e r e n c e s i n t h e r a t e of h a b i t u a t i o n between sexes as measured by comparing t h e s l o p e s o f the males and t h e hermaphrodites [t(19)=-3.803, p=0.1457]. The response t o t a p 7 was s i g n i f i c a n t l y lower than the response t o t h e i n i t i a l t a p [males: t(19)=7.763, p<0.0001; hermaphrodites: t(91)=3.583, p=0.0011], i n d i c a t i n g t h a t both sexes showed h a b i t u a t i o n a f t e r 7 t a p s . Furthermore, the response t o t a p 7 d i d not d i f f e r between males and hermaphrodites [t(38)=-0.406, p=0.3435]. A f t e r 60 s t i m u l i the response magnitude i n hermaphrodites was s i g n i f i c a n t l y lower than i n males. T h i s was determined by Figure 10A. Habituation curve for reversal magnitude of males (n=20) showing the mean magnitude of reversals + SEM a t each stimulus presentation. Reversal magnitude was standardized as a percentage of the i n i t i a l reversal of individual animals. Shock administration was represented by v e r t i c a l l i n e after stimulus 60. Males showed rapid habituation of response magnitude. Males did not show dishabituation i n response to a 60 V shock, possibly due to the large amount of v a r i a b i l i t y i n response magnitude from stimulus 50-60. Figure 10B. Habituation curve for reversal magnitude of hermaphrodites (n=20) showing the mean magnitude of reversals + SEM at each stimulus presentation. Reversal magnitude was standardized as a percentage of the i n i t i a l reversal of individual animals. Shock administration was represented by v e r t i c a l l i n e after stimulus 60. Hermaphrodites showed a rapid decline i n response magnitude, reaching asymptote near zero. Signifi c a n t dishabituation (response at stimulus 62) was seen following shock administration. 140 H LU (f) 120 -z STIMULUS comparing t h e mean response f o r both males and hermaphrodites f o r t h e f i n a l 10 s t i m u l i p r i o r t o shock a d m i n i s t r a t i o n [males: mean=23.32, SEM=5.382; hermaphrodites: mean=0.692, SEM=0.165, t(18)=4.185, p=0.0003]. Furthermore, a t t h e end o f t h e h a b i t u a t i o n s t i m u l i , males showed a s i g n i f i c a n t l y h i g h e r degree o f v a r i a b i l i t y i n t h e i r response magnitudes a t h a b i t u a t e d l e v e l s , as determined by comparing the mean v a r i a n c e o f males w i t h t h a t o f hermaphrodites over the l a s t 10 s t i m u l i [F(20,20)=892.064, p<0.0001]. Males d i d not show s i g n i f i c a n t d i s h a b i t u a t i o n o f response magnitude f o l l o w i n g the shock [t(19)=0.522, p=0.304] whereas hermaphrodites d i d show d i s h a b i t u a t i o n [t(19)=1.188, p=0.0376]. T h i s was determined by a v e r a g i n g t h e magnitude o f t h e l a s t 10 responses and comparing t h i s mean t o the response i m m e d i a t e l y f o l l o w i n g t h e shock u s i n g a p a i r e d t - t e s t . D i s c u s s i o n : Both males and hermaphrodites showed h a b i t u a t i o n t o r e p e a t e d s t i m u l a t i o n . Rankin and B r o s t e r (1991) suggested t h a t two p r o c e s s e s may u n d e r l i e h a b i t u a t i o n , one g o v e r n i n g t h e d e s c e n d i n g p o r t i o n o f the c u r v e , and t h e o t h e r g o v e r n i n g t h e a s y m p t o t i c l e v e l of h a b i t u a t i o n reached. S i n c e males and hermaphrodites showed the same o v e r a l l r a t e of h a b i t u a t i o n , i t i s l i k e l y t h a t 1) the mechanisms u n d e r l y i n g t h e d e s c e n d i n g p o r t i o n o f t h e h a b i t u a t i o n curve are the same i n both sexes and t h a t 2) t h e a d d i t i o n a l sensory s t r u c t u r e s i n t h e t a i l o f t h e males do not a f f e c t those mechanisms. However, s i n c e t h e males showed a s i g n i f i c a n t l y h i g h e r a s y m p t o t i c response magnitude t h a n hermaphrodites, th e mechanisms of h a b i t u a t i o n g o v e r n i n g t h e a s y m p t o t i c l e v e l may d i f f e r between the sexes. The f i n d i n g of no s i g n i f i c a n t d i s h a b i t u a t i o n i n males s h o u l d be i n t e r p r e t e d w i t h c a u t i o n . C l o s e i n s p e c t i o n o f t h e l a s t t e n t a p s p r e c e d i n g the shock a d m i n i s t r a t i o n r e v e a l e d a l a r g e amount o f v a r i a b i l i t y i n the r e s p o n s i v e n e s s o f males ( F i g u r e 10A); t h e magnitude of some responses observed were l a r g e r t h a n t h a t observed a f t e r the a d m i n i s t r a t i o n o f shock. . Thus, i t i s p o s s i b l e t h a t no s i g n i f i c a n t d i s h a b i t u a t i o n was seen because the magnitude of the responses p r e c e d i n g shock had a h i g h degree of v a r i a b i l i t y . F u r t h e r r e s e a r c h u s i n g a l a r g e r number of animals might decrease the response v a r i a b i l i t y d u r i n