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UBC Theses and Dissertations

Isolation and study of two mutants affecting motor activity in Drosophila melanogaster. Williamson, Rodney, L.M. 1971

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THE ISOLATION AND STUDY OF TWO MUTANTS AFFECTING MOTOR ACTIVITY I N DROSOPHILA MELANOGASTER by Rodney L. M. Williamson B.Sc., Uni v e r s i t y of B r i t i s h Columbia, 1968 A' THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in Genetics in the Department of Zoology We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA May, 1971 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced deg ree a t the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r a g r ee t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my Depar tment o r by h i s r e p r e s e n t a t i v e s , i t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f i i ABSTRACT Mitants of Prosophila melanogaster which are paralysed by exposure to one temperature, but recover mobility at another temperature may aid i n the i n v e s t i g a t i o n of the neural and muscular components which govern motor a c t i v i t y . With the help of a mechanical screening method, a recessive sex-linked temperature-sensitive p a r a l y t i c mutant (para^ s -53°9) was discovered among the progeny of e t h y l methane-t«? sulphonate-treated males and attached-X females, para f l i e s which had been r a i s e d at 22°C were paralysed within 5 seconds a f t e r t r a n s f e r to 29°C, but quickly regained m o b i l i t y when returned to 22°C When l e f t at 29°C for prolonged periods, the f l i e s gradually regained mob i l i t y . Further studies indicated that the time required f o r recovery following an increase i n temperature was d i r e c t l y r e l a t e d t o the magnitude and rate of the temperature r i s e . Temperature-sensitive p a r a l y s i s was seen only i n adult f l i e s . The abnormal movements which are c h a r a c t e r i s t i c of the behavioural mutants Hk , Hk , Sh , when lin k e d t o para i n males,were quickly stopped and st a r t e d by temperature s h i f t s from 22°C t o 29°C and 29°C to o 22 C, r e s p e c t i v e l y . The pos s i b l e s i g n i f i c a n c e of these observations i s discussed. para^ s/M(T)0 females exhibited temperature-sensitive p a r a l y s i s . The p o s s i b i l i t y that the chromosome bearing the M(l)0 mutation might ts a l s o carry a d e l e t i o n or mutant a l l e l e of para has not yet been inv e s t i g a t e d . A sex-linked dominant mutation which caused abnormal movements of the head and appendages under ether anaesthesia as w e l l as shuddering i i i movements in unetherised flies was also discovered. The mutation was called Shuddering (Shu - 5 5 « 1 ) . The shuddering movements could be temporarily suppressed by feeding the flies media containing LiCl but not NaCl, NH4.CI or KC1. The evidence presented in this and other studies suggests that ts the effects of para and Shu mutations upon motor activity are mediated through their effects upon the nervous system,, i v TABLE OF CONTENTS Page Introduction 1 Methods and Materials ^ Results 24 Discussion ^ Summary 60 Bibliography 6 2 V LIST OF TABLES TABLE Page 1„ Frequency of spontaneous shuddering in Shu/FM6 flies 2^ after different salt treatments. 2„ Total numbers of flies tested for shuddering induced 28 by mechanical stimulation after different salt treatments„ 3« Estimated number of flies screened for sex-linked . 31 recessive and autosomal dominant temperature-sensitive paralysiso 4„ Types of movements which may be seen during recovery 36 from temperature induced paralysis,, t s 5 . Recombination data for para and different Neuro kk mutants„ 6. Reisolation of single mutation chromosomes by ^ 5 recombination of double mutants„ LIST OF FIGURES Figure Page 1, Type I-a screening device for isolating paralysed 5 flies, 2, Type I-b screening device for isolating paralysed ? flie s . 3o Type II screening device for isolating paralysed 9 flies. 4, End views of the type II screening device during 10 the isolation of paralysed flies, 5o Procedure for detection of a dominant temperature 12 sensitive paralytic mutant. 6, Procedure for detection of a sex-linked recessive 13 or autosomal dominant temperature-sensitive paralytic mutant. 7, Twin water-heated chambers for comparing behaviour 18 at constant temperatures. 8. A semi-immersible chamber for observing 19 behaviour during temperature changes. 9. Protocol for generating and testing Neuro para^s/Y 2 2 males, 10, Reduction of shuddering of Shu/FM6 flies subjected 2 9 to mechanical stimulation, 11, Recovery of motor competence with time after increases in temperature. 12, The number of para t s f l i e s , preconditioned at 22°G 38 (solid line) and 17°C (dashes) which were able to stand after being transferred to 29.5°G. 13, The number of para t s flies able to stand when temperatures were changed from (22.2°-22.4°C) to (27.0o-27o6°C) within (a) 14 minutes and (b) 6 minutes. Solid line - number of flies able to. stand; dashes - temperature, ts 14, The number of para flies able to stand when 41 temperatures were changed from (21.6°-22,0 C) to (28,5°-29,5°C) within (a) 24 minutes (b) 6 minutes. Solid line - number of flies standing; dashes -temperature. V 1 X Figure Page Genetic locat: X chromosome. ts 5 15. Genetic locations of HK.para and Sh on the 43 v i i i ACKNOWLEDGEMENT I am very grateful to Dr, David T. Suzuki for providing me with a most interesting research problem, and for his continued guidance, faith and help. I am very grateful to Dr. Leonie K. Piternick for her enthusiastic encouragement and help in the early stages of this work. It has been a great pleasure to work with Mr. Thomas Grigliatti who shared equally in the task of finding and characterising the para^s mutant. I would like to thank my father, Dr. Bruce Williamson for bringing lithium ion therapy to my attention and providing me with his professional view of its efficacy in the treatment of manic-depression and related disorders. I wish to thank a l l the members of Dr, Suzuki's laboratory whose help and encouragement have made this work both possible and enjoyable. 1 INTRODUCTION Comparative neuroanatomy was founded in the Alexandrian schools of medicine in 300 B.C. Since that time, knowledge of the nervous systems of numerous animals has been enlarged through many disciplines. The current interest in neurobiology represents a renewed awareness of the diversity of nervous systems which can now be analysed with a large assortment of technical and biological tools. In view of the dramatic success of genetic analyses of microbial systems, many investigators now wonder whether the mysteries of the nervous system may also be amenable to a similar strategy. Although the effects of single mutations upon behaviour have been studied for some time (McEwen, 1918), an organised pursuit of single mutations which affect a specific nervous function was f i r s t launched in Drosophila melanogaster by Benzer (I967), His search has yielded many nonphototactic mutants. Those which are characterisable by abnormalities in their electroretinograms have been assigned to five cistrons of the X chromosome by genetic mapping and complementation tests (Hotta and Benzer, 1970), Brenner (1970) pointed out that the morphogenesis of a nervous system probably requires extensive genetic regulation. Mutations affecting regulation are therefore likely to be found among behavioural mutants. Accordingly, Brenner has conducted a search for nonspecified behavioural deviants using a Nematode which possesses 50 axons and a ganglion comprised of 20 neurons. 350 mutants have been characterised and 20 functional groups have been defined. Electron microscopic studies of some of the mutants have revealed alterations of the nerve 2 circuitry. Unfortunately, the species has not been accessible to electrophysiological probes. Whiting (1932) was able to determine that the expression of sex specific behaviour in the wasp Habrobracon juglandis depended upon the genetic composition of the head, by studying gynandromorphs. Genetic mosaics have also been analysed by Ikeda and Kaplan (1970b) and Hotta and Benzer (1970) to determine the regional specificity and autonomy of behavioural mutants of Drosophila. In the latter studies, the frequency of genetic mosaics was increased by using the somatically vC unstable ring chromosome In(l)w (Ronton, 1955) or the mutation claret-nondisjunctional (Lewis and Gencarella, 1952). The genetic manipulations made possible by the variety of chromosome aberrations and mutations in Drosophila melanogaster. together with the accumulated knowledge of the organism, sustain its usefulness as a tool for genetic study. Furthermore, the neuro-physiology of the organism is now accessible to intracellular electrical recording techniques (Ikeda and Kaplan, 1970a). The use of mutations to probe the adult motor nervous system was initiated by Kaplan and Trout (1969) and Ikeda and Kaplan (1970a,b). The existing knowldge of this system and its relationship to muscular physiology make i t a promising area for molecular investigation. The first step towards a genetic study of neural and muscular function is the isolation of mutants. Paralysis is both a logical and a feasible mutant phenotype to look for. The extensive documentation of conditional mutations whose phenotypes are temperature-dependent (Suzuki, 1970) suggested a search for temperature-dependent paralysis, A mutation which allows mobility at one temperature, but is reversibly 3 paralysed by another provides a basis for large scale screening methods for its detection. If the fly recovers mobility after i t is returned to the "permissive" temperature, the mutation may be perpetuated as a stock. By screening for temperature-sensitive paralysis, individuals exhibiting various kinds of debilitated motor activity might also be detected. With the recovery of each behavioural mutant, the probability that one of them will be accessible to an existing means of investigation increases. The same rationale applies to the para-lytic phenotype itself. From a variety of defects which cause paralysis, a new access to the neuromuscular system may be found. METHODS AND MATERIALS General Procedures. Complete descriptions of the mutations affecting behaviour will be given; other stocks used routinely have been described by Lindsley andGraLl (1968). Unless specified, the medium was the standard mixture used at the California Institute of Technology. In experiments requiring media containing various molarities of LiCl, NH^ .C1, KC1 and NaCl the appropriate salt solution was diluted ten fold with standard media and blended with a Sorvall Omnimixer. Crosses were made in shell vials or quarter and half pint milk bottles. The standard laboratory temperature was 21.5 - 1°C« Screening devices for recovery of paralysed flies. Theoretically a paralysed individual should be easy to find within a group of active flies. Unfortunately, a caged population of normal flies usually includes many which stay perfectly s t i l l at any one moment. Because each fly goes through its own cycles of activity and inactivity, the membership of the motionless segment of the population is constantly changing. For this reason, direct attempts to determine which motionless fly was actually paralysed proved to be impractical. Two types of apparatus were designed to overcome this problem. Considerable success in separating mobile from immobile flies was achieved by placing the flies in an empty shell vial which was capped with a paper funnel leading upward into another shell vial containing medium (Figure 1). The flies, driven by their tendency to move upward and the attraction of the medium, climbed into the upper FIGURE 1 Type I-a screening device for isolating paralysed fli e s . The device consists of an upper and lower vial facing mouth to mouth separated by an upwardly directed paper funnel. The upper vial contains media. An active fly (A) climbs into the upper vial. After many flies have left the lower vial, the paralysed fly (P) is more easily noticed among the diminished population. 