- Email: [email protected]
Developmental process of visual pattern discrimination in the fly
Brain Research, 512 (1990) 75-80
75
Elsevier BRES 15291
Developmental process of visual pattern discrimination in the fly Keiichi Mimura Nagasaki University, Faculty of Liberal Arts, Nagasaki (Japan) (Accepted 15 August 1989)
Key words: Fly; Pattern discrimination; Experience-dependent development; Protein synthesis inhibitor; Chilling
It has been found that the development of visual pattern discrimination in the fly is strongly influenced by visual experience in the early stage of post-emergence. Behavioral experiments were performed to determine the details of the temporal conditions of the visual experience and to obtain a lead to the determination of the molecular mechanism underlying the developmental process. By anesthetizing the flies with chilling at various periods post-emergence, it was demonstrated that visual experience during 5 h post-emergence must be followed by normal neuronal activity within a very short time of 15 min for the normal development of visual pattern discrimination to occur. The subsequent development of normal pattern discrimination required the maintenance of normal neuronal activity for at least 5 h. Injection of some protein synthesis inhibitors to the fly head before or immediately after visual experience resulted in impairment of the development of visual pattern discrimination, thus supporting the developmental process revealed by the chilling experiment. Moreover, both the chilling experiment and injection of inhibitors of protein synthesis showed that the neuronal mechanisms were different for phototaxis and for pattern discrimination, suggesting that the former is a natural function while the latter is an acquired function after emergence. INTRODUCTION B e h a v i o u r a l e x p e r i m e n t s on flies h a v e d e m o n s t r a t e d t h a t visual e x p e r i e n c e w i t h a v a r i e t y of p a t t e r n s during the early p e r i o d of p o s t - e m e r g e n c e is i n d i s p e n s a b l e for the n o r m a l d e v e l o p m e n t of visual p a t t e r n discrimination 12-14. T h i s was s u p p o r t e d by a h i s t o c h e m i c a l study using c y t o c h r o m e o x i d a s e 15, suggesting plasticity of synaptic t r a n s m i s s i o n in the d e v e l o p m e n t o f the h i g h e r visual function. T h e p r e s e n t study was u n d e r t a k e n
to e x a m i n e the
details of the c o n d i t i o n s of visual e x p e r i e n c e n e e d e d for the n o r m a l d e v e l o p m e n t o f visual p a t t e r n d i s c r i m i n a t i o n a n d , in an a t t e m p t to a p p r o a c h the e l u c i d a t i o n of the m o l e c u l a r m e c h a n i s m s u n d e r l y i n g the plasticity of neur o n a l activity, was u n d e r t a k e n to d e m o n s t r a t e the effect of inhibitors of p r o t e i n synthesis on the d e v e l o p m e n t of visual p a t t e r n d i s c r i m i n a t i o n . MATERIALS AND METHODS Flesh flies (Boettcherisca peregrina) of both sexes were used. They were all raised in a 12 h light-dark cycle (LD) until the beginning of pupation, after which they were kept in continuous darkness (DD). After emergence, the flies were raised at 25 °C in DD except during visual experience for 5 h immediately after emergence. This 5 h duration of visual experience immediately after emergence is the minimum required for the normal development of pattern discrimination 13. Exposure to visual experience was done by keeping the flies in a transparant cage so as to allow them to see everything in their surroundings. The cage was placed in a lighted room.