g t h e f i n a l t a p s , t h e r e b y i n c r e a s i n g t h e p r o b a b i l i t y o f f i n d i n g s i g n i f i c a n t d i s h a b i t u a t i o n i n males. Experiment V I I : Spontaneous Recovery from H a b i t u a t i o n F o l l o w i n g t h e h a b i t u a t i o n of a response, t h e magnitude o f t h e h a b i t u a t e d response r e c o v e r s back t o b a s e l i n e l e v e l s w i t h t h e passage of t ime i n the a b s e n c e y o f f u r t h e r h a b i t u a t i n g s t i m u l i . T h i s p r o c e s s i s r e f e r r e d t o as spontaneous r e c o v e r y from h a b i t u a t i o n (Groves & Thompson, 1970). I t has been h y p o t h e s i z e d t h a t s t u d y i n g spontaneous r e c o v e r y from h a b i t u a t i o n would l e a d t o i n s i g h t s about p r o c e s s e s u n d e r l y i n g t h e h a b i t u a t i o n process i t s e l f (Rankin & B r o s t e r , 1991). As s uch, R a n k i n and B r o s t e r (1991) i n v e s t i g a t e d t h e f a c t o r s a f f e c t i n g spontaneous r e c o v e r y from h a b i t u a t i o n , i n c l u d i n g t h e 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 i n i t i a l h a b i t u a t i o n p r o c e s s as w e l l as t h e number of s t i m u l i l e a d i n g t o h a b i t u a t i o n . F o l l o w i n g 60 t r a i n s of taps w i t h an i n t e r s t i m u l u s i n t e r v a l o f 10 s, t h e y found t h a t hermaphrodites showed s i g n i f i c a n t r e c o v e r y t o about 70% of i n i t i a l response l e v e l s 10 min a f t e r t h e l a s t h a b i t u a t i o n s t i m u l u s . Recovery remained a t t h i s l e v e l 20 and 30 min a f t e r the l a s t h a b i t u a t i o n s t i m u l u s . The c u r r e n t experiment e x p l o r e d t h e t e m p o r a l parameters t h a t govern spontaneous r e c o v e r y from h a b i t u a t i o n i n d u c e d by s i n g l e t a p s i n males and hermaphrodites. A l t h o u g h males showed a h i g h e r f i n a l a s y m p t o t i c magnitude of response t h a n t h e hermaphrodites i n Experiment V I , t h i s was not a concern i n t h i s experiment s i n c e Rankin and B r o s t e r (1991) have shown t h a t r e c o v e r y r a t e i s a f f e c t e d o n l y m i n i m a l l y by t h e degree o f decrement p r i o r t o r e c o v e r y . S u b j e c t s ; Twenty male and twenty h e r m a p h r o d i t i c C. elegans were used i n t h i s experiment. A l l animals were a t l e a s t f o u r but not e x c e e d i n g f i v e days i n age. P r o c e d u r e : A s e r i e s o f 60 s i n g l e t a p s w i t h an i n t e r s t i m u l u s i n t e r v a l o f 10 seconds were d e l i v e r e d t o e s t a b l i s h h a b i t u a t i o n . To t e s t f o r spontaneous r e c o v e r y from h a b i t u a t i o n , a s i n g l e t a p was a d m i n i s t e r e d a t 30 s, 10, 20 and 30 min f o l l o w i n g t h e l a s t t a p o f t h e i n i t i a l s e r i e s of 60 t a p s . 58 S c o r i n g and S t a t i s t i c a l A n a l y s i s Only animals which showed a r e v e r s a l t o t h e i n i t i a l t a p were s c o r e d . A response on which an animal showed no r e v e r s a l was a s s i g n e d a v a l u e o f z e r o , w h i l e a response i n which an an i m a l showed an a c c e l e r a t i o n was r e p l a c e d w i t h t h e group mean. Only t h o s e responses o b t a i n e d f o r t h e i n i t i a l t a p , t h e l a s t t a p of t h e h a b i t u a t i o n s e r i e s , and the 30 s, 10, 20 and 30 min p o s t - h a b i t u a t i o n taps were a n a l y z e d . P r o p o r t i o n a l d a t a were a n a l y z e d w i t h a one f a c t o r repeated measures ANOVA u s i n g dichotomous d a t a f o r t h e f i r s t and l a s t t a p i n t h e h a b i t u a t i o n s e r i e s , a l o n g w i t h the t a p a d m i n i s t e r e d a t 30 s, 10, 20, and 30 min. Magnitude d a t a were a n a l y z e d w i t h t r e n d and r e g r e s s i o n a n a l y s e s . R e s u l t s : R e v e r s a l Frequency: The p r o p o r t i o n of males r e v e r s i n g t o t a p changed d u r i n g t h e c o u r s e o f re p e a t e d taps [ F i g u r e 11A, F{5,95)=8.018, p<0.0001, r e p e a t e d measures ANOVA on i n i t i a l t a p , f i n a l h a b i t u a t i o n t a p , and t a p a t 30 s, 10, 20 and 30 m i n ] , d e m o n s t r a t i n g t h e presence of h a b i t u a t i o n and r e c o v e r y from h a b i t u a t i o n . A l l males r e v e r s e d t o the i n i t i a l t a p ; by t h e f i n a l h a b i t u a t i o n t a p , s i g n i f i c a n t l y fewer worms were showing r e v e r s a l s (p<0.01). Recovery was e v i d e n t by 10 min p o s t -h a b i t u a t i o n : t h e number of males r e v e r s i n g a t 10, 20 and 30 min was s i g n i f i c a n t l y l a r g e r than t h e number r e v e r s i n g a t t a p 60 (p<0.01). 