6 vial. The cone was a fairly efficient valve which prevented migrants from returning. Immobile individuals were selected from the diminished population of the lower vial. This assembly will be referred to as the type I-a screener. A modification of this design is shown in Figure 2, Vials positioned above and below funnels cut out of -§•" plexiglass were held in place by foam rubber cushions. This device (type I-b) permitted easy handling of the vials and provided an unobstructed view of the flies. When a large population of flies is confined to a small space, the flies tend to cluster, their activity seems aimless and their anti-geotactic behaviour diminishes. Consequently, the type I designs imposed a limit of a few hundred flies per vial for optimal screening efficiency, Benzer (19^7) found that nonphototactic mutants could be selected by counter current distribution from a population of several hundred flies placed in an 18 x 150 mm test tube. However, each group of flies to be tested must be put through several operations and tubes, A second limitation of the type I device was the dependence on a constant behaviour pattern, namely migration through the funnel into the upper vial. Many flies were capable of climbing, but simply remained in the lower vial. Some showed no antigeotactic tendency while other flies stopped inside the funnel at its lower edge. The efficiency of the screener was somewhat improved by selecting for a strain of flies characterised by its ability to reach the upper vial quickly. Mutants were induced and selected from this stock which will be referred to as F,C, for fast climbers. However, heterogeneous behaviour was never eliminated. Consequently, a more efficient 7 FIGURE 2 i Type I-b screening device for i s o l a t i n g paralysed f l i e s . The device works on the same pr i n c i p l e as the type I-a. In this case, the upper and lower v i a l s are pushed against funnels of plexiglass with foam rubber cushions. 8 separating device was needed. The type II screener (Figures 3 and 4) is basically an elongated box with a diagonal barrier (b) extending the length of the box and a drawer (d) f i t t i n g into the acute angle between the barrier and the bottom of the box. The diagrams of the end view of the box, shown in Figure 4, i l l u s t r a t e the sequence of operations used to isolate an immobilised individual (P) from a large population. Rotation of the box clockwise 90° and then counterclockwise 90° (i,e,, from position 1 to 2 to 3) causes P to f a l l into the drawer. The activity of the population is greatly increased by introducing a small quantity of vinegar and detergent solution onto the l e f t side of the barrier (3)» A l l f l i e s which are not inside the drawer can be eliminated by rotating the box 90° and shaking the active f l i e s into the solution (4), Finally, P can be selected from among the small number of individuals which remain in the drawer (5), The design minimises the deleterious effects of crowding, as well as the number of physical obstacles which tend to inhibit free movement. Six thousand f l i e s at a time could be readily screened i n the type II box shown in Figure 3« Screening procedure for dominant temperature-sensitive mutants. The efficiency of the type I screener depended upon the speed with which a large proportion of the f l i e s would enter the upper v i a l . In this respect, Oregon-R was better suited to the apparatus than Samarkand. The efficiency of separation was further improved by occasionally regenerating the stock with f l i e s which had reached the upper v i a l most quickly. 9 FIGURE 3 Type I I screening dev ice f o r i s o l a t i n g para lysed f l i e s . Outside dimensions 6" x 6" x 18" (^ -" p l e x i g l a s s ) . Vinegar Feed Pipe F l y Entrance Vinegar I n l e t Drawer Stop V e n t i l a t i o n Hole Diagonal 3 a r r i e r 10 FIGURE 4 End views of the type I I screening device during the i s o l a t i o n of paralysed f l i e s . Steps 1 to 5 represent successive stages i n the procedure. The l a b e l b refers to a diagonal ba r r i e r ; d i s a drawer P i s an immobilised f l y . The hatched area represents a vinegar and detergent so lut ion. The operation of th i s device i s explained on page 8 . 11 F.C, Oregon-R males were l e f t f o r 24 hours i n h a l f p i n t milk b o t t l e s containing two discs of Whatman No. 1 f i l t e r paper dampened with 1 ml of 0.025M e t h y l methanesulphonate (EMS) i n a 1$ sucrose s o l u t i o n (Lewis and Bacher, 1968). The mutagen treated males were then mated with F.C. Oregon-R females i n quarter and h a l f p i n t milk b o t t l e s (10 to 15 p a i r s per b o t t l e ) . These f l i e s were tra n s f e r r e d t o f r e s h b o t t l e s every 3 to 8 days. Eggs were never c o l l e c t e d beyond 16 days a f t e r treatment. The progeny were r a i s e d at 22°C, A f t e r eclosion, they were placed i n t o a type I or type II screener which had been prewarmed to o o 29 C„ A f t e r 10 minutes to 2 hours at 29 C, the population was screened f o r immobile f l i e s . Each immobile f l y which recovered mobility at o 22 was then crossed to 3 F.C. Oregon-R f l i e s i n a s h e l l v i a l . The progeny of the f e r t i l e i n d i v i d u a l s were again r a i s e d at 22°C and then tested f o r the inheritance of p a r a l y s i s by d i r e c t t r a n s f e r to a 29°C v i a l . This procedure i s outlined i n Figure 5. Screening procedure f or sex-linked recessive and autosomal dominant  mutations. A search f o r sex-linked mutants was also conducted (Figure 6). EMS-treated Oregon-R males were crossed to F.C. females carrying an progeny carrying mutagenised paternal X chromosomes. A l l screening was c a r r i e d out i n a type II apparatus. The selected males and females were i n d i v i d u a l l y crossed to F.C, XX/Y females and F.C, Oregon-R males, r e s p e c t i v e l y . Their progeny were tested f o r i n h e r i t e d temperature-s e n s i t i v e p a r a l y s i s as described above. attached-X chromosome This permitted the recovery of male 12 FIGURE 5 Procedure for detection of a dominant temperature-sensitive paralytic mutant. F o C . 0 r e g o n - R^ ( 0 o 0 2 5 M E K 3 ) x F 0 C . Oregon-R 2 2 ° C Fi adults shifted from 2 2 ° C to 2 9 ° C Immobile flies selected with a type I or type II screener Flies selected are returned to 22 C Flies recovering mobility at 22 C are mated A F . C , O r e g o n - R o r F . C . Oregon-R 2 2 ° C 2 2 ° C Tested for paralysis induced by a 2 2 ° C to 2 9 ° C shift Tested for recovery by returning flies to 22 C 13 FIGURE 6 Procedure for detection of a sex-linked recessive or autosomal dominant temperature-sensitive paralytic mutant. F.C. 0regon-R^/(0.025M EMS) x F8C. f\/Y,+ ^ 22°C Fi adults shifted from 22°C to 29°C Immobile flies selected with a type II screener • o Immobile flies returned to 22 C Flies which recover are mated <5*xF.C. XX/Y,+ or F.C. Oregon-R^ 22°C 22°C O a Tested for paralysis induced by a 22 C to 29 C shift Tested for recovery by returning flies to 22 C 14 Recovery and genetic mapping of a shuddering mutant. In the screen f o r dominant temperature-sensitive p a r a l y s i s using a type I-b device, a female which occasionally shuddered was noticed i n the lower v i a l . The behaviour was found to be i n h e r i t e d as a sex-l i n k e d dominant; consequently, the mutant chromosome was balanced over the multiply inverted chromosome, FM6, While anaesthetised under ether, the shuddering f l i e s s c i s s o r e d t h e i r wings and shook t h e i r legs i n quick vigorous spasms. In order to l o c a l i s e the genetic s i t e responsible f o r the behaviour under ether, females were crossed to f l i e s carrying the following markers (followed by t h e i r symbols and genetic l o c a t i o n ) ; yellow - y_ (0.0), c r o s s v e i n l e s s -cv (13.7), vermilion - v (33.0), forked - f (56,7), carnation - car (62,5). Heterozygous females were testcrossed and a l l male and female progeny were scored. The shuddering males were extremely weak and uncoordinated and survived only f o r one to three days under good culture conditions. In order to reduce the severe e f f e c t s of crowding and competition on these f l i e s , s i n g l e females were mated to three males i n s h e l l v i a l s and were s e r i a l l y t r a nsferred t o new v i a l s each day f o r 10 days before being discarded. This mutant w i l l hereafter be r e f e r r e d to as Shuddering or Shu, The e f f e c t s of monovalent cations on shuddering behaviour. The p o s s i b i l i t y that l i t h i u m ions might suppress shuddering behaviour was suggested by t h e i r therapeutic e f f e c t s upon nervous disorders i n guinea pigs and man (Cade, 1949). The s i m i l a r i t i e s between shuddering and these disorders w i l l be discussed l a t e r . Ammonium ions were also tested because the aberrant leg shaking of neurological 15 mutants of Drosophila under ether anaesthesia was reversibly suppressed by ammonia (Kaplan, personal communication). Four pairs of Shu/FM6 flies were placed in quarter pint bottles (1 pair per bottle) containing various concentrations of LiCl, KCi, NaCl