Behavioural experiments of pattern discrimination were done at various periods of post-emergence. The behavioural experiment was a test of choice of two visual patterns. The method was the same as in previous experiments, the details of which have been described TM 13. Only two patterns, one a radial, star-shaped pattern and the other an oblique bar pattern (Fig. 1), were used for the choice tests, because the star-shape is the most attractive for flies and the oblique bar is the least attractive ~°. The pair of patterns was presented at one end of the arena as the target. The target was made of thin, white paper on a black background, and evenly transiUuminated from behind. The dewinged fly was placed in the arena at the opposite end and allowed to walk freely toward the pattern targets. This choice behavior was reinforced phototactically, but the clear choice between the two patterns suggested pattern discrimination 1°-12. Tests were begun from the 5th day of post-emergence, when the normal development was completed lz, and were continued every day till the 8th day of post-emergence. One test consisted of 20 choice trials. Then, evaluation of whether the discrimination was well developed or not was based on a statistically significant difference between the number of times of arrival at these two patterns in choice behavior. This statistical method was the same as used in our cytochrome oxidase experiment 15. Flies were kept in a low-temperature environment during certain periods of post-emergence in order to determine the important and essential periods for the normal development of pattern discrimination, because the low temperature environment produces a lowering of the general activity, especially the nervous activity, of the fly. The low temperature environment was made by placing the cage in an incubator. The effect of inhibitors of protein synthesis on pattern dicrimination was examined using three kinds of inhibitors: anisomycin, cycloheximide and puromycin. The dose of these inhibitors to be injected was determined based on the experiment on crickets by Nagao TM. By considering the fly's volume as 41.5 mm 3, 1.38/.d was injected. The inhibitor solution was prepared by dissolving 0.5 mg of inhibitor in 0.5 ml of Ringer's solution. The amount of inhibitor injected to each fly was 1.4 or 0.14/~g. The inhibitors were injected using a glass pipette microelectrode with a relatively thick tip fitted
Correspondence: K. Mimura, Nagasaki University, Faculty of Liberal Arts, Nagasaki 852, Japan. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
76
20
Ed15 >
"E-
Fig. t. Two visual patterns used for pattern discrimination.
10
-a 5
on a microsyringe. Ringer's solution of the same volume as the inhibitor solution was injected as the control. After the fly was anesthetized by chilling on ice, the inhibitor solution was injected from a small incision made in the cuticle of the back of the head. As the incision was made at a location just covering the brain, injection of the solution first resulted in immersion of the brain. No treatment was done to cover the incision after injection, but this did not cause any trouble in the fly's behavior.
RESULTS
Influence of low temperature upon pattern discrimination behavior Post-emergence visual experience is necessary for the normal development of pattern discrimination. Therefore, interruption of the experience by lowering the general activity including the activity of the central nervous system of the fly would presumably impair the normal development of pattern discrimination. As illustrated in the inset of Fig. 2, flies were kept in a low-temperature environment during visual experience
~
rnerg
5th day postemerg
L
_1~1 1/,
D
0
I
I
o
I
I
1o
I
20
T e m p e r a t u r e (*C)
Fig. 3. Total number of arrivals at the two different patterns, regardless of pattern shape, in vision-experienced flies at various temperature. Experimental conditions were the same as shown in Fig. 2.
for 5 h immediately after emergence. Various temperatures were used, and pattern discrimination was tested from the 5th day of post-emergence to the 8th day. As shown in Fig. 2, when flies were kept at 0-10 °C during visual experience, the development of pattern discrimination did not occur at all. In proportion to rise of temperature, pattern discrimination gradually developed, and normal development was seen at 20 °C and above. These results suggest that lowering of normal activity of the nervous system has influences upon the normal development of neuronal function. As arrival at lighted patterns is reinforced with phototaxis, regardless of the target patterns, total number of arrivals at both patterns was considered as an index of phototaxis. As seen in Fig. 3, the total number of arrivals was scarcely influenced by lowering the temperature. Thus, contrary to pattern discrimination, photo
.
chilling
~
/
E o •-
12
c
8
E
6
12
a
2
10
10
14
emerg
5th dsy postllmerg
I / - - -
kt~>t
tests
ch i l l i n g
E 8
-2
o I
I
I
I0
I
I
20
c 6
Temperature ('C)
Fig. 2. The effect of temperature during visual experience upon the development of pattern discrimination. As illustrated in the inset, chilling was performed during exposure to light for 5 h immediately after emergence, following which the flies were kept in the dark. Behavioral tests for pattern discrimination was performed on 4 days, from the 5th day to 8th day of post-emergence. Since discrimination is shown as the difference between the number of arrivals at the star shape and the oblique bar, positive values indicate formation of normal discrimination, emerg and 5th day posternerg show emergence and 5th day of post-ermergence, respectively. Vertical bars indicate standard deviation. L and D, lighted and dark conditions, respectively.
£3
2
-2
J
i
I
L
i
I
L
I
I
I
i
~
0
1
2
3
4
S
6
7
8
9
10
11
t~
I]~ 12
P 24
(h)
Fig. 4. The effect of anesthesia by chilling for various lengths of time (t~) immediately after visual experience upon the development of pattern discrimination. For abbreviations, see the legend of Fig. 2.