59 The number of hermaphrodites showing r e v e r s a l s a l s o demonstrated h a b i t u a t i o n and r e c o v e r y d u r i n g t h e c o u r s e o f t h e experiment [ F i g u r e 11A, F(5,95)=15.299, p<0.0001]. The number of worms r e v e r s i n g by t a p 60 was v e r y s m a l l (p<0.01). Recovery o c c u r r e d by 30 s as t h e number of animals r e v e r s i n g was l a r g e r a t 30 s, 10 min (p<0.05) and a t 20 and 30 min (p<0.01). There was no d i f f e r e n c e i n the number o f males and hermaphrodites r e v e r s i n g a t any of t h e times t e s t e d [F(l,38)=1.185, p=0.2832]. R e v e r s a l Magnitude: To compare r e c o v e r y magnitude d a t a f o r males and hermaphrodites, r e v e r s a l magnitudes were s t a n d a r d i z e d by e x p r e s s i n g each worm's response as a percentage o f i t s i n i t i a l r e sponse t o t a p , which i t s e l f was s e t a t 100%. The r e v e r s a l magnitudes of the males i n d i c a t e d h a b i t u a t i o n t o r e p e a t e d t a p s , as t h e response t o t h e f i n a l h a b i t u a t i n g s t i m u l u s was s i g n i f i c a n t l y s m a l l e r than t h e response t o t h e i n i t i a l t a p [t(19)=83.918, p<0.0001]. Spontaneous r e c o v e r y from h a b i t u a t i o n i n males o c c u r r e d i n a p o s i t i v e l i n e a r f a s h i o n o v e r t h e 30 min p o s t - h a b i t u a t i o n p e r i o d [ t r e n d a n a l y s i s i n c l u d e d t a p #60, 10, 20 and 30 min t a p s , F(1,57)=10.338, p<0.01]. As w e l l , t h e r e was s i g n i f i c a n t r e c o v e r y above h a b i t u a t e d l e v e l s , s i n c e t h e mean s l o p e f o r t h e r e g r e s s i o n l i n e i n the l i n e a r t r e n d was s i g n i f i c a n t l y d i f f e r e n t than ze r o [ F i g u r e 11B, t(15)=-2.745, p=0.0075]. I n hermaphrodites, h a b i t u a t i o n and s i g n i f i c a n t r e c o v e r y were a l s o e v i d e n t [ F i g u r e 11B, i n i t i a l t a p v s . f i n a l t a p 60 F i g u r e 11A. Number of males and hermaphrodites (n=20 f o r each sex) showing a r e v e r s a l i n response t o t h e i n i t i a l t a p , t h e h a b i t u a t i o n t a p (tap # 60), and 30 s, 10, 20 and 30 min f o l l o w i n g t h e l a s t h a b i t u a t i o n t a p . S i g n i f i c a n t l y fewer r e v e r s a l s were seen by t a p 60 compared t o the i n i t i a l t a p i n both s e x e s , i n d i c a t i n g s i g n i f i c a n t h a b i t u a t i o n . Males and hermaphrodites showed s i g n i f i c a n t l y more r e v e r s a l s a t 30 s, 10, 20 and 30 min p o s t -h a b i t u a t i o n than t o t h e 60th t a p . There were no s i g n i f i c a n t d i f f e r e n c e s between males and hermaphrodites i n t h e number of r e v e r s a l s shown. i F i g u r e 11B. Magnitude of r e v e r s a l s shown by males and hermaphrodites (n=20 f o r each sex) + SEM f o r r e v e r s a l s i n response t o t h e i n i t i a l t a p , the h a b i t u a t i o n t a p ( t a p # 6 0 ) , and a t 30 s, 10, 20 and 30 min f o l l o w i n g t h e l a s t h a b i t u a t i o n t a p . Both males and hermaphrodites showed r e c o v e r y from h a b i t u a t i o n i n a l i n e a r f a s h i o n . There were no sex d i f f e r e n c e s i n t h e r a t e o f spontaneous r e c o v e r y from h a b i t u a t i o n . 61 A M A L E • TAP 1 E3 TAP 60 M 30 s 0 10 MIN • 20 MIN • 30 MIN HERM B M A L E HERM response: t(16)=-2.511, p=0.0116; p o s i t i v e l i n e a r t r e n d , F(l,57)=6.727, p<0.025; s l o p e of r e g r e s s i o n l i n e , t(16)=-2.511, p=0.0116]. There were no s i g n i f i c a n t d i f f e r e n c e s between sexes i n t h e r a t e o f r e c o v e r y from h a b i t u a t i o n , which was determined by comparing t h e mean s l o p e o f the r e g r e s s i o n l i n e f o r t a p #60, and t h e 10, 20 and 30 min t a p [males: mean slope=25.963, SEM=9•459; hermaphrodite: mean slope=18.2877, SEM=1.283; t(13)=0.526, p=0.3038]. D i s c u s s i o n : S i n c e t h e r e were no s i g n i f i c a n t d i f f e r e n c e s between t h e males and hermaphrodites i n e i t h e r the response f r e q u e n c y o r t h e r a t e o f r e c o v e r y as measured by response magnitude, i t i s h y p o t h e s i z e d t h a t the processes which govern r e c o v e r y from h a b i t u a t i o n a r e comparable i n the two sexes o f C. elegans. G e n e r a l D i s c u s s i o n : The g e n e r a l purpose of t h e seven p r e c e d i n g experiments was t o t e s t whether the presence of a d d i t i o n a l s e n s o r y r e c e p t o r s i n t h e C. elegans males r e s u l t e d i n d i f f e r e n c e s between t h e males and hermaphrodites i n spontaneous b e h a v i o u r , r e f l e x i v e r e v e r s a l behaviour i n response t o t a p , as w e l l as i n n o n - a s s o c i a t i v e l e a r n i n g . Spontaneous a c t i v i t y d i f f e r e d i n males and hermaphrodites, p r i m a r i l