or NH^Cl, and twelve pairs were placed on standard media. The number of jerks and shudders produced by each pair of flies during one minute was recorded daily. Shudders lasting for about three seconds or more were counted as two shudders. Prior to each observation, the bottle cap was replaced with a transparent plastic petri plate. Following this disruptive manipulation, the bottle was left undisturbed. Under these conditions, flies treated with 0,32 M to 0,35 M LiCl ceased shuddering after three and four days of treatment; however, the mortality rate was high. Consequently, a second series of experiments was performed to test the capacity of LiCl to suppress shuddering induced by mechanical stimulation, and to determine whether or not the flies would continue to live, i f they were returned to standard media at critical times. Groups of 5 to 40 Shu/FM6 flies were placed in quarter pint milk bottles on standard media and media containing LiCl, NaCl and NH/j,Cl at 0,2M, 0.32M and 0.33M concentrations. Every 12 or 24 hours, flies in each bottle were shaken down repeatedly. By one and a half days of age, Shu/FM6 flies kept on standard media would inevitably give a shuddering response to this type of mechanical stimulation. Two types of responses were recorded: "no shuddering" or "slight shuddering". The latter class was defined by a few slight jerks seen within a group. Two groups of 40 flies were transferred from 0.32M LiCl to standard media after three days. The rest of the flies were returned to standard media 16 after six days or at various earlier times when the f i r s t deaths occurred in each bottle. Bottles of media which were not used immediately were stored in plastic bags to reduce desiccation and consequent changes in salt concentration. The f l i e s were usually transferred to new media every three days in order to minimise exposure of the f l i e s to desiccated and microbially infected media. Virgin females were used i n most experiments to obviate changes in the behaviour of adults on media which had become softened and dampened by developing larvae. In cultures containing many larvae, Oregon-R f l i e s walked slowly with a slight side to side wobble. The same behaviour was accentuated i n Shu/FM6 f l i e s in which the wobble frequently became a more vigorous shudder. Observation of a temperature-sensitive paralytic mutant. A male whose paralysis was temperature-dependent was isolated from an mother using a type II screening device. Upon crossing the male to XX/Y females, a l l male offspring showed temperature-sensitive paralysis. Females heterozygous for the mutation and FM6 were unaffected by temperature, thereby showing the mutation to be recessive. Homozygous females were also paralysed at 29°C. Males carrying the mutation were crossed to y cv v f car and their o F-j_ daughters were testcrossed at 22 C„ The F 2 progeny were scored for the v i s i b l e markers and the males were screened for paralysis at 29°C, The mutation was called paralytic temperature-sensitive and designated ts as para . Preliminary observations of the effects of temperature upon the 17 ts behaviour of para were made by simply transferring the f l i e s to vials which had been preconditioned to various temperatures. In order to examine behaviour at constant temperatures, the f l i e s were placed into twin observation chambers which were b u i l t into a trans-parent temperature-controlled water bath (Figure 7)*» The temperature in the chambers was maintained by constantly exchanging water between the observation bath and a Blue M constant temperature bath. The temperature was monitored with a Yellow Springs Instrument Company general purpose thermistor probe and registered on a Rustrak recorder,, ts Generally 5 to 10 para and an equal number of Oregon-R f l i e s were observed over a two hour period following transfer of the f l i e s to the observation chamber. Most experiments required one person to monitor behaviour continuously while another recorded the changes in behaviour with temperature and time. A second type of chamber was constructed i n order to observe the f l i e s while the temperature was being changed (Figure 8). The f l i e s were placed in the bottom of three chambers which were formed by stacking four 3.5cm plastic petri plates on top of each other. The chambers were sealed together with plastic insulation tape. A 1mm air hole was made i n each of the upper three plates, each hole having a staggered position, with respect to i t s vertical axis, i n relation to the other two. A thermistor wrapped i n insulation tape above the sensing resistor was seated snugly into the lower chamber. By partially submerging this assembly i n a water bath, the temperature of the bottom chamber was quickly altered and equilibrated within the variations of the bath i t s e l f . * This apparatus was designed and bu i l t by Tom G r i g l i a t t i . 18 F I G U R E 7 Twin water-heated chambers f o r comparing behaviour at constant temneratures. p h e r m i s t o r Observation chambers 19 F I G U R E 8 A semi-immersible chamber f o r observing behaviour during temperature changes. 2 0 The quickest temperature changes were produced by moving the chamber from one bath to another. More gradual temperature increases were achieved by immersing the chamber i n a Haake F,S, constant temperature bath and r a i s i n g the bath temperature e l e c t r i c a l l y . Observations were made through a d i s s e c t i n g microscope with sub-stage i l l u m i n a t i o n by l i g h t r e f l e c t e d from the bottom of the bath. Overhead l i g h t could not be used because the r e f l e c t i o n s obscured the ts view, and the heat r a d i a t i o n was s u f f i c i e n t l y high t o immobilise para flies„ Because the f l i e s were anaesthetised with ether i n order to place them i n the chamber, and because the chamber temperature was a l t e r e d by handling during i t s assembly, the f l i e s were maintained f o r an hour at o 2 2 C following t h e i r recovery from the anaesthetic, ts Interactions between para and n e u r o l o g i c a l mutations. IP i p Under ether anaesthesia, the sex-linked mutants Hyperkinetic -HK ( 3 0 . 9 ± 0 6 ) , H y p e r k i n e t i c 2 1 - HK 2 T ( 3 0 . 4 + . 7 ) and Shaker 5 - S h 5 ( 5 8 , 2 1 , 6 ) shake t h e i r legs vigorously and r a p i d l y (Kaplan and Trout, 1 9 6 9 ) . In the case of S h 5 . the l e g movements are accompanied by repeated s c i s s o r i n g of the wings. These tremulous f l i e s were c a l l e d " n e u r o l o g i c a l mutants" by Kaplan and Trout ( 1 9 & 9 ) a m * w i l l hereafter be generalised by the abbreviation Neuro. Ikeda and Kaplan ( 1 9 7 0 a ) demonstrated that the c h a r a c t e r i s t i c IP r a p i d l eg shaking of HK originated as impulse bursts i n the three paired motor areas of the t h o r a c i c ganglion. The rhythmic bursts were t r u l y endogenous and received no contributions from the cephalic, sensory or neuromuscular systems. Studies of b i l a t e r a l thoracic mosaics 21 containing HK1P/+ * and HK1 P/0 tissues (Ikeda and Kaplan, 1970b) IP IP indicated that the electrophysiology of HK /0 and HK /+ tissues and the associated behaviour was bi l a t e r a l l y autonomous within the thorax and also independent of the rest of the body. The interactions of Neuro and para"*-'5 mutations could be best analysed i n Neuro para^5/Y males, / ts Single Neuro +/+ para females were placed with wild type males for 7 days at 22°C and then discarded (Figure 9, step l ) . The male progeny were scored for temperature-sensitive paralysis at 29°C and either-induced leg shaking at 22°C and 29°C0 The phenotypes were ts compared with Neuro, para and Oregon-R stocks. Newly arising ts phenotypes which exhibited characteristics of both Neuro and para were designated Neuro para^S/Y. The genetic integrity of the phenotype was tested by crossing each male to females carrying attached-X chromosomes and scoring for the same phenotype i n male progeny (Figure 9, step 2). The double mutation condition of the chromosomes could be verified by re-isolating the single mutations by recombination. Therefore, single Neuro para^S/++ females derived from each of the original putative recombinant double mutant males were crossed to Oregon-R males (step 4), The male progeny were then scored for the ts occurrence of para and Neuro behaviour separately. ts Interaction of para and Minutes. The possibility that the mutation M(l)0. located at 56.6, might be a deletion which would span the paralytic region, prompted a test for ts pseudodominance of para when heterozygous with M(l)0. A pa r t i a l t<? paralysis distinct from that of para homozygotes was observed in * For a l l practical purposes HK can be regarded as recessive. 22 F I G U R E 9 Protocol for generating and testing Neuro para t s/Y males Steps 1. + + / l / x + para t s/Neuro + ^ 2. Select putative | Neuro para^/Ytf 7 ts 3o + +/+ x Neuro para /Y & k. + +/Yd* x Neuro para t s/ + + |» Score for recovery of Neuro+ZY^1 t p a r a ^ / Y ^ 23 M(1)0 f/para^ s females, but not i n f/para^ s controls. This suggested that Minute mutations in general might affect the expression of para^ s. ts Male para f l i e s were crossed to single females of the following genotypes: M(l)0 f/FM6, M(2)eS/SM5; M(2)S7/SM5; M(3)w 1 2*Vln(3R)C. e l(3)e; M(3)wB/ln(3R)C. e l(3)e. Five matings of each type were made on two separate occasions. The progeny were raised to 22°C, transferred directly into 29°C vials and observed for any change i n behaviour. 