77
14
tD
18
5th post day erff~g
~msrg Sh ~< t2
•tests
chilling
12
I
14
w6
lO
1.4pg • 0.14pg
0
16
12
> b.10
~8 ".2,
~6 E "r" u 4
-
8
o ~"
6
2 4
0 -2
I" "
5
10
g
t2 ( h )
Fig. 5. The effect of anesthesia by 24 h chilling at various times (¢2) after cessation of visual experience upon the development of pattern discrimination. For abbreviations, see the legend of Fig. 2.
~
Fig. 7. The effect of inhibitors of protein synthesis upon total number of arrivals at the two different patterns regardless of their shapes. Injection of inhibitors and other experimental conditions were the same as in Fig. 6.
taxis was not influenced by chilling.
Chilling experiment to investigate the need for normal neuronal activity in the development of visual function Firstly, since absence of visual experience immediately after emergence disturbs the normal development of pattern discrimination, a study was done to determine the
14
duration of normal neuronal activity required for formation of pattern discrimination due to the visual experience (Fig. 4). It was found that an extremely short duration was needed. When the normal activity was lowered immediately after visual experience, impairment of development rapidly increased with length of chilling. When chilling was continued for 30 min, development of discrimination decreased to only a third of normal.
emsrg 5th day
I~ection
postemrg
14
tests
12
gh
10
DL ' j ' - ~ O 1.4~g
8
•
6
0'14P1
10
5th .day pos=emerg
emergnjection • II
Sh
•
I.~
tests
~8
0 1.4pg • 0.14yg
4
c 6 E
2
~4 2
0 -2
.c_
_
=o
o=
_
c
~
oE
Fig. 6. The effect of 3 inhibitors of protein synthesis upon the development of pattern discrimination. Ringer's solution as control, anisomycin, cycloheximide and puromycin were injected immediately after emergence and just before visual experience. Doses were 1.4/tg (open circles) and 0.14/~g (closed circles). For abbreviations, see the legend of Fig. 2.
-2
~,
E
_¢
E Fig. 8. The effect of 3 inhibitors of protein synthesis upon the development of pattern discrimination. Ringer's solution as control, anisomycin, cycloheximide and puromycin were injected immediately after visual experience for 5 h. For abbreviations, see the legend of Fig. 2.
78
18
18 0 1 . 4 ,ug
16
16
00.14pg
c) 14
14
c
d
c12 > "r-
E ~o
~
10
~
8
ill
-a
8
o F--
i-
0 1.4pg
6
6
I 0.14pg
4
oT
•-~
c
'~
s
x o
u
Fig. 9. The effect of inhibitors of protein synthesis upon total number of arrivals at the two patterns regardless of their shapes. Experimental conditions were the same as in Fig. 8.
With chilling for 1 h or more, dicrimination was reduced to a fourth of full development and, with further increase of the length of chilling, the discrimination decreased very slowly. This indicates that short-term, normal activity is absolutely needed immediately after visual experience for the development of pattern discrimination. Secondly, the minimum duration of normal activity needed immediately after visual experience for normal
E o
~
g
Fig. 11. The effect of inhibitors of protein synthesis upon total number of arrivals at the two patterns regardless of their shapes. Experimental conditions were the same as in Fig. 10.
development was investigated (Fig. 5). The minimum duration was found to be 5 h. Pattern discrimination developed hardly in the presence of normal activity for less than 4 h. Discrimination became abruptly welldeveloped from 5 h of normal activity. This shows that, for the development of pattern discrimination, normal activity must by maintained for at least 5 h after visual experience.
Injection of inhibitors of protein synthesis at various stages of post-emergence 16
J,mcrlg
Sth in , c t i o n pos~4Lir~n,rgl
14
tests
12 10
o ~ 8 •u
6
,-~ 4
0
1.4 pg
•
0-14 p g
-2 c
-
o
oE
Fig. 10. The effect of 3 inhibitors of protein synthesis upon the development of pattern discrimination. Injection was done at 5 h after cessation of visual experience. For abbreviations, see the legend of Fig. 2.