y due t o the e x i s t e n c e o f t h e s e l f -f e e l i n g r e v e r s a l i n males. When the s e l f - f e e l i n g r e v e r s a l was e x c l u d e d from t h e a n a l y s i s , the magnitude o f t r u e spontaneous r e v e r s a l s was t h e same i n both sexes, however males s t i l l e x h i b i t e d more r e v e r s a l s . I t i s h y p o t h e s i z e d t h a t the mechanisms which r e s u l t i n t h e h i g h e r frequency o f spontaneous r e v e r s a l s a r e an a d a p t i v e response f o r a s p e c i a l b e h a v i o u r a l r o l e p l a y e d by t h e male, namely mating w i t h the hermaphrodite t o ensure g e n e t i c d i v e r s i t y i n f u t u r e p o p u l a t i o n s . • By showing a l a r g e number of r e v e r s a l s , t h e p r o b a b i l i t y of c o n t a c t i n g an o b j e c t w i t h t h e s e n s o r y r e c e p t o r s o f t h e c o p u l a t o r y bursa are g r e a t l y enhanced. Thus, one f u n c t i o n a l r o l e of spontaneous r e v e r s a l a c t i v i t y i n t h e male i s t o make c o n t a c t w i t h another worm i n an attempt t o i n i t i a t e m ating. I n response t o t a p , males showed r e f l e x i v e r e v e r s a l s . T h i s response was f i r s t e x p l o r e d i n t h e graded d u r a t i o n experiment by v a r y i n g the number of taps a d m i n i s t e r e d . Both sexes e x h i b i t e d a graded response as t h e number o f t a p s i n c r e a s e d , a l t h o u g h hermaphrodites showed a l a r g e r average r e v e r s a l magnitude a t a l l t a p c o n d i t i o n s . While hermaphrodites responded w i t h t h e same r e v e r s a l frequency as t h e number of t a p s i n c r e a s e d , males showed an i n c r e a s i n g tendency t o respond w i t h an a c c e l e r a t i o n response. T h i s f i n d i n g l e a d t o t h e h y p o t h e s i s o f an asymmetrical s e n s i t i v i t y and c o n s e q u e n t l y a * p o s i t i v e b i a s t o i n p u t from the t a i l of t h e male. The h y p o t h e s i s of asymmetrical s e n s i t i v i t y was e x p l o r e d i n t h e graded i n t e n s i t y experiment, i n which s i n g l e t a p s o f d i f f e r e n t i n t e n s i t i e s were a d m i n i s t e r e d t o males and hermaphrodites. I t was h y p o t h e s i z e d t h a t by g i v i n g a s i n g l e weak o r s t r o n g t a p , t h e r e s u l t a n t responses would a l l o w t h e e s t a b l i s h m e n t of s e n s i t i v i t y t o t a p i n males and hermaphrodites. Both males and hermaphrodites showed t h e same fre q u e n c y o f r e v e r s a l s t o a l l t a p i n t e n s i t i e s , s u g g e s t i n g t h a t b o t h sexes c o u l d respond t o the d i f f e r e n t i n t e n s i t i e s . Both sexes a l s o showed a graded response t o i n c r e a s i n g t a p i n t e n s i t y . S u r p r i s i n g l y , hermaphrodites demonstrated s m a l l e r r e v e r s a l magnitudes t h a n males t o a l l t a p i n t e n s i t i e s , i n c o n t r a s t t o t h e spontaneous r e v e r s a l and graded d u r a t i o n e x p e r i m e n t s , where the response magnitude shown by hermaphrodites was l a r g e r than t h a t shown by males. I t was suggested t h a t t h e s e c o n t r a s t i n g r e s u l t s might be t h e r e s u l t o f d i f f e r e n t h a n d l i n g c o n d i t i o n s s i n c e animals i n t h e graded d u r a t i o n experiment were t e s t e d 1-2 minutes a f t e r b e i n g p l a c e d on t h e t e s t p l a t e w h i l e animals i n t h e graded i n t e n s i t y experiment were t r a n s f e r r e d t o the t e s t p l a t e 2 hours p r i o r t o t e s t . The e f f e c t s of h a n d l i n g were f u r t h e r e x p l o r e d by l o o k i n g a t d a t a from s e v e r a l experiments i n which h a n d l i n g c o n d i t i o n s d i f f e r e d . The mean of the response magnitude t o a s i n g l e 60 V t a p was compared between animals which were t e s t e d 1-2 minutes a f t e r t r a n s f e r t o the t e s t p l a t e (from experiments on graded d u r a t i o n , s i n g l e t a p c o n d i t i o n n=20 males, 20 hermaphrodites; 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 , i n i t i a l t a p , n=20 males, 20 hermaphrodites; r e c o v e r y from h a b i t u a t i o n , i n i t i a l t a p , n=20 males, 20 hermaphrodites; t o t a l # a n i m a l s , 60 males and 60 hermaphrodites) and animals which were p l a c e d on t h e t e s t p l a t e 2 o r 24 hours p r i o r t o t e s t i n g (from experiments on graded i n t e n s i t y , n=20 males, 20 hermaphrodites, 60 V t a p c o n d i t i o n ; i n h i b i t i o n , i n i t i a l t a p c o n d i t i o n , n=20 males, 20 hermaphrodites; t a i l - t o u c h h a b i t u a t i o n , i n i t i a l t a p c o n d i t i o n , n=20 males, 20 hermaphrodites; t o t a l # a n i m a l s , 60 males and 60 h e r m a p h r o d i t e