2h RESULTS Shuddering: phenotype and map p o s i t i o n There were no obvious behavioural differences between Shu/FM6 and Shu/+ f l i e s i n e i t h e r the •'conscious" or anaesthetised s t a t e s . Since the Shuddering mutation was conveniently maintained i n Shu/FM6 females, they were the main subjects of study, A four day old Shu/FM6 f l y shuddered on an average of 8 times a minute, although i n d i v i d u a l s v a r i e d from le s s than 1 to more than 50 times a minute. The shuddering phenotype was produced by a r a p i d sequence of leg jerks which caused the f l y to lunge i n various d i r e c t i o n s . The re s u l t a n t "shudder" l a s t e d f o r a f r a c t i o n of a second to about four seconds, and v a r i e d i n i n t e n s i t y from a s l i g h t jerk to a shudder which was strong enough t o throw the f l y on i t s back. Although shuddering occurred spontaneously, i t could also be e l i c i t e d by gently moving a b o t t l e containing Shu/FM6 f l i e s which had previously been l e f t undisturbed. Drosophila can often be induced to f l y when they are l i f t e d from a surface by means of a toothpick glued to the d o r s a l side of the thorax. Under t h i s tethered f l i g h t condition, Shu/FM6 f l i e s v ibrated t h e i r legs continuously. In rare instances, Oregon-R f l i e s a l so v i b r a t e d t h e i r legs, but th i s movement was i n e v i t a b l y stopped during f l i g h t . When Shu/FM6 f l i e s were anaesthetised with ether, they remained s t i l l f o r only a b r i e f period before shaking t h e i r legs vigorously and r a p i d l y . The shaking was generated i n the t i b i a e and tarsae while the femurs remained r e l a t i v e l y s t i l l . The i n t e n s i t y of the shaking was 25 spasmodic, u n l i k e the continuous tremor of the legs i n tethered flight,, Eventually wing s c i s s o r i n g began, accompanied by movements of the halte r e s , antennae and head. These l a t e r movements d i d not normally begin simultaneously, but they were often observed to be i n synchrony with one another. Very s i m i l a r movements were seen i n etherised f l i e s which were decapitated. When these headless f l i e s recovered from ether, they r i g h t e d themselves and then shuddered occasionally, although the shuddering seemed infrequent when compared with whole f l i e s . None of these patterns of behaviour was observed i n whole or decapitated FM6 or Oregon-R f l i e s . Shu/Y males were extremely shaky. They were thrown onto t h e i r backs frequently. Under ether they shook t h e i r legs more vigorously than the heterozygotes. When they were able to walk, the males u s u a l l y dragged t h e i r wings which were s h r i v e l l e d and held l a t e r a l l y , drooping towards the t i p s . Shuddering mapped at 55.1 - 0.4*. This p o s i t i o n was calculated by the r e l a t i v e p o s i t i o n of the mutation observed between v and f and placed i n t o the book distance. The book value f o r the v to f distance i s 23.7 u n i t s whereas the observed distance was 19.6 u n i t s . The behaviour of recombinants a f t e r recovery from ether ind i c a t e d that shuddering, leg shaking and wing s c i s s o r i n g were not separable by crossing over and therefore were phenotypic manifestations of the same mutation. E f f e c t s of monovalent cations on shuddering behaviour Table 1 summarises the r e s u l t s of two experiments which tested * The 95$ confidence l i m i t s were calculated, by the method of Stevens (19^2) TABLE 1 Frequency of spontaneous shuddering i n Shu/FM6 f l i e s af ter d i f fe rent s a l t treatments. Frequency i s expressed as shudders per f l y per minute. Where death has occurred, the survivors are presented i n brackets as a f r a c t i on of the o r i g i n a l number of f l i e s . Experiment 1 Experiment 2 Age i n days 0-2 1-3 2-4 1-1 1/2 2-2 1/2 3-3 1/2 4-4 1/2 Days on treatment 0 1 2 1 2 3 4 Molarity Molarity 0.0 7.1 13.1 12,4 0.0 2 4.7 6.8 8.1 S a l t /0.12 4,4 7.5 11,4 0.32 0.4(7/8) 0.5(6/8) 0.0(6/8) 0,0(6/8) [0.18 7.3 12.5 9.1 0.33 0.5(6/8) 5.3(6/8) 0.0(6/8) 1.0(2/8) L i C l Jo . 24 7.0 11.5 17.8 0,34 0.0(7/8) 0.0(4/8) 0.0(4/8) (0/8) /0.30 6.1(7/8) 5.6(7/8) 9.7(7/8) 0.35 0.1(6/8) 1.0(5/8) 0.0(5/8) 0.0(4/8) \ 0,36 5.5(7/8) 2.2(5/8) 0.0(5/8) 0.36 0.8 3.2 1.6 0,0(5/8) /0,12 9.7 15.8 13.0 0.32 1.6 4.5 6,3 8.9 10.18 10,1 14,9 9.9 0.33 4,0 7.7 8.8 8.8 NaCl "S0.24 7.9 7.9 12,1 0.34 2,6 8,8 10.3 11.1 J0„30 9.9 13.5 15.3 0.35 1.5 4.3 10.1 10,9 \0.36 12,3 12.4 12.3(6/8) 0.35 3.2 6.0 9.3 9.0 /0,12 8,8 9.4 0.8 0.32 3.7 12.6 6.8 9.0 0,18 3.5 14.9 5.5 0.33 1.1 6.9 5.9 9.0 N%C1 { 0.24 12.4 7.9 7.3 0.34 4.3 5.0 4.9 3.9 0.30 12.1 13.5 14,5 0.35 2,6 2.8 3.6 3.8 \0.36 9.8 12.4 4.0 0.36 2.6 8,6 1.8 5.8 / 0.32 5.3 2.1 0.5 4.4 0.33 3.2 3.1 3.1 5.0(7/8) KCl \ 0.34 3.6 0.8 5.9 5.8 0.35 1.7 0.4 3.^ 2.0 \ 0.36 1.0 0.9 2.3 1.9 27 the e f f e c t s of various s a l t s on spontaneous shuddering i n undisturbed Shu/FM6 f l i e s . The experiments d i f f e r e d with respect t o the ages of the f l i e s and the times at which observations were made r e l a t i v e to the beginning of treatments. The shuddering frequency of f l i e s placed on standard media and NaCl media increased with time. No cl e a r trends were seen i n f l i e s placed on NH^Cl and KC1. 0„32M to 0,35M L i C l seemed to suppress shuddering a f t e r three or four days of treatment, however many f l i e s died during t h i s time, A second set of experiments tested the capacity of L i C l to suppress shuddering induced by mechanical stimulation and attempted to increase s u r v i v a l by returning the f l i e s t o standard media at various c r i t i c a l times. The number of f l i e s placed on each treatment together with the number which subsequently died i s presented i n Table 2, The r e s u l t s of the treatments which suppressed shuddering are presented i n Figure 10. Although many f l i e s died on 0.32M and 0.33M L i C l , the number of survivors (102 out of 131) d i d not change from day 6 to day 13. The tests c l e a r l y indicated a reduction or complete suppression of shudder-ing a f t e r three days of treatment with 0.32M or 0.33M L i C l . The previous data ind i c a t e d that even under les s disturbing conditions, untreated Shu/FM6 f l i e s of an age comparable to the treated se r i e s would be shuddering at an average rate of 7 times a minute. Shuddering was also suppressed i n a group of 5 f l i e s placed on 0.2M L i C l and 5 f l i e s on 0.33M NaCl. Shuddering could not be e l i c i t e d from the l a t t e r group when they were f i r s t placed on the treated media. This nonrespon-s i v e behaviour was occasionally seen i n very young f l i e s . Shuddering was e a s i l y e l i c i t e d on subsequent days. Therefore, the i n i t i a l observation probably d i d not r e f l e c t an e f f e c t of the NaCl, 28 TABLE 2 Total numbers of f l i e s tested for shuddering induced by mechanical stimulation after different salt treatments. 0.2M 0.32M 0.33M L i C l 25 66 (15 died) 65 (14 died) NaCl 5 ( 1 died) 5 5 NH^Cl 10 10 10 Standard Media 45 FIGURE 10 Reduction of shuddering in Shu/FM6 f l i e s subjected to mechanical stimulation. White squares indicate slight shuddering, black squares no shuddering. Arrows indicate duration of treatment. Survivors are presented as a fraction of the original sample size, M and N signify observations made at midnight and noon, respectively. Time in days # Observations Survivors/ Age of f l i e s per 24 hours f l i e s tested Treatment 0 1 2 3 4 5 6 M M 0-1 1 29/40 ,32M L i C l .. n » " 0-1 1 30/40 ,33M LiCl 1 / 2 - 1 2 4/6 .32M LiCl 1 / 2 - 1 2 4/5 .33M LiCl 1 - 1 1 / 2 2 9/10 ,32M L i C l 1 - 1 1/2 2 8/10 .33M L i C l 1 - 1 1/2 2 9/10 ,32M L i C l M M HMD N M N • • • • • • M • . N M N M mm m M N M • • • M N M N N M • • • • • • • M N M M M • • M N M 1 - 1 1/2 2 9/10 ,33M LiCl • % • N N M M N 1 / 2 - 1 2 5/5 .2 M LiCl • • a UP t 1/2-1 2 5/5 ,33M NaCl 30 Lethargic behaviour and a f i n e tremor which are symptoms of t o x i c l e v e l s of l i t h i u m i n rats and men (Schou, 1958, 1959), were •oe:casi«nal.]^ seen i n Shu/FM6 i n d i v i d u a l s and were u s u a l l y followed by death. However, most f l i e s i n which shuddering had been suppressed showed neither of these symptoms. In f a c t , they appeared to be more ac t i v e than untreated Shu/FM6 f l i e s of the same age. ts I s o l a t i o n of para An estimate of the number of f l i e s screened f o r sex-linked recessive or autosomal dominant temperature-sensitive p a r a l y s i s was obtained by counting the number of f l i e s i n one or two b o t t l e s out of every t h i r t y screened. The r e s u l t s are summarised i n Table 3° Of the estimated 250,000 f l i e s screened, 293 were immobilised at 29°C, 200 of these were found t o be dead. I t i s not known whether death occurred before, during or a f t e r the screening process. Of the 93 f l i e s which recovered mobility at 22°C, 34 were f e r t i l e . One f l y was found to transmit h e r i t a b l e temperature-sensitive p a r a l y s i s . The sex-linked mutation, p a r a ^ s . mapped at 53<>9 (Suzuki et a l , , 1971). I t i s thought that a much la r g e r number of f i i e s was screened f o r dominant temperature-sensitive p a r a l y s i s , although estimates of t h i s number were not made. The chances of f i n d i n g the dominant mutation may have been a l t e r e d by the use of two types of screening devices rather than one. For these reasons, a v a l i d comparison of the success of the two screening procedures cannot be made. ts E f f e c t s of d i f f e r e n t temperatures on para f l i e s o t s At 22 C, para f l i e s exhibited normal walking, climbing and 31 TABLE 3 Estimated number of f l i e s screened f o r sex-linked recessive and autosomal dominant temperature-sensitive p a r a l y s i s . Type of f l i e s Number T o t a l f l i e s screened 250,000 Immobilised at 29°C 293 Dead 200 Recovered mobility at 22°C 93 F e r t i l e at 22°C Temperature-sensitive p a r a l y t i c mutations 1 32 f l y i n g a b i l i t i e s . When shaken i n t o v i a l s which had been preheated to o 0 29 C, they became paralysed within 5 seconds. Upon returning to 22 C, they regained very a c t i v e mobility within 2 or 3 seconds. Paralysis and recovery could be induced repeatedly i n t h i s way with no apparent harm o ts to the f l i e s . When kept at 29 C f o r prolonged periods, para f l i e s d i d not remain paralysed. Af t e r 30 minutes, most f l i e s were able to get to t h e i r f e e t . At s t i l l l a t e r times, they were able to walk and climb. However, these f l i e s never exhibited the same degree of coordination o o and a c t i v i t y that they had shown at 22 C, At any time, a s h i f t to 22 C would r e s u l t i n an immediate recovery of wild type a c t i v i t y . The mutation d i d not appear to a f f e c t v i s c e r a l muscles. After a s h i f t from 22 WC to 29 C, heart pumping could s t i l l be seen through the t<5 d o r s a l abdominal c u t i c l e of para f l i e s . A f t e r being t r a n s f e r r e d from a 22°C to a 29°C Drosophila Ringers s o l u t i o n (Ephrussi and Beadle, 1936), the para^ s ejaculatory duct also continued t o pulsate. None of the l a r v a l i n s t a r s was a f f e c t e d by t r a n s f e r from a 22°C culture to Drosophila Ringers solutions kept at 29°C, However, a comparative study of the development of para^ s/Y and X^,p_ara^f +/Y s i b l i n g s indicated that the number of successful eclosions by the males was normal when pupae r a i s e d at 29°C were s h i f t e d to 22°C p r i o r to eclosion, but g r e a t l y reduced when pupae were maintained at 29°C (Suzuki et a l , , 1971)„ The behavioural responses to increased temperature were studied i