Three inhibitors of protein synthesis were injected into the fly's head in order to confirm the above results and to obtain a lead to the molecular basis underlying the developmental process. Injections were given at three stages of post-emergence: immediately after emergence, just before visual experience; immediately after 5 h of visual experience; and 5 h after cessation of visual experience. When the inhibitors were injected immediately after emergence and immediately after visual experience, pattern discrimination did not develop regardless of the kind of inhibitor (Figs. 6 and 8). The injection of inhibitors immediately after visual experience was more effective than just before visual experience, because slight discrimination was seen with injections of low concentration (0.14 /~g) just before visual experience, while discrimination did not develop at all with injections immediately after visual experience except with 0.14/~g of anisomysin injection. In contrast to the above results, injections at 5 h after cessation of visual experience did not influence at all the
79 development of pattern discrimination (Fig. 10). These results support the findings of the chilling experiments. One more important finding involved the influence of inhibitors on phototactic behavior. Regardless of the stage of injection, the phototactic behavior was hardly influenced by the injection of inhibitors, although the number of arrivals decreased slightly (Figs. 7, 9 and 11). This suggests that the visual mechanisms for pattern discrimination and for phototaxis are two separate functions from the viewpoint of the developmental process. DISCUSSION It has been demonstrated in vertebrates that postnatal signals from sensory surfaces modulate neuronal activity and hence interfere with the self-organizing processes 3'2°. Even in invertebrates such as the fly, the same phenomenon has been demonstrated from the development of visual pattern discrimination 12. In the mammalian visual cortex, these experiencedependent modifications are restricted to a critical period of postnatal development 1'2°. As shown also in this study of the fly, visual experience had to be given within a very restricted period post-emergence for the normal development of pattern discrimination. Moreover, it was demonstrated that brief, normal neuronal activity immediately after visual experience plays a role in driving the development, and that the normal activity of the nervous system had to be maintained for at least 5 h for the normal development, suggesting identification of short-term and long-term memory, which may correspond to neuronal activity and molecular modification, respectively. In this experiment, the normal activity level was lowered in the fly anesthetized by chilling. The anesthetizing influence upon the development of pattern discrimination was evident when the fly was chilled in the suitable stage and period. Similar phenomena were seen in the development of cortical functions of the kittens, in which visual behavior failed to develop when they are paralyzed and/or anesthetized while exposed to light 1'6'19. Hence, for the normal development of higher functions such as pattern discrimination in the fly and such as the complicated visual behavior in the kitten, it is a prerequisite that the neuronal circuits are active2°. As one example, this was demonstrated from the effect of a REFERENCES 1 Buisseret, P., Gary-Bobo, E. and Imbert, M., Ocular motility and recovery of orientational properties of visual cortical neurones in dark-reared kittens, Nature (Lond.), 272 (1987)
certain level of norepinephrine in the extracellular space 7,8. The effect of several inhibitors of protein synthesis upon memory formation has been demonstrated: anisomycin influenced memory formation in miceS; retrograde amnesia for a passive avoidance response was produced in mice by cycloheximideXS; and puromycin caused loss of memory of avoidance discrimination learning in mice 4. These 3 kinds of inhibitors of protein synthesis impair 'long-term' but not 'short-term' memory 21. In fact, Castellucci et al. 2 detected, using 2-D gel analysis, 4 proteins, which changed in their rates of synthesis in long-term memory. The present experiment demonstrated the effect of 3 kinds of inhibitors of protein synthesis upon formation of pattern discrimination in flies. The effective time of injection was coincident with the period of normal activity, which was necessary for formation of pattern discrimination. It has been proposed that long-term memory depends on protein synthesis in synaptic membrane 17. Furthermore, it has been demonstrated that the formation of imprinting behavior accompanies protein synthesis at special locations of the brain and morphological changes of synapse z2. In flies, Kral and Meinertzhagen 9 observed electronmicroscopically that light experience relates to formation of retina-lamina synaptic connection. The development of pattern discrimination may imply formation of neuronal circuits after emergence including modification of synaptic connection, which accompanies molecular changes. The other important finding in the present experiment was the separation of the two visual functions for phototaxis and pattern discrimination. The former mechanism is already completed at emergence as a natural function and the latter develops after emergence as an acquired function. This has been previously suggested in the first experiment on experience-dependent development of pattern discrimination in the fly12. That is, pattern dicrimination did not develop when flies were kept in the dark for more than 5 days after emergence, while phototactic behavior was not influenced even under such condition. Acknowledgements. I wish to thank Dr. T. Nagao of Hokkaido University for helpful advice on injection of inhibitor of protein synthesis and also Dr. K. Yorichika for improving the English. This work was supported by Grants-in-Aid from the Ministry of Education, Science and Culture of Japan (60304011, 63540578 and 63304005).