s ) . Males t h a t were t e s t e d 1-2 minutes p o s t -t r a n s f e r showed a s i g n i f i c a n t l y s m a l l e r average r e v e r s a l magnitude than those which were t e s t e d 2-24 hours p o s t - t r a n s f e r [ F i g u r e 12; a l l responses were s t a n d a r d i z e d t o worm l e n g t h ; 1-2 min w a i t : mean=0.877, SFw=0.048; 2-24 hr w a i t : mean=1.064, SEM=0.015; t(59)=2.017, p=0.0242]. C o n v e r s e l y , hermaphrodites showed t h e o p p o s i t e p a t t e r n , worms t h a t were t e s t e d 1-2 minutes p o s t - t r a n s f e r showed a s i g n i f i c a n t l y l a r g e r average response magnitude than those which were t e s t e d 2-24 hours p o s t - t r a n s f e r [1-2 min w a i t : mean=1.209, SF.jipO.083; 2-24 hr w a i t : mean=0.72, SFM=0.056; t(59)=-4.978, p=0.0001]. Thus, h a n d l i n g i m m e d i a t e l y p r i o r t o e x p e r i m e n t a t i o n appears t o have a s e n s i t i z i n g e f f e c t on hermaphrodites, w h i l e an i n h i b i t o r y e f f e c t i s observed i n males, p r o v i d i n g an e x p l a n a t i o n of t h e d i f f e r e n c e s observed between hermaphrodites and males i n the graded d u r a t i o n and graded i n t e n s i t y experiments. I t i s u n c l e a r a t t h i s p o i n t as t o why d i f f e r e n c e s i n h a n d l i n g l e a d t o i n c r e a s e d r e v e r s a l magnitudes i n hermaphrodites and decreased magnitudes i n males. F i g u r e 12. The e f f e c t s of h a n d l i n g on r e v e r s a l magnitude. Magnitude o f r e v e r s a l s + SEM shown by males and hermaphrodites when t e s t e d e i t h e r 1-2 min (n=60 males, 60 h e r m a p h r o d i t e s ) , 2 hr (n=20 males, 20 hermaphrodites) o r 24 h r (n=40 males, 40 hermaphrodites) f o l l o w i n g t r a n s f e r t o t e s t p l a t e . R e v e r s a l magnitude f o r each animal was s t a n d a r d i z e d by d i v i d i n g t h e r e v e r s a l observed by t h e l e n g t h of t h e animal showing t h a t r e v e r s a l . Males showed s i g n i f i c a n t l y s m a l l e r responses i f t e s t e d 1-2 min p o s t - t r a n s f e r than i f t e s t e d 2-24 h r s p o s t -t r a n s f e r . Hermaphrodites showed the o p p o s i t e p a t t e r n o f response, s i g n i f i c a n t l y l a r g e r responses were observed i f t h e worm was t e s t e d 1-2 min p o s t - t r a n s f e r than i f t e s t e d 2-24 hr p o s t - t r a n s f e r . WAIT T IME 68 F u r t h e r e x p e r i m e n t a t i o n on t h e e f f e c t s o f h a n d l i n g are needed t o f u l l y understand t h i s phenomenon. S i n c e Experiment I I I (Graded I n t e n s i t y ) d i d not c o m p l e t e l y r e s o l v e t h e i s s u e of asymmetric s e n s i t i v i t y i n males, t h i s h y p o t h e s i s was f u r t h e r t e s t e d i n Experiment IV i n whi c h i n p u t from head and t a i l r e c e p t o r s was p l a c e d i n d i r e c t c o m p e t i t i o n . I f males are a s y m m e t r i c a l l y s e n s i t i v e w i t h a response b i a s t o i n p u t from t h e t a i l r e g i o n , then a l a r g e r amount o f i n h i b i t i o n would be p r e d i c t e d i n males. A t t h e 1 and 5 s t a i l - t o u c h / t a p i n t e r v a l s , males and hermaphrodites showed t h e same amount o f i n h i b i t i o n of r e v e r s a l f requency and magnitude, s u g g e s t i n g t h a t the a d d i t i o n a l s e n s o r y r e c e p t o r s i n t h e c o p u l a t o r y b u r s a o f the male do not render t h e a n i m a l more s e n s i t i v e t o t a i l - t o u c h than the hermaphrodite. T h i s i s su p p o r t e d by d a t a i n d i c a t i n g no s i g n i f i c a n t sex d i f f e r e n c e s a t any o f t h e t a i l - t o u c h t a p i n t e r v a l s . Furthermore, t h e l a c k o f any sex d i f f e r e n c e s suggests t h a t t a i l - t o u c h a c t i v a t e s t h e same n e u r a l c i r c u i t s which l e a d t o i n h i b i t i o n i n both sexes. Thus, t h e h y p o t h e s i s o f asymmetrical s e n s i t i v i t y i s not su p p o r t e d . An i n t e r e s t i n g s i m i l a r i t y between t h e r e s u l t s f o r d i f f e r e n t h a n d l i n g c o n d i t i o n s and f o r the i n h i b i t i o n experiment i s t h a t hermaphrodites show f a c i l i t a t i o n w h i l e males do n o t . H a b i t u a t i o n t o repe a t e d s i n g l e taps o f 10 s I S I was p r e s e n t i n both males and hermaphrodites. The r a t e o f h a b i t u a t i o n d i d not d i f f e r between the two sexes i n e i t h e r t h e h a b i t u a t i o n o r r e c o v e r y experiments. The f i n a l l e v e l o f h a b i t u a t i o n and t h e v a r i a n c e a s s o c i a t e d w i t h t h a t l e v e l was s i g n i f i c a n t l y h i g h e r i n males than i n hermaphrodites i n t h e 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; t h e s e t r e n