n f l i e s which had been r a i s e d at 22°C by shaking f l i e s from 22°C v i a l s i n t o separate water-heated chambers. Observations of changes i n behaviour were made on 20 of each sex of p a r a t s and Oregon-R f l i e s at one degree i n t e r v a l s over a 22°G to 35°C range. Behaviour was monitored 33 continuously for the f i r s t hour and then once every 10 minutes over the second hour. For purposes of general comparison, specific types of behaviour were classified in the following order of increased motor competence; paralysis, leg shaking, self-righting to a standing position, walking, climbing, flying. Figure 11 is a graph of these motor a c t i v i t i e s , exhibited by at least 50$ of the flies,plotted on an arbitrary scale against time. Recovery rates were far from uniform within a group; i n i t i a t i o n of the same activities being separated by as much as 50 minutes in different f l i e s . Although the average mobility clearly improved with time, the recovery of each f l y was not always progressive. Flies which had demonstrated good walking and climbing a b i l i t i e s would occasionally revert to a very weak stance for several minutes. Even within a single f l y , recovery did not appear to be uniformly progressive. Often one or two legs on one side of the body would move while the legs on the opposite side remained s t i l l . Differences in the extension of the legs frequently caused the f l y to l i s t while standing and pitch and r o l l while walking. The debilitating effects of progressively higher temperatures were manifested in both the extent of the i n i t i a l response and the length of time taken to recover further activity at various ts temperatures, para f l i e s were noticeably debilitated by shifts to the 26°C to 28°C range. At higher temperatures they were completely o ts paralysed. At 29 C, para f l i e s were able to climb after 70 minutes; o at 31 C, 105 minutes were needed to regain the same activity. After two hours at any temperature up to 33°C, the f l i e s were able to recover mobility upon returning to 22°C„ However, a two hour exposure to 34° C resulted i n 90$ of the f l i e s dying. Oregon-R f l i e s were unaffected by 34 FIGURE 11 Recovery of motor competence with time a f t e r increases i n temperature. FLYING PARALYSIS 60 TIME (MINUTES) 90 120 35 two hour exposures to temperatures ranging from 22°C to 37°C, A l i s t of the types of movements observed during recovery at immobilising temperatures i s presented i n roughly chronological order i n Table 4, A l l of the movements l i s t e d were r a r e l y seen i n a s i n g l e f l y . However, they were crude ind i c a t o r s of l e v e l s of recovery, o Although 22 C had been chosen as the permissive temperature f o r screening and most s h i f t experiments, i t was subsequently discovered "t s o that para f l i e s r a i s e d at 17 C became slow or immobile when tra n s f e r r e d to 22°C 0 Upon returning to 17°C, they became a c t i v e within ts a few seconds. Thus i t appeared that the para mutation had rendered the f l y s e n s i t i v e to temperature increases over at l e a s t a 17°C to o 29 C range. The experiments which followed the discovery of 22°C s e n s i t i v i t y were designed to t e s t a simple model which had been used to explain t s p a r a l y s i s and recovery phenomena. For h e u r i s t i c purposes, para might be assumed to produce an enzyme whose c a t a l y t i c e f f i c i e n c y was an inverse function of temperature ( i , e , , Km i s a d i r e c t function of temperature) and whose rea c t i o n product permitted mobility of the f l y when the product was formed at a s u f f i c i e n t r a t e . Given equal substrate concentrations, the rate of product formation would diminish at high temperatures and motor a c t i v i t y would consequently diminish a l s o . However, the reduced e f f i c i e n c y of the enzyme would a l s o cause the substrate concentration to increase i f the substrate were supplied to the enzyme at a s u f f i c i e n t r a t e . I f the substrate l e v e l then became high enough to overcome the increased Km of the enzyme, product formation might again reach a rate which would permit mobility. This hypothesis i s presented as an example of a basic concept, namely a "damming up" 36 TABLE 4 Types of movement which may be seen during recovery from temperature-induced p a r a l y s i s . The l i s t i s i n roughly chronological order, 1, P a r a l y s i s (legs contracted), 2, Extension and contraction of the femurs. 3, T a r s a l twitching. 4, Extension and contraction of the t i b i a s , 5, Slow movements of the wings l a t e r a l l y or d o r s a l l y and v e n t r a l l y , 6, Ventral f l e x i n g of the p o s t e r i o r abdomen. Movements of the g e n i t a l i a . Excretion. Head nodding. 7, Leg extension followed by pawing movements. 8, Righting. 9, Walking, 10. Preening, 11. Climbing. 12. F l y i n g . 37 e f f e c t . The accumulation of a neural transmittor might have f i t the observations as w e l l , i f t h i s process were more c l e a r l y understood. I f the notion of damming up substrate were correct, f l i e s conditioned at 17°C with a r e l a t i v e l y low substrate concentration, would be expected to take longer to recover mobility at 29°C than f l i e s conditioned to 22°C. Accordingly, 44 para^ s f l i e s (0 to 3 days of age) which had been r a i s e d at 22°C were divided i n t o two equal groups. One group was l e f t at 22°C, while the other was placed at 17°C f o r three days. 22°C and 17°C groups 4- c O of para f l i e s were then simultaneously transferred to 29.5 C. A d e t a i l e d comparison was made of two groups of 3 f l i e s while the remaining two groups of 19 f l i e s were observed more casually f o r the recovery of basic parameters of mobility, such as standing, walking and climbing. By a l l measured c r i t e r i a , 17°C-conditioned f l i e s recovered more slowly than f l i e s conditioned to 22°C. The d i f f e r e n c e i s i l l u s t r a t e d by the graph shown i n Figure 12 i n which the numbers of f l i e s able to stand i s p l o t t e d against time. Only a f t e r more than an hour do the two curves approach each other. Although these experiments support the "substrate accumulation" model, they also suggest that the time taken f o r recovery i s i n v e r s e l y r e l a t e d to the temperature shock.* While temperature shock i s an i n t u i t i v e l y understandable term, f o r purposes of c l a r i t y i t s h a l l be defined here as increase i n body temperature per u n i t of time. Thus i t s magnitude at any inst a n t i s a d i r e c t function of the d i f f e r e n c e i n temperature between the f l y ' s body and i t s environment. * I am g r a t e f u l to Dr. Hans S t i c h f o r r a i s i n g t h i s important point. 38 FIGURE 12 The number of para t s f l i e s , preconditioned at 22°C (solid line) and 17°C (dashes) which were able to stand after being transferred to 2 9 . 5°C 22 2 0 Time (Minutes) 39 I f the temperature were ra ised at d i f fe rent rates over the same i n t e r v a l , and wi th in a short enough time to induce para ly s i s , the rates of recovery should d i s t ingu i sh between the v a l i d i t i e s of the two arguments. According to the "shock hypothesis", the fas ter r i s e i n temperature should prolong recovery. On the other hand, the quicker a r r i v a l at the high temperature should cause the substrate to accumulate at i t s maximum rate from an ea r l i e r time, thus shortening the time needed for recovery by the "accumulation hypothesis". The e f fec t of temperature shock was studied i n four groups of ts para f l i e s , each group being comprised of 5 males and 5 females, 0 to 2 days o ld . Observations of the number of f l i e s able to stand were made once a minute during and fol lowing each temperature r i s e . When the temperature was ra ised from (22,2°- 22.4°C) to (27,0°- 27,6°C) wi th in a 14 (Figure 13 a) and 6 minute (Figure 13b) i n t e r v a l , the response was dramatical ly d i f f e ren t . The same effects can be seen i n Figures 14a and 14b, although th i s comparison may not be v a l i d since the faster temperature r i s e was also greater by one degree, t s These experiments suggest that para f l i e s are sens i t i ve not only to temperature, but also to temperature shock. The ef fect of shock was seen i n the f l y ' s i n i t i a l response and i n i t s rate of recovery. The resu l t s contradict a "substrate accumulation hypothesis", ts The responses of para f l i e s i n the above experiments strongly suggest that the monitored temperatures were underestimates of the actua l chamber temperatures. The source of error was probably heat loss from the thermistor r e s i s t o r by conduction along 1 cm,of probe wire. Fortunately the s ign i f i cance of these experiments i s not changed by th i s error. 40 FIGURE 13 The number of para u s f l i e s able to stand when temperatures were ehan from (22.2°- 22,4°C) to (27.0° - 27.6°C) within (a) 14 minutes, (b) 6 minutes. Solid line - number of f l i e s able to stand; dashes -temperature. 0 20 — I — 40 60 80 Time (Minutes) Z o z < I -(0 OT UJ (b) 28° 2.T v. 2501 •«««. * 24' 22 A u . o cz Hi CO z 41 FIGURE 14 .ts The number of para J f l i e s able to stand when temperatures were changed from (21.6° - 22 ucT~to (28.5° - 29.5°C) within (a) 24 minutes, (b) 6 minutes. S o l i d l i n e - number of f l i e s standing; dashes -temperature. I to I 0 20 40 60 80 100 - f — (b) 29°. i l i 26°J / 27 26*1 2S5 2,4° 23? 22° T i m e (Minutes) f- ' ^ 120 A r\ jo 9 8 7 6 5 4 3 2 I 1 0 a z < co co u. o ce Ul CO 2 z t s Interactions between para and neu r o l o g i c a l mutants t s / Putative Neuro para /Y males ex h i b i t i n g the c h a r a c t e r i s t i c s of ts both mutations were recovered from Neuro/para females. The standard positions of the Neuro mutations can be seen i n Figure 1 5 , The number ts / of HK para /Y recombinants was less than expected, while the number of para ^ Sh 5/Y males agreed w e l l with t h e o r e t i c a l recombination distances (Table 5 ) . These discrepancies probably r e f l e c t differences i n s u r v i v a l under crowded conditions. The double mutant nature of the phenotypically selected recombinants was confirmed i n a l l but two cases. In both cases, the exceptions r e s u l t e d from c l a s s i f i c a t i o n of HK 1 P/Y males as HK 1 P para t s/Y. Each double mutant was v e r i f i e d by r e i s o l a t i n g s i n g l e mutant chromosomes by recombination (Table 6), The response of Neuro para t s/Y males to a 2 2 ° C t o 2 9 ° C s h i f t was a quick cessation of a l l movement whether induced by ether anaesthetisa-t i o n or not. A l l movements could be restored at 2 2 ° C and stopped again at 2 9 ° C within a few seconds, HK /Y f l i e s were a l s o somewhat s e n s i t i v e to s h i f t s from 2 2 ° C to 2 9 ° C , Ether-induced leg shaking would cease within 1 5 seconds to a minute of exposure to 2 9 ° C , Unlike the legs of HK"*"Ppara^S/Y, these legs began to shake again a f t e r about a minute. Leg shaking could a l s o be r e i n i t i a t e d by returning the HK 1 P/Y f l i e s to 2 2 ° C , but could not be stopped again by returning them to 2 9 ° C , I t should a l s o be emphasised IP that the s e n s i t i v i t y of HK /Y f l i e s t o temperature could u s u a l l y , but 1P not always, be demonstrated. In short, the response of HK /Y d i f f e r e d from that of HK para t s/Y by being slow, u n r e l i a b l e , unsustained and n onr epeatable. 