816-817. 2 Castellucci, V.E, Kennedy, T.E., Kandel, E.R. and Goelet, P., A quantitative analysis of 2-D gels identifies proteins in which labeling is increased followinglong-term sensitization in aplysia, Neuron, 1 (1988) 321-328.
80 3 Chow, K.L., Neuronal changes in the visual system following visual deprivation. In R. Jung (Ed.), Central Processing of Visual
Information A: Intergrative Functions and Comparative Data, Handbook of Sensory Physiology, Vol. VII/3A, Springer, Berlin, pp. 599-627. 4 Flexner, J.B., Flexner, L.B. and Stellar, E., Memory in mice as affected by intracerebral puromycin, Science, 141 (1963) 57-59. 5 Flood, J.F., Bennett, E.L. and Orme, A.E., Relation of memory formation to controlled amounts of brain protein synthesis, Physiol. Behav., 15 (1975) 97-102. 6 Freeman, R.D. and Bonds, A.B., Cortical plasticity in monocularly deprived immobilized kittens depends on eye movement, Science, 206 (1979) 1093-1095. 7 Kasamatsu, T. and Pettigrew, J.D., Preservation of binocularity after monocular deprivation in the striate cortex of kittens treated with 6-hydroxydopamine, J. Comp. Neurol., 185 (1979) 139-162. 8 Kasamatsu, T., Pettigrew, J.D. and Ary, M., Restoration of visual cortical plasticity by local microperfusion of norepinephrine, J. Comp. NeuroL, 185 (1979) 163-181. 9 Kral, D. and Meinertzhagen, I.A., Functional plasticity in the fly's visual system, Soc. Neurosci. Abstr., 12 (1987) 930. 10 Mimura, K., Phototactic behaviour of walking flies in response to some visual patterns: correlation with receptive field patterns of photoreceptors, J. Comp. Physiol., 144 (1981) 75-82. 11 Mimura, K., Discrimination of some visual patterns in Drosophila melanogaster, J. Comp. Physiol., 146 (1982) 229-233. 12 Mimura, K., Development of visual pattern discrimination in the
fly depends on light experience, Science. 232 (1986) 83-85. 13 Mimura, K., Persistence and extinction of the effect of visual pattern deprivation in the fly, Exp. Biol., 46 (1987) 155-162. 14 Mimura, K., The effect of partial covering of the eye on the results of selective deprivation of visual pattern in the fly, Brain Research, 437 (1987) 97-102. 15 Mumura, K., Cytochrome oxidase histochemistry in the effect of light deprivation on the fly visual system, Brain Research, 445 (1988) 228-233. 16 Nagao, T., personal communication. 17 Nestler, E.J. and Greengard, P., Protein phosphorylation in the brain, Nature (Lond.), 305 (1983) 583-588. 18 Quartermain, D., McEwen, B.S. and Azmitia, Jr., E.C., Amnesia produced by electroconvulsive shock or cycloheximide: conditions for recovery, Science, 169 (1970) 683-686. 19 Singer, W., Central core control of developmental plasticity in the kitten visual cortex: I. Diencephalic lesions, Exp. Brain Res., 47 (1982) 209-222. 211 Singer, W., Learning to see: mechanisms in experience-dependent development. In P. Marler and H.S. Terrace (Eds.), The Biology of Learning, Springer, Berlin, pp. 461-477. 21 Squire, L.R. and Barondes, S.H., Anisomycin, like other inhibitors of cerebral protein synthesis, impairs "long-term' memory of a discrimination task, Brain Research, 66 (1974) 301-308. 22 Takamatsu, K. and Tsukada, Y., Neurochemical studies on imprinting behavior in chick and duckling, Neurochem. Res., 10 (1985) 1371-1391.