d s were a l s o p r e s e n t i n t h e r e c o v e r y experiment but d i d not r e a c h s i g n i f i c a n c e . T h i s suggests t h a t t h e mechanisms u n d e r l y i n g t h e d e s c ending p o r t i o n of the h a b i t u a t i o n curve are s i m i l a r i n males and hermaphrodites but t h a t t h e mechanisms g o v e r n i n g t h e a s y m p t o t i c p o r t i o n of the h a b i t u a t i o n curve may be d i s s i m i l a r between t h e sexes. I t i s c o n c e i v a b l e t h a t t h e l a r g e r number and magnitude of r e v e r s a l s d i s p l a y e d by males a t asymptote i s r e l a t e d t o t h e importance of t a c t i l e s t i m u l a t i o n i n t h e males' mating b e h a v i o u r . I f the animal was c o m p l e t e l y h a b i t u a t e d t o t a c t i l e s t i m u l a t i o n , whether from a t a p o r from c o n t a c t w i t h a n o t her worm, v e r y few o r no r e v e r s a l s would o c c u r . The absence o f r e v e r s a l a c t i v i t y would p r e v e n t , o r a t the v e r y l e a s t s e r i o u s l y hamper, the mating b e h a v i o u r of t h e males. P r o t e c t i n g t h e t o u c h c i r c u i t from complete h a b i t u a t i o n would a l l o w t h e male t o c o n t i n u e t o r e v e r s e , t h e r e b y a f f o r d i n g t h e male the o p p o r t u n i t y t o i n v e s t i g a t e s t i m u l a t i o n o f t h e p o s t e r i o r s e n s o r y r e c e p t o r s t h a t might be i n d i c a t i v e o f a p o t e n t i a l mating p a r t n e r . I t was suggested i n the d i s c u s s i o n of Experiment VI ( 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 ) t h a t the l a c k o f d i s h a b i t u a t i o n i n males may have been due t o t h e h i g h v a r i a b i l i t y o f t h e t e n responses p r e c e d i n g t h e a d m i n i s t r a t i o n of t h e shock. S i n c e d i s h a b i t u a t i o n was not shown by males, i t i s p o s s i b l e t h a t t h e 70 h a b i t u a t i o n shown by males might be due t o se n s o r y a d a p t a t i o n o r muscular f a t i g u e (Groves & Thompson, 1970). However, t h i s does not seem t o be t h e case, i n l i g h t of t h e o b s e r v a t i o n t h a t males r e v e r s e t o t a p a f t e r t a i l - t o u c h h a b i t u a t i o n (Experiment V ) , s u g g e s t i n g t h a t f a t i g u e was not the cause o f h a b i t u a t i o n . Furthermore, i f two d i s t i n c t p r ocesses u n d e r l i e h a b i t u a t i o n i n males and hermaphrodites, t h a t i s , i f f a t i g u e o r se n s o r y a d a p t a t i o n are the cause of h a b i t u a t i o n i n males w h i l e l e a r n i n g i s t h e cause of h a b i t u a t i o n i n hermaphrodites, then t h e two sexes would not be expected t o show t h e same r a t e s o f r e c o v e r y from h a b i t u a t i o n . However, Experiment VI demonstrated t h e same r a t e o f r e c o v e r y from h a b i t u a t i o n i n males and hermaphrodites, t h e r e f o r e , i t i s u n l i k e l y t h a t d i f f e r e n t p r o c e s s e s u n d e r l i e h a b i t u a t i o n i n t h e male and t h e hermaphrodite. On the b a s i s of d a t a o b t a i n e d from Experiment V I , i t i s a l s o p o s t u l a t e d t h a t the same p r o c e s s e s may u n d e r l i e r e c o v e r y from h a b i t u a t i o n i n the males and hermaphrodites. C o n c l u s i o n s and D i r e c t i o n s f o r Future Research: The beh a v i o u r of male C. elegans d i s p l a y s many s i m i l a r i t i e s and d i f f e r e n c e s from t h e beh a v i o u r o f t h e hermaphrodite. Both sexes d i s p l a y a graded response t o t h e number and i n t e n s i t y o f taps a d m i n i s t e r e d ; an i n c r e a s e i n magnitude o f response i s shown t o an i n c r e a s e i n t a p number o r i n t e n s i t y . A d d i t i o n a l l y , both sexes are c a p a b l e o f d i s p l a y i n g i n h i b i t i o n , t a i l - t o u c h h a b i t u a t i o n , h a b i t u a t i o n t o t a p , and r e c o v e r y from h a b i t u a t i o n . Thus, males d i s p l a y t h e same 71 c a p a c i t y f o r b e h a v i o u r a l p l a s t i c i t y as has been demonstrated f o r t h e hermaphrodite, s u g g e s t i n g t h a t the c i r c u i t r y u n d e r l y i n g such p l a s t i c i t y i s common i n both sexes. Furthermore, t h e s e experiments found no evidence f o r d i f f e r e n c e s i n s e n s i t i v i t y due t o t h e presence o f the a d d i t i o n a l sensory r e c e p t o r s i n t h e c o p u l a t o r y b u r s a o f the male. D i f f e r e n c e s between the male and hermaphrodite do e x i s t i n spontaneous a c t i v i t y ; males showed a l a r g e r number o f spontaneous r e v e r s a l s of a l l t y p e s . Males do not h a b i t u a t e t o as low a l e v e l as hermaphrodites, d e s p i t e both sexes showing t h e same i n i t i a l r a t e of h a b i t u a t i o n . F i n a l l y , males respond d i f f e r e n t l y t o h a n d l i n g c o n d i t i o n s (1-2 min p r e - t e s t v s . 