4 3 FIGURE 15 t s 5 Genetic locations of Hk5 para and Sh_ on the X chromosome. iJK-30.5 I pord-53.9 v-33.0 -56.7 TABLE 5 ts Recombination data fo r para and d i f fe rent Neuro mutants, Maternal parent + para t s /HK 1 P + # female progeny 5 2 3 Male progeny recovered Non-crossovers HK' IP 2 6 1 + p a r a t s 1 0 2 Crossovers + + 4 9 HK 1 P p a r a t s 11 Crossing over 1 4 , 2 ( 2 3 . 4 ) 1 tS lrjV21 , + para /HK + 421 HK' 2T + 176 + o a r a t s 108 + + 4 5 HK 2 T p a r a t s 1 7 1 7 o 9 ( 2 3 . 9 ) -3-ts , n 1 5 para +/+ Sir 5 9 0 + SJr ts ^ para + 1 5 4 1 1 6 + + 4 ts 5 e para SIT 5 3 o 2 ( 3.8) * f igure i n bracket i s standard distance. TABLE 6 R e i s o l a t i o n of sing l e mutation chromosomes by recombination of double mutants. Male progeny recovered $ # female Crossing Maternal parent progeny Non-crossovers Crossovers over HK 1 Ppara t s/++ 911 HjT^para^. 210 H T ^ + 92 24.4 (23.4) + + 268 + p a r a t s 62 HK 2 Tpara t s/++ 2,261 H K 2 T p a r a t s 489 HK 2 T + 224 23.5 (23.4) + + 7 3 5 + P a r a t s 152 p a r a t s Sh5/++ 706 p a r a t s S h 5 193 + Sh£ 18 5.1 (3.8) + + 314 p a r a t s + 7 * f i g u r e i n bracket i s standard distance. 46 The behaviour of HK 1 P/Y and HK 1 Ppara t s/Y f l i e s were s i m i l a r at 22°C 0 Both showed a kinetogenic response (jumping i n response to hand movements) and both shook t h e i r legs vigorously under ether anaesthesia, HK para /Y f l i e s d i d not show a kinetogenic response to hand movements at 22°C„ Under ether, l e g shaking was very subdued when 2T / compared with HK IT and i n many cases, was reduced to a r a p i d tremor of the tarsae. At 22°C, S]£ para /Y males exhibited the c h a r a c t e r i s t i c l eg shaking seen i n Sh^/Y mutants under ether anaesthesia, but the wings d i d not s c i s s o r , 2T 5 The vigorous l e g shaking of HK /Y and the wing s c i s s o r i n g of ShPJj ts were restored when the para mutation was removed from the respective Neuro para^ s chromosomes by recombination, ts I n t e r a c t i o n between para and Minutes Of the Minutes tested i n combination with p a r a t s . only M(l ) 0 i n t e r a c t e d to produce a change i n behaviour following a 22°C to 29°C s h i f t , M(l ) 0 f / p a r a ^ s females assumed stationary positions on the f l o o r and walls of the observation chamber f o r 3 minutes or more while FM6/Y and FM6/para controls remained highly a c t i v e . Unlike para homozygotes, M(l ) 0 f / p a r a ^ s f l i e s were able to remain on t h e i r f e e t a f t e r the r i s e i n temperature, although the stances of the f l i e s seemed weak and the f i r s t movements were extremely slow. The recovery of normal ts walking and climbing a c t i v i t y took about two hours. As with para f l i e s , m o b i l i t y could be qu i c k l y and completely restored by returning the f l i e s to 22°C, 47 DISCUSSION The search for i n h e r i t e d temperature-sensitive p a r a l y s i s has l e d to the discovery of two sex-linked mutations which a f f e c t motor a c t i v i t y ; p a r a t s -(53.9) and Shu -(55.1). A cross of Shu/para t S females by para^ s/Y males f a i l e d to y i e l d w i l d types or double mutant recombinants among 2,256 progeny. Therefore, the Shu and p a r a t s mutations may be much clo s e r than f i r s t estimates had indicated. ts A t h i r d mutation, M(1)0 -56.5), appeared to i n t e r a c t with para ts to produce an al t e r e d p a r a l y t i c phenotype i n para /M(1)0 females. ts However, a d i r e c t comparison of such females with para f l i e s has not yet been made. Furthermore, the M(l)Q-bearing chromosome was not studied g e n e t i c a l l y or c y t o l o g i c a l l y . Thus, i t i s not known whether a pre- e x i s t i n g para a l l e l e or del e t i o n of the para"1" locus exists on the chromosome. U n t i l further information i s obtained on the genetic and f u n c t i o n a l ts r e l a t i o n s h i p s of the Shu and para mutations, an understanding of shuddering and pa r a l y s i s can only be i n f e r r e d by phenotypic symptoms and the a l t e r a t i o n of t h e i r expression by genetic, chemical and p h y s i c a l manipulation. Shu/FM6 f l i e s are s i m i l a r to the homozygous ne u r o l o g i c a l mutants, IP 2T 5 HK , HK and S h , i n that they a l l exhibit abnormally vigorous and IP r a p i d l e g shaking under ether anaesthesia. The behaviour of HK /+ 2T and HK /+ females was found to be very d i f f i c u l t to d i s t i n g u i s h from ei t h e r Canton-S or Oregon-R w i l d type females. The leg shaking of Sh 5/+ was subdued compared with the homozygous females, but not completely suppressed. Comparisons were drawn between the homozygous neuro l o g i c a l 4 8 mutants (HK 1 P, HK 2 T and Sh 5) and Shu/FM6 females because of the i r phenotypic closeness; a l be i t the genetic comparison i s inconsistant. The movements of Shu/FM6 and Sh 5 f l i e s under ether are s imi la r i n that they both sc i ssor the i r wings and halteres as w e l l as nodding t he i r heads and extending the i r antennae. However, when Shu/FM6 and Sh 5 were etherised simultaneously and examined together, Sh 5 f l i e s were found to begin shaking t he i r legs much sooner than Shu/FM6 f l i e s d i d . The wing sc i ssor ing of Sh 5 f l i e s involved shorter, quicker movements. I t was also noted that the leg shaking movements of Sh 5 f l i e s involved a l l segments of leg while the movements of Shu/FM6 f l i e s were mainly confined to the t i b i a and tarsus. Thus, Sh 5 and Shu/FM6 were pheno-t y p i c a l l y d i s t inguishable. 5 IP 2T Sh , HK and HK f l i e s jump i n response to the v i s ua l stimulus of a hand movement or to mechanical ag i tat ion (Kaplan and Trout, 1969). However, Shu/FM6 f l i e s responded only to mechanical st imulat ion and d id so by shuddering while standing i n one spot. Ind iv idual Shu/FM6 females continued to shudder pe r i od i ca l l y , even when l e f t fo r long periods i n undisturbed i s o l a t i o n , IP The pattern of rapid leg shaking i n HK f l i e s has been demonstrated to r e su l t from rapid bursts of neural a c t i v i t y (Ikeda and Kaplan, 1970a), The fact that the movements of the wings, halteres, head and antennae of Sh 5 f l i e s and Shu/FM6 females were usual ly i n phase with one another a lso suggests they were e l i c i t e d by a common neurogenic a c t i v i t y rather than independent myogenic impulses. The p o s s i b i l i t y that l i t h i um might suppress the shuddering behaviour of Shu/FM6 f l i e s was suggested by i t s therapeutic ef fects on analogous disorders i n guinea pig and man (Cade, 1949), The analogies drawn 49 between f l i e s and man were crude. Yet the v a r i e t y of behavioural disorders which t h i s ion had been shown to suppress suggested that i t acted upon a common fa c t o r which might even be found i n f l i e s . Cade (1949) discovered that l i t h i u m s a l t s were able to suppress the severe convulsant e f f e c t of i n t r a p e r i t o n e a l urea i n j e c t i o n s upon guinea pigs. He also discovered that the same treatment seemed to eliminate the expression of chronic and recurrent mania i n man. According to Van der Velde (1970) subsequent studies have involved more than 39000 p a t i e n t s . C r i t i c a l evidence f o r the succ e s s f u l treatment of mania has been supplied by Maggs (I963). Strong evidence f o r i t s prophylactic a c t i o n against the recurrence of depression i n manic depressive psychoses has also been presented (Baasturp and Schou, 1967; Goodwin et a l , , 1969)o Shou (1959; 1963) noted that u n l i k e other psycho-therapeutic drugs, l i t h i u m d i d not suppress normal mental, emotional or p h y s i c a l a c t i v i t y . Thus, the ion seemed t o act upon the disorder per se. The s i m i l a r i t y between the shuddering behaviour of Shu/FM6 f l i e s and urea-induced c l o n i c convulsions i n guinea pigs i s fair!Ly obvious. Both are sudden, vigorous, uncontrolled motor a c t i v i t i e s . The syndromes of recurrent mania and manic depression can also be compared with the shuddering of Shu/FM6 i n that they are both characterised by r i s e s and f a l l s i n motor a c t i v i t y (Kraepelin, 1906; A r i e t y , 1959); a l b e i t the r i s e i n a c t i v i t y of the f l y i s more sudden and uncontrolled. In man the r i s e s i n motor a c t i v i t y are accompanied by a loss of con t r o l over the large r e p i t o i r e of behaviour which i s c h a r a c t e r i s t i c of his species. Thus he becomes verbose, i n t r u s i v e and occasionally destructive (Kraepelin, 1906; A r i e t y , 1959). Manic and depressed states may occur 50 repeatedly (Lundquist, 1945) and often cannot be a t t r i b u t e d to environmental stress (Hudgens et a l . , 1967)= The shuddering of Shu/FM6 f l i e s i s also recurrent and sponeous although the shuddering response can a l s o be e l i c i t e d by mechanical stimulation. Among the chloride s a l t s tested, L i C l demonstrated a unique capacity to supress both induced and spontaneous shuddering a c t i v i t y i n Shu/FM6 f l i e s . However, i t was not possible to sustain doses which would supress the increased nervous a c t i v i t y of older f l i e s without k i l l i n g them. I t i s i n t e r e s t i n g to note that older people are also l e s s l i k e l y to respond to l i t h i u m therapy (Van der Velde, 1970). The observed t o x i c i t y of l i t h i u m i n f l i e s suggests that the supression of shuddering might be a p a t h o l o g i c a l prelude to death. This was r u l e d out by the s u r v i v a l of lithium-treated f l i e s which had ceased to shudder f o r one or more days, when they were placed on standard medium. Most f l i e s i n which shuddering had been suppressed exhibited wild type le v e l s of a c t i v i t y and showed no t o x i c symptoms. The attempt to suppress shuddering i n a l l of the treated f l i e s probably required the use of s a l t concentrations which exceeded the tolerance of some i n d i v i d u a l s i n the sample. The uptake, excretion and and s e n s i t i v i t i e s to l i t h i u m have been found to d i f f e r among i n d i v i d u a l s i n both man and animals (Schou, 1958, 1959; Weischer, 1969). Conse-quently, during l i t h i u m therapy i n man, the dosage must be co n t r o l l e d while blood l e v e l s and behaviour are monitored (Schou, 1959). Shuddering was suppressed i n a group of f i v e f l i e s treated with a r e l a t i v e l y low dose of L i C l (0.2M) (Figure 10) which had no e f f e c t on s u r v i v a l . However, higher concentrations would have exceeded the therapeutic l e v e l and may have k i l l e d the f l i e s . 