75
Elsevier BRES 15291
Developmental process of visual pattern discrimination in the fly Keiichi Mimura Nagasaki University, Faculty of Liberal Arts, Nagasaki (Japan) (Accepted 15 August 1989)
Key words: Fly; Pattern discrimination; Experience-dependent development; Protein synthesis inhibitor; Chilling
It has been found that the development of visual pattern discrimination in the fly is strongly influenced by visual experience in the early stage of post-emergence. Behavioral experiments were performed to determine the details of the temporal conditions of the visual experience and to obtain a lead to the determination of the molecular mechanism underlying the developmental process. By anesthetizing the flies with chilling at various periods post-emergence, it was demonstrated that visual experience during 5 h post-emergence must be followed by normal neuronal activity within a very short time of 15 min for the normal development of visual pattern discrimination to occur. The subsequent development of normal pattern discrimination required the maintenance of normal neuronal activity for at least 5 h. Injection of some protein synthesis inhibitors to the fly head before or immediately after visual experience resulted in impairment of the development of visual pattern discrimination, thus supporting the developmental process revealed by the chilling experiment. Moreover, both the chilling experiment and injection of inhibitors of protein synthesis showed that the neuronal mechanisms were different for phototaxis and for pattern discrimination, suggesting that the former is a natural function while the latter is an acquired function after emergence. INTRODUCTION B e h a v i o u r a l e x p e r i m e n t s on flies h a v e d e m o n s t r a t e d t h a t visual e x p e r i e n c e w i t h a v a r i e t y of p a t t e r n s during the early p e r i o d of p o s t - e m e r g e n c e is i n d i s p e n s a b l e for the n o r m a l d e v e l o p m e n t of visual p a t t e r n discrimination 12-14. T h i s was s u p p o r t e d by a h i s t o c h e m i c a l study using c y t o c h r o m e o x i d a s e 15, suggesting plasticity of synaptic t r a n s m i s s i o n in the d e v e l o p m e n t o f the h i g h e r visual function. T h e p r e s e n t study was u n d e r t a k e n
to e x a m i n e the
details of the c o n d i t i o n s of visual e x p e r i e n c e n e e d e d for the n o r m a l d e v e l o p m e n t o f visual p a t t e r n d i s c r i m i n a t i o n a n d , in an a t t e m p t to a p p r o a c h the e l u c i d a t i o n of the m o l e c u l a r m e c h a n i s m s u n d e r l y i n g the plasticity of neur o n a l activity, was u n d e r t a k e n to d e m o n s t r a t e the effect of inhibitors of p r o t e i n synthesis on the d e v e l o p m e n t of visual p a t t e r n d i s c r i m i n a t i o n . MATERIALS AND METHODS Flesh flies (Boettcherisca peregrina) of both sexes were used. They were all raised in a 12 h light-dark cycle (LD) until the beginning of pupation, after which they were kept in continuous darkness (DD). After emergence, the flies were raised at 25 °C in DD except during visual experience for 5 h immediately after emergence. This 5 h duration of visual experience immediately after emergence is the minimum required for the normal development of pattern discrimination 13. Exposure to visual experience was done by keeping the flies in a transparant cage so as to allow them to see everything in their surroundings. The cage was placed in a lighted room.