2-24 hr p r e - t e s t w a i t ) than hermaphrodites; males showed a decrement i n r e s p o n s i v e n e s s immediately a f t e r h a n d l i n g w h i l e hermaphrodites showed f a c i l i t a t i o n . To more p r e c i s e l y determine t h e r o l e o f t h e i n d i v i d u a l r e c e p t o r s i n t h e males i n the d i f f e r e n c e s i n b e h a v i o u r observed i n t h e s e experiments, mutants m i s s i n g c e r t a i n s t r u c t u r e s c o u l d be t e s t e d . A l t e r n a t i v e l y , i t i s p o s s i b l e t o a b l a t e by l a s e r t h e c e l l s g i v i n g r i s e t o each of t h e sensory r e c e p t o r s d u r i n g t h e development of the male. These animals c o u l d t h e n be t e s t e d t o determine whether a p a r t i c u l a r s t r u c t u r e p l a y e d a r o l e i n t h e d i f f e r e n t b ehaviours shown by male C. elegans. A t h i r d p o s s i b i l i t y i s t o study, the changes i n t h e b e h a v i o u r of t h e a n i m a l a t d i f f e r e n t developmental s t a g e s , n o t i n g how t h e a d d i t i o n o f new s t r u c t u r e s a f f e c t s the behaviour o f t h e a n i m a l . 72 A second paradigm u t i l i z i n g mating as a b e h a v i o r a l measure h o l d s promise f o r d e t e r m i n i n g the r o l e p l a y e d by each of t h e s e n s o r y r e c e p t o r s i n t h e c o p u l a t o r y b u r s a o f t h e males i n response t o mechanical s t i m u l a t i o n . I n males, a r e c i p r o c a l r e l a t i o n s h i p e x i s t s between th e mating response and t h e response t o mechanical s t i m u l a t i o n . I f a male r e c e i v e s a t a p t o i t s body immediately p r i o r t o e n c o u n t e r i n g a hermaphrodite, mating w i l l be i n h i b i t e d . I f mating has a l r e a d y been i n i t i a t e d , t h e male w i l l not show an o v e r t response t o m e c h a n i c a l s t i m u l a t i o n (Hodgkin, 1974). T h i s suggests t h a t t h e r e c e p t o r s o f t h e c o p u l a t o r y bursa have an i n f l u e n c e i n both t h e mating and t a p w i t h d r a w a l response. Perhaps f u t u r e r e s e a r c h s t u d y i n g t h e r e c e p t i v e p r o p e r t i e s o f the sensory r e c e p t o r s o f t h e c o p u l a t o r y b u r s a would be w e l l served by e x p l o r i n g t h e r o l e of t h e r e c e p t o r s i n mating behaviour. S i n c e mating b e h a v i o u r i n v o l v e s a complex c o o r d i n a t i o n of a l l t h e p o s t e r i o r s e n s o r y r e c e p t o r s , i t may be p o s s i b l e , t o determine more p r e c i s e l y what r o l e each s e n s o r y r e c e p t o r p l a y s i n response t o m e c h a n i c a l s t i m u l a t i o n . I n a d d i t i o n , a b e h a v i o u r a l a n a l y s i s o f t h e i n t e r a c t i o n between mating and the t a p w i t h d r a w a l response might expand our u n d e r s t a n d i n g of t h e way t h e u n d e r l y i n g n e u r a l c i r c u i t s f o r each behaviour can i n t e r a c t . 73 References Beck, C. D. O. (1991). The e f f e c t s of a g i n g on non- a s s o c i a t i v e l e a r n i n g i n the nematode Caenorhabditis elegans. U n p u b l i s h e d master's t h e s i s , U n i v e r s i t y o f B r i t i s h Columbia, Vancouver. Brenner, S. T. (1974). The g e n e t i c s o f Caenorhabditis elegans. G e n e t i c s . 77. 71-94. B y e r l y , L., Cassada, R. C , & R u s s e l l , R. L. (1976). The l i f e c y c l e o f t h e nematode Caenorhabditis elegans. Developmental  B i o l o g y , 51, 23-33. C h a l f i e , M., S u l s t o n , J . E., White, J . G., Southgate, E., Thomson, J . N., & Brenner, S. (1985). The n e u r a l c i r c u i t f o r t o u c h s e n s i t i v i t y i n Caenorhabditis elegans. The J o u r n a l o f  N e u r o s c i e n c e . 5, 956-964. C h i b a , C. M., & Rankin, C. H. (1990). A developmental a n a l y s i s o f spontaneous and r e f l e x i v e r e v e r s a l s i n t h e nematode Caenorhabditis elegans. J o u r n a l of N e u r o b i o l o g y . 21. 543-554. C o u l s o n , A., S u l s t o n , J . , Brenner, S., & Karn, J . (1986). Towards a p h y s i c a l map o f the genome of t h e nematode Caenorhabditis elegans. Proceedings of t h e N a t i o n a l Academy  of S c i e n c e s . 83, 7821-7825. Groves, P. M., & Thompson, R. F. (1970). H a b i t u a t i o n : A d u a l - p r o c e s s t h e o r y . P s y c h o l o g i c a l Review, 77. 419-450. Hodgkin, J . (1983). Male phenotypes and mating e f f i c i e n c y i n Caenorhabditis elegans. G e n e t i c s , 103. 