51 Lithium has also been found to supress induced aggression i n r a t s , mice and golden hamsters as w e l l as the n a t u r a l aggression of male Siamese f i g h t i n g f i s h (Weischer, 1969; Sheard, 1970). Once again, these treatments d i d not suppress normal l e v e l s of a c t i v i t y . These experiments were probably suggested by aggressive behaviour which i s often displayed i n the manic state. At t h i s time i t does not seem p r o f i t a b l e to examine the v a l i d i t y of t h i s comparison or the analogies which have been drawn between f l i e s and man. On the other hand, the a e t i o l o g i c a l s i g n i f i c a n c e of these experiments cannot be ignored. The r e s u l t s suggest that among the mechanisms which give r i s e to these various forms of behaviour i n such widely separated organisms, there may be one which i s common to them a l l . I t follows that future i n v e s t i g a t i o n s i n t o the causes of shuddering behaviour i n Drosophila might b e n e f i t from information concerning the e f f e c t s of l i t h i u m on manic depressive biochemistry i n man and r e l a t e d biochemistry i n other animals. Before reviewing the biochemistry of these psychoses, i t i s important to d i s t i n g u i s h at l e a s t two i n h e r i t e d disorders. Manic depression appears to be transmitted as a sex-linked, dominant mutation a with p a r t i a l penetrance. I t s linkage with colour blindness and the Xg blood group has been demonstrated (Reich et a l . , 1969; Winokur and Tanna, I969). Manic depressive males have fathers who are not af f e c t e d whereas 6ji of t h e i r mothers are af f e c t e d (Winokur et a l . , I969). F i r s t degree r e l a t i v e s of manic depressive probands often experience only depression. Another disorder i n which both probands and t h e i r r e l a t i v e s show only depression appears not to be sex-linked (Winokur and P i t t s , 1965; Winokur et a l . , 1969). 52 Lithium has been shown to act against mania and manic depression. While l i t h i u m seems to r e l i e v e some types of recurrent depression (Baastrup and Schou, 196?; Gooodwin et a l . , 1969) Its use as a general treatment of depressed patients has been questioned (Van der Velde, 1970). The discovery, by Coppen et a l . , (1965) that l i t h i u m treatment caused a drop i n i n t r a c e l l u l a r sodium, gained s i g n i f i c a n c e i n view of the l a t e r discovery that i n t r a c e l l u l a r sodium was r e l a t e d to mood i n + + + the following way: Normal Na < Depressive Na < Manic Na (Coppen et a l . , 1966), I t i s l i k e l y that the change i n i n t r a c e l l u l a r sodium concentration occurs i n nerve c e l l s as w e l l as other t i s s u e s . Lithium has been shown t o compete s u c e s s f u l l y with sodium f o r the " e a r l y " i n f l u x channels i n the formation of action p o t e n t i a l s (Meves and Chandler, 1965; Cole, I968). At the same time, the ac t i v e removal of l i t h i u m from the c e l l occurs more slowly than sodium (Araki et al..1965). The l i n k i n g of depression to low l e v e l s of seratonin and nonadrenaline, and of mania to high l e v e l s of these biogenic amines has l a r g e l y been i n f e r r e d from the e f f e c t s of reserpine, imipramine and the manoamine oxidase i n h i b i t o r s on mood and body biochemistry (Bunney and Davis, 1965; Schildkraut, 1965). Lithium ions have been shown to accelerate the uptake of noradrena-l i n e i n t o preparations of synaptosomes i n v i t r o (Colburn et a l . , 1967). Uptake of seratonin i n t o the red blood c e l l s of manic deppressive patients i s a l s o increased by l i t h i u m (Murphey et a l . , 1970). Katz et a l . , (1968) discovered that l i t h i u m seemed to reduce the r a t e at which noradrenaline and seratonin were released from e l e c t r i c a l l y - s t i m u l a t e d b r a i n s l i c e s . These r e s u l t s may i n d i c a t e that l i t h i u m increases the 53 e f f i c i e n c y of the reuptake mechanism for biogenic amines, Schandberg et a l , , (196?) found that l i t h i u m accelerated norepinephine catabolism within a few hours of treatment. However, B l i s s and A i l i o n , (1970) maintained that a f t e r two weeks of l i t h i u m treatment no change i n noradrenaline metabolism could be detected. I t i s known that i n t r a c e l l u l a r l e v e l s of adenosine 3 ' 5 9 - c y c l i c monophosphate (cyclic-AMP) r i s e i n response to the catecholamines and seratonin i n many target t i s s u e s (Sutherland, 1968), Therefore, i t i s not too s u r p r i s i n g to f i n d that cyclic-AMP i s excreted at higher than normal rates i n manic patients and lower than normal rates i n depressed patients (Paul et a l , , 1971a). The t r a n s i t i o n from the depressed to the manic state i s also marked by a t r a n s i t o r y increase i n cyclic-AMP excretion which i s even greater than that of the manic state (Paul et a l . , 1971b), These authors have a l s o shown that l e v e l s of cyclic-AMP f o l l o w the d i r e c t i o n of c l i n i c a l change brought about by l i t h i u m . Thus cyclic-AMP dropped i n patients recovering from mania and rose i n patients recovering from depression (Paul et a l , . 1971a), This f i n d i n g tends t o broaden rather than confine the problem, since cyclic-AMP i s a common intermediate i n hormal c o n t r o l (Sutherland, et a l . , 1968). Investigations of two other systems i n which cyclic-AMP i s involved — g l y c o l y c i s (Sutherland and Robinson, 1966) and s t e r o i d production (Sutherland et a l . , I968) — have not yet revealed c l e a r chemical r e l a t i o n s h i p s with manic depressive disorders (Coppen, 1967; Rubin, 1967; Van der Velde, 1969; Heninger and Meuller, 1970), Thus f a r , the mechanism upon which l i t h i u m operates to produce i t s e f f e c t has not been discovered. To t h i s end i t may be u s e f u l to compare the biochemistry of manic depressive patients with that of Shuddering 54 f l i e s . Whereas the patterns of behaviour seen i n Shuddering f l i e s suggested a nervous disorder, the temperature-induced cessation of a l l behaviour ts patterns i n para f l i e s could have any number of po s s i b l e bases f o r the e f f e c t . The r e q u i s i t e i n s i g h t i n t o the t i s s u e s p e c i f i c i t y of the mutation was gained by examining genetic mosaic i n d i v i d u a l s derived from y para^ S/ln(l)w V^.-H- zygotes (Suzuki et a l . , 1971). Loss of the vC somatically unstable r i n g X chromosome, l n ( l ) w (Hinton, 1955) produced i n d i v i d u a l s which c a r r i e d both y para^ s/++ and y para^ s/0 c e l l s . Tissue derived from X/0 c e l l s could be recognized e x t e r n a l l y by the expression of the recessive c u t i c u l a r mutation, y_. I t was discovered that f l i e s with yellow legs and w i l d type bodies were able to move t h e i r legs back and f o r t h at high temperature, but were unable to coordinate or p o s i t i o n t h e i r legs properly. F l i e s with yellow thoraces contracted t h e i r legs i n response to a temperature r i s e , whereas f l i e s with yellow heads assumed a normal stance but were unable to walk. F l i e s with yellow abdomens were unaffected by temperature. B i l a t e r a l , mosaic f l i e s moved t h e i r wild type legs at high temperature, but were unable to move t h e i r yellow legs. The r e s u l t s of these ts experiments i n d i c a t e that the para mutation probably has a d i r e c t e f f e c t upon the nervous system which i s not mediated through a f r e e l y c i r c u l a t i n g f a c t o r . Furthermore, the presence of the mutation i n d i f f e r e n t parts of the nervous system appears to lead t o behaviourally d i s t i n c t consequences. The observation that the recovery of mobility i n ts d i f f e r e n t parts of the body of para f l i e s at high temperature often seems to occur at d i f f e r e n t rates may also i n d i c a t e the gene's autonomy. In addition to walking and climbing a c t i v i t i e s , tethered f l i g h t was 55 also found to be s e n s i t i v e to temperature change (Suzuki et a l M 1971)» F l i g h t could be qu i c k l y i n i t i a t e d or stopped by exposure to 22°C and o 29 C, r e s p e c t i v e l y . IP ts The f a c t that the temperature s e n s i t i v i t y of HK para /y i s c h a r a c t e r i s t i c of para^ S rather than HK^ suggests that para^ s d i r e c t l y or i n d i r e c t l y supresses the i n i t i a t i o n or subsequent conduction of impulses burst from the tho r a c i c ganglion. The temperature-dependence 2T ts of HK para /Y males supports t h i s contention since the expression of 2T the HK a l l e l e was not subject to temperature change. Some of the mosaic f l i e s who had mutant t i s s u e around one eye were observed t o follow a h e l i c a l path at 29°C when climbing up the i n s i d e surface of a cyl i n d e r (Suzuki et a l . , 1971). Since the mutant eye was fac i n g upward during the climb i t was thought that v i s i o n i n that eye might be impaired. These mosaic i n d i v i d u a l s showed a normal optomotor response ( i . e . , they turned i n the d i r e c t i o n of moving s t r i p e s ) at 22°C and 29°C. When paint, which had been shown to e f f e c t i v e l y shut out l i g h t , was used to cover the wild type eye and o c e l l i , the f l i e s s t i l l showed a normal optomotor response at 22°G, but behaved ambiguously at 29°C. Normal changes i n p o t e n t i a l of the surface of the eye i n response t s to stimulation by l i g h t (Hotta and Benzer, 19&9) were recorded f o r para at both 22°C and 3 0 ° C * Studies of microbial systems have indicated that a temperature-induced loss of function may be caused by the thermal i n s t a b i l i t y of a p a r t i c u l a r molecule. The i n s t a b i l i t y can be caused by a s i n g l e amino a c i d change within a pr o t e i n (Jockusch, 1964, 1966) or a s i n g l e base change * The electroretnagrams were obtained f o r us through the kindness of Drs. Y o s h i k i Hotta and Seymour Benzer. 