Behavioural experiments of pattern discrimination were done at various periods of post-emergence. The behavioural experiment was a test of choice of two visual patterns. The method was the same as in previous experiments, the details of which have been described TM 13. Only two patterns, one a radial, star-shaped pattern and the other an oblique bar pattern (Fig. 1), were used for the choice tests, because the star-shape is the most attractive for flies and the oblique bar is the least attractive ~°. The pair of patterns was presented at one end of the arena as the target. The target was made of thin, white paper on a black background, and evenly transiUuminated from behind. The dewinged fly was placed in the arena at the opposite end and allowed to walk freely toward the pattern targets. This choice behavior was reinforced phototactically, but the clear choice between the two patterns suggested pattern discrimination 1°-12. Tests were begun from the 5th day of post-emergence, when the normal development was completed lz, and were continued every day till the 8th day of post-emergence. One test consisted of 20 choice trials. Then, evaluation of whether the discrimination was well developed or not was based on a statistically significant difference between the number of times of arrival at these two patterns in choice behavior. This statistical method was the same as used in our cytochrome oxidase experiment 15. Flies were kept in a low-temperature environment during certain periods of post-emergence in order to determine the important and essential periods for the normal development of pattern discrimination, because the low temperature environment produces a lowering of the general activity, especially the nervous activity, of the fly. The low temperature environment was made by placing the cage in an incubator. The effect of inhibitors of protein synthesis on pattern dicrimination was examined using three kinds of inhibitors: anisomycin, cycloheximide and puromycin. The dose of these inhibitors to be injected was determined based on the experiment on crickets by Nagao TM. By considering the fly's volume as 41.5 mm 3, 1.38/.d was injected. The inhibitor solution was prepared by dissolving 0.5 mg of inhibitor in 0.5 ml of Ringer's solution. The amount of inhibitor injected to each fly was 1.4 or 0.14/~g. The inhibitors were injected using a glass pipette microelectrode with a relatively thick tip fitted
Correspondence: K. Mimura, Nagasaki University, Faculty of Liberal Arts, Nagasaki 852, Japan. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
76
20
Ed15 >
"E-
Fig. t. Two visual patterns used for pattern discrimination.
10
-a 5
on a microsyringe. Ringer's solution of the same volume as the inhibitor solution was injected as the control. After the fly was anesthetized by chilling on ice, the inhibitor solution was injected from a small incision made in the cuticle of the back of the head. As the incision was made at a location just covering the brain, injection of the solution first resulted in immersion of the brain. No treatment was done to cover the incision after injection, but this did not cause any trouble in the fly's behavior.
RESULTS
Influence of low temperature upon pattern discrimination behavior Post-emergence visual experience is necessary for the normal development of pattern discrimination. Therefore, interruption of the experience by lowering the general activity including the activity of the central nervous system of the fly would presumably impair the normal development of pattern discrimination. As illustrated in the inset of Fig. 2, flies were kept in a low-temperature environment during visual experience
~
rnerg
5th day postemerg
L
_1~1 1/,
D
0
I
I
o
I
I
1o
I
20
T e m p e r a t u r e (*C)
Fig. 3. Total number of arrivals at the two different patterns, regardless of pattern shape, in vision-experienced flies at various temperature. Experimental conditions were the same as shown in Fig. 2.
for 5 h immediately after emergence. Various temperatures were used, and pattern discrimination was tested from the 5th day of post-emergence to the 8th day. As shown in Fig. 2, when flies were kept at 0-10 °C during visual experience, the development of pattern discrimination did not occur at all. In proportion to rise of temperature, pattern discrimination gradually developed, and normal development was seen at 20 °C and above. These results suggest that lowering of normal activity of the nervous system has influences upon the normal development of neuronal function. As arrival at lighted patterns is reinforced with phototaxis, regardless of the target patterns, total number of arrivals at both patterns was considered as an index of phototaxis. As seen in Fig. 3, the total number of arrivals was scarcely influenced by lowering the temperature. Thus, contrary to pattern discrimination, photo
.
chilling
~
/
E o •-
12
c
8
E
6
12
a
2
10
10
14
emerg
5th dsy postllmerg
I / - - -
kt~>t
tests
ch i l l i n g
E 8
-2
o I
I
I
I0
I
I
20
c 6
Temperature ('C)
Fig. 2. The effect of temperature during visual experience upon the development of pattern discrimination. As illustrated in the inset, chilling was performed during exposure to light for 5 h immediately after emergence, following which the flies were kept in the dark. Behavioral tests for pattern discrimination was performed on 4 days, from the 5th day to 8th day of post-emergence. Since discrimination is shown as the difference between the number of arrivals at the star shape and the oblique bar, positive values indicate formation of normal discrimination, emerg and 5th day posternerg show emergence and 5th day of post-ermergence, respectively. Vertical bars indicate standard deviation. L and D, lighted and dark conditions, respectively.
£3
2
-2
J
i
I
L
i
I
L
I
I
I
i
~
0
1
2
3
4
S
6
7
8
9
10
11
t~
I]~ 12
P 24
(h)
Fig. 4. The effect of anesthesia by chilling for various lengths of time (t~) immediately after visual experience upon the development of pattern discrimination. For abbreviations, see the legend of Fig. 2.