43-64. Hodgkin, J . (1988). Sexual Dimorphism and Sex D e t e r m i n a t i o n . In W. B. Wood ( E d . ) , The Nematode Caenorhabditis elegans (pp. 243-279). C o l d S p r i n g Harbour: C o l d S p r i n g Harbour L a b o r a t o r y . Hodgkin, J . , H o r v i t z , H. R., & Brenner, S. (1979). N o n d i s j u n c t i o n mutants of the nematode Caenorhabditis elegans. G e n e t i c s . 91. 67-94. Hodgkin, J . A. (1974). G e n e t i c and a n a t o m i c a l a s p e c t s o f t h e  Caenorhabditis elegans male. Unpublished d o c t o r a l d i s s e r t a t i o n , Darwin C o l l e g e , Cambridge. 74 H o r v i t z , H. R., S t e r n b e r g , P. W., Greenwald, I . S., F i x s e n , W., & E l l i s , H. M. (1983). M u t a t i o n s t h a t a f f e c t n e u r a l c e l l l i n e a g e s and c e l l f a t e s d u r i n g development of t h e nematode Caenorhabditis elegans. C o l d S p r i n g Harbour Symposia on  Q u a n t i t a t i v e B i o l o g y , 48, 453-463. R a n k i n , C. H. (1991). I n t e r a c t i o n between two a n t a g o n i s t i c r e f l e x e s i n Caenorhabditis elegans. J o u r n a l of Comparative  P h y s i o l o g y A. i n p r e s s . R a n k i n , C. H., Beck, C. D. 0., & C h i b a , C. M. (1990). Caenorhabditis elegans: A new model system f o r t h e s t u d y o f l e a r n i n g and memory. B e h a v i o r a l B r a i n Research. 37. 89-92. R a n k i n , C. H., & B r o s t e r , B. S. (1991). F a c t o r s a f f e c t i n g h a b i t u a t i o n and r e c o v e r y from h a b i t u a t i o n i n t h e nematode Caenorhabditis elegans, B e h a v i o r a l N e u r o s c i e n c e . i n p r e s s . R a n k i n , C. H., & C h i b a , CM. (1988). Short and l o n g term l e a r n i n g i n t h e nematode C. elegans. S o c i e t y f o r  N e u r o s c i e n c e A b s t r a c t s , 14, 607. , R a n k i n , C. H., S t o p f e r , M., Marcus, E. A., & Carew, T. J . (1987). Development of l e a r n i n g and memory i n Aplysia. I . F u n c t i o n a l assembly of g i l l and s i p h o n w i t h d r a w a l . The  J o u r n a l of N e u r o s c i e n c e . 1, 120-132. Rose, A. M., & B a i l l i e , D. L. (1979). The e f f e c t o f temperature and p a r e n t a l age on r e c o m b i n a t i o n and n o n d i s j u n c t i o n i n Caenorhabditis elegans. G e n e t i c s . 92, 409-418. S u l s t o n , J . E., A l b e r t s o n , D. G., & Thomson, J . N. (1980). The Caenorhabditis elegans male: Postembryonic development of nongonadal s t r u c t u r e s . Developmental B i o l o g y . 78., 542-576. S u l s t o n , J . E., S c h i e r e n b e r g , E., White, J . G., & Thomson, J . N. (1983). The embryonic c e l l l i n e a g e of t h e nematode Caenorhabditis elegans. Developmental B i o l o g y . 100. 64-119. S u l s t o n , J . E., & White, J . G. (1980). R e g u l a t i o n and c e l l autonomy d u r i n g postembryonic development o f Caenorhabditis elegans. Developmental B i o l o g y . 78., 577-597. Ward, S., & C a r r e l , J . S. (1979). F e r t i l i z a t i o n and sperm c o m p e t i t i o n i n t h e nematode Caenorhabditis elegans. Developmental B i o l o g y . 73. 304-321. 75 Ward, S., Thomson, N., White, J . G., & Brenner, S. (1975). E l e c t r o n m i c r o s c o p i c a l r e c o n s t r u c t i o n o f the a n t e r i o r s e n s o r y anatomy o f t h e nematode Caenorhabditis elegans. J o u r n a l o f  Comparative Neurology. 160, 313-338. White, J . G., Southgate, E., Thomson, E., & Brenner, S. (1986). The s t r u c t u r e o f the nervous system o f Caenorhabditis elegans. P h i l o s o p h i c a l T r a n s a c t i o n s of the Ro y a l S o c i e t y o f  London. S e r i e s B. B i o l o g i c a l S c i e n c e s , 314. 1-340. Winer, B. J . (1971). S t a t i s t i c a l P r i n c i p l e s i n E x p e r i m e n t a l  Design (2nd e d . ) . New York: McGraw-Hill Book Company. Winer, B. J . , Brown, D. R., & M i c h e l s , K. M. (1991). S t a t i s t i c a l P r i n c i p l e s i n E x p e r i m e n t a l Design ( 3 r d e d . ) . New York: M c G r a w - H i l l , I n c . Wood, W. B. (1988). I n t r o d u c t i o n t o C. elegans b i o l o g y . I n W. B. Wood ( E d . ) , The Nematode Caenorhabditis elegans (pp. 1-16). C o l d S p r i n g Harbour, NY: C o l d S p r i n g Harbour L a b o r a t o r y . Appendix I 76 Fic. 4. Ventral view of adult male tail; anaesthetised animal mounted on agar with fan spread against cover slip. Nomarski optics; bar - 20 jun. Adapted from Sul s t o n & White (1980) U t f f t I M Fio. I. Left subventral view of young adult tail.^ o show general anatomy. Adapted from S u l s t o n , A l b e r t s o n & Thomson (1980) Adapted from Wood (1988) Appendix I I I 78 STIMULUS GENERATOR O oo o o O o o o o i i O ^ . VIDEO CAMERA VIDEO CASSETTE RECORDER TIME-DATE GENERATOR v MICROSCOPE I M P A CAENORHABDITIS ELEGANS \ \ \ \ / \ v J V \ VIDEO \ \ MONrroR \ \ \ \ MICRO- \ MANIPULATOR / & STAND \ \ PETRI PLATE HOLDER SHOCK ELECTRODE -

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.831.1-0100715/manifest

Comment

Related Items