56 within a t r a n s f e r RNA species (Smith et a l „, 1970), The capacity of some mutant molecules to function normally depends upon the temperature at the time of t h e i r synthesis and assembly, but does not depend on subsequent temperatures (Edgar and L i e l a u s i s , 1964; Sadler and Novick, 1964), The functioning of other mutant molecules i s temperature-dependent subsequent to syntheses (Maas and Davis, 1952; Igarashi, 1969; Sadler and Novick, 1965). t s The speed of response of para f l i e s to temperature change precludes the p o s s i b i l i t y of much macromolecular synthesis and suggests that a l t e r a t i o n s i n behaviour are mediated through a pre - e x i s t i n g thermo-l a b i l e product. Although mutant proteins have often been sighted as the basis of temperature s e n s i t i v i t y , the nature of the thermolabile t s f a c t o r i n para f l i e s cannot be assumed. The s e n s i t i v i t y of a mutant function to a p a r t i c u l a r temperature change i s often an exaggeration of the wild type s e n s i t i v i t y to the same change, Guthrie et a l . , (1969) a t t r i b u t e d the enhanced recovery of ribosomal assembly defects among co l d - s e n s i t i v e mutants to the normal s e n s i t i v i t y of t h i s process to cold. The s e n s i t i v i t y of catalase a c t i v i t y (Igarashi, 1969) and the h a l f l i f e of pantothenate synthesising enzyme (Horowitz and F l i n g , 1952) to increased temperature i s grossly exaggerated i n the mutant condition, O'Donnovan and Ingraham (I965) discovered that a mutant phosphoribosyl ATP pyrophosphoralase i n E, c o l i which had a greatly increased s e n s i t i v i t y to feedback i n h i b i t i o n by h i s t i d i n e . The a d d i t i o n a l s e n s i t i v i t y of the mutant enzyme to i n h i b i t i o n at low temperatures was about the same as that of the wild type enzyme, but the consequences to the growth of the mutant were much greater. The process of screening f o r heat-sensitive mutants may tend to 57 s e l e c t systems which are already somewhat s e n s i t i v e to heat p r i o r to mutation. I f the phenomenon of temperature-sensitive p a r a l y s i s i s viewed i n t h i s way, the demonstrated s e n s i t i v i t y of the c e n t r a l nervous system to increased temperatures acquires a s p e c i a l i n t e r e s t . Kerkut and Taylor (1958) observed that the maximum impulse frequency emitted by an i s o l a t e d t h o r a c i c or abdominal cockroach ganglion occurred at the temperature at which the animal had been preconditioned and f e l l o f f gradually above and below that temperature. These readings were obtained by changing the temperature of the ganglion slowly and permitting time f o r the frequency to reach a steady state. A rapid temperature increase of l6°C caused impulses to cease almost immediately. A f t e r eleven seconds of i n a c t i v i t y , the impulses suddenly resumed at a higher frequency than before. The same e f f e c t was achieved by a 13°C increase i n c r a y f i s h abdomenal ganglia and a 3°C increase i n slug pedal ganglia (Kerkut and Taylor, 1958). In a l l these systems, quick drops i n temperature induced t r a n s i e n t increases i n impulse frequency. These tra n s i e n t responses to temperature were c h a r a c t e r i s t i c of some, but not a l l c e n t r a l nervous elements (Kerkut and Taylor, 1958). Nevertheless, the speed of re a c t i o n of the ganglia which c o n t r o l mobility to cold t s and heat shock was very reminiscent of para behaviour. The biochemical l i m i t s which define an organism's temperature range as w e l l as i t s means of a c c l i m a t i s i n g to a new temperature have been extensively studied. In t h e i r review of t h i s subject, Hochachka and Somero (1971) have stressed the importance of enzyme substrate a f f i n i t y , as implied by low Km values,in determining the range of immediately exploi t a b l e temperatures. Adaptation to a new range may require de novo synthesis of an enzyme whose lowest Km values extend 58 over the new range. Thus, when Rainbow t r o u t were tr a n s f e r r e d from V?°C to 2°C they were immobilized. Af t e r a few weeks, a return of mobility was accompanied by the appearance of a new low temperature a c e t y l -cholenesterase and the disappearance of a high temperature form (Baldwin and Hochachka, 1970), I t appears that t h i s i s a simple example of a common phenomenon (Hochachka and Somero, 1971) <> Long term adaptation i s also accompanied by i o n i c changes (Heiniche and Houston, 19&5) a^d a l t e r a t i o n s i n the phospholipid species of the c e n t r a l nervous system (Roots, 1968), Preliminary t e s t s of short ts and long term acclimation of para f l i e s t o high temperature suggested the existence of short and long term mechanisms f o r regaining mobility (Suzuki et a l , , 1971), The short term advantage appears to be quickly l o s t a f t e r a b r i e f s h i f t down followed by a return to high temperature. The long term gain i s not so e a s i l y l o s t and may, therefore, be accom-plish e d by an acclimation process such as de novo synthesis of a new ts f a c t o r t o replace or a i d the para f a c t o r . An organism's i n s e n s i t i v i t y t o temperature need not always depend on the i n s e n s i t i v i t y of systems within i t . Hochachka and Samero (1971) have named f i v e mechanisms which are probably required t o balance the s e n s i t i v i t y of fructose diphosphatase t o AMP i n h i b i t i o n at low tempera-tures. By mutation t h i s kind of balance could e a s i l y be upset (O'Donnovan and Ingraham, I965). The positions and nature of the temperature-sensitive elements of ts the para nervous system are yet unknown and must await the r e s u l t s of * e l e c t r o p h y s i o l o g i c a l i n v e s t i g a t i o n . The discovery of the dramatic r i s e i n acetylcholenesterase and These studies are being conducted by Dr, Kazuo Ikeda, 59 choleneacetyltransferase a c t i v i t i e s i n Drosophila melanogaster during adult metamorphosis (Dewhurst et a l , , 1970) provides a p l a u s i b l e explanation f o r the a d u l t - s p e c i f i c f u nction of the para mutation. However, the a c t i v i t i e s of these enzymes were not found to be temperature-ts * s e n s i t i v e i n para f l i e s , T u n n i c l i f f et a l . (1970) discovered that dopamine concentrations are below normal i n an a c t i v e s t r a i n of Drosophila melanogaster and above normal i n an i n a c t i v e s t r a i n . The opposite was true f o r noradrenaline. Thus, noradrenaline has become one of the candidates ts f o r biochemical study i n both para and Shuddering f l i e s . However, i f a l t e r e d l e v e l s of noradrenaline were discovered i n these f l i e s , the problem of primary cause would s t i l l remain. The i n v e s t i g a t i o n of primary causes of behavioural defects i s best c a r r i e d out i n s i n g l e mutant systems. The s e l e c t i v e recovery of s i n g l e gene mutants a f f e c t i n g the motor system i s f e a s i b l e and has, i n t h i s instance, y i e l d e d two mutants of general n e u r o l o g i c a l i n t e r e s t . This biochemical analysis was conducted by Dr„ Linda H a l l , 6o SUMMARY With the i n t e n t i o n of recovering mutations a f f e c t i n g neural and/or muscular systems, a search f o r a temperature-sensitive mutant among Drosophila melanogaster adults was conducted. Mechanical screening devices f o r the separation of a c t i v e from immobilised f l i e s were constructed. Two mating schemes were used t o recover chromosomes from males treated with e t h y l methanesulphonate. In one scheme the treated males were crossed with w i l d type females so that only dominant mutations would be detected i n the F]_ generation. In the second scheme, the males were crossed t o attached-X (XX/Y) females, thus permitting the detection of sex-linked recessive or autosomal dominant mutations. The F]_ generations were r a i s e d to adulthood at 22°C and then screened f o r i n d i v i d u a l s which were immobilised at 29°C, From approximately 250,000 progeny of XX/Y females, one male which exhibited h e r i t a b l e temperature-sensitive p a r a l y s i s was recovered. The mutation (symbolised as para^ s f o r paralytic-temperature-sensitive) was found to be recessive and sex-linked, mapping g e n e t i c a l l y at 53«9o p a r a ^ s f l i e s which had been r a i s e d at 22°C were paralysed within 5 seconds of exposure t o 29°C and recovered f u l l m obility within a few seconds of exposure t o 22°G, S e n s i t i v i t y to temperature change was detected only i n adult f l i e s . Prolonged exposure of p a r a t s f l i e s to 29°C r e s u l t e d i n a gradual recovery of mobility. In general, the length of the recovery time was d i r e c t l y r e l a t e d to the magnitude and rate of the temperature r i s e . 61 IP t The movements of etherised and non-etherised HK para /Y, HK^para^ 5/Y and para^ sSh~VY males were subject to the same temperature-ts s e n s i t i v e p a r a l y s i s that para f l i e s exhibited. The observation that IP ts leg shaking of HK para /Y males was r a p i d l y supressed and r e i n i t i a t e d ts by temperature change suggests that para may have d i r e c t l y or i n d i r e c t l y supressed the i n i t i a t i o n or subsequent conduction of impulse bursts which have been shown t o cause the c h a r a c t e r i s t i c l eg shaking of HK^ f l i e s . In the search f o r a dominant mutation causing temperature-sensitive p a r a l y s i s a f l y carrying a dominant mutation which caused p e r i o d i c shuddering was discovered. The sex-linked mutation,called Shuddering (Shu), mapped g e n e t i c a l l y at 55.1. I t was possible to suppress the shuddering behavior by feeding the f l i e s media containing L i C l but not NaCl, KCl, or NH^Cl. The s i g n i f i c a n c e of these observations has been considered i n the l i g h t of other e f f e c t s of l i t h i u m ions upon the biochemistry, physiology and behaviour of man and other animals. 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