77
14
tD
18
5th post day erff~g
~msrg Sh ~< t2
•tests
chilling
12
I
14
w6
lO
1.4pg • 0.14pg
0
16
12
> b.10
~8 ".2,
~6 E "r" u 4
-
8
o ~"
6
2 4
0 -2
I" "
5
10
g
t2 ( h )
Fig. 5. The effect of anesthesia by 24 h chilling at various times (¢2) after cessation of visual experience upon the development of pattern discrimination. For abbreviations, see the legend of Fig. 2.
~
Fig. 7. The effect of inhibitors of protein synthesis upon total number of arrivals at the two different patterns regardless of their shapes. Injection of inhibitors and other experimental conditions were the same as in Fig. 6.
taxis was not influenced by chilling.
Chilling experiment to investigate the need for normal neuronal activity in the development of visual function Firstly, since absence of visual experience immediately after emergence disturbs the normal development of pattern discrimination, a study was done to determine the
14
duration of normal neuronal activity required for formation of pattern discrimination due to the visual experience (Fig. 4). It was found that an extremely short duration was needed. When the normal activity was lowered immediately after visual experience, impairment of development rapidly increased with length of chilling. When chilling was continued for 30 min, development of discrimination decreased to only a third of normal.
emsrg 5th day
I~ection
postemrg
14
tests
12
gh
10
DL ' j ' - ~ O 1.4~g
8
•
6
0'14P1
10
5th .day pos=emerg
emergnjection • II
Sh
•
I.~
tests
~8
0 1.4pg • 0.14yg
4
c 6 E
2
~4 2
0 -2
.c_
_
=o
o=
_
c
~
oE
Fig. 6. The effect of 3 inhibitors of protein synthesis upon the development of pattern discrimination. Ringer's solution as control, anisomycin, cycloheximide and puromycin were injected immediately after emergence and just before visual experience. Doses were 1.4/tg (open circles) and 0.14/~g (closed circles). For abbreviations, see the legend of Fig. 2.
-2
~,
E
_¢
E Fig. 8. The effect of 3 inhibitors of protein synthesis upon the development of pattern discrimination. Ringer's solution as control, anisomycin, cycloheximide and puromycin were injected immediately after visual experience for 5 h. For abbreviations, see the legend of Fig. 2.
78
18
18 0 1 . 4 ,ug
16
16
00.14pg
c) 14
14
c
d
c12 > "r-
E ~o
~
10
~
8
ill
-a
8
o F--
i-
0 1.4pg
6
6
I 0.14pg
4
oT
•-~
c
'~
s
x o
u
Fig. 9. The effect of inhibitors of protein synthesis upon total number of arrivals at the two patterns regardless of their shapes. Experimental conditions were the same as in Fig. 8.
With chilling for 1 h or more, dicrimination was reduced to a fourth of full development and, with further increase of the length of chilling, the discrimination decreased very slowly. This indicates that short-term, normal activity is absolutely needed immediately after visual experience for the development of pattern discrimination. Secondly, the minimum duration of normal activity needed immediately after visual experience for normal
E o
~
g
Fig. 11. The effect of inhibitors of protein synthesis upon total number of arrivals at the two patterns regardless of their shapes. Experimental conditions were the same as in Fig. 10.
development was investigated (Fig. 5). The minimum duration was found to be 5 h. Pattern discrimination developed hardly in the presence of normal activity for less than 4 h. Discrimination became abruptly welldeveloped from 5 h of normal activity. This shows that, for the development of pattern discrimination, normal activity must by maintained for at least 5 h after visual experience.
Injection of inhibitors of protein synthesis at various stages of post-emergence 16
J,mcrlg
Sth in , c t i o n pos~4Lir~n,rgl
14
tests
12 10
o ~ 8 •u
6
,-~ 4
0
1.4 pg
•
0-14 p g
-2 c
-
o
oE
Fig. 10. The effect of 3 inhibitors of protein synthesis upon the development of pattern discrimination. Injection was done at 5 h after cessation of visual experience. For abbreviations, see the legend of Fig. 2.
Three inhibitors of protein synthesis were injected into the fly's head in order to confirm the above results and to obtain a lead to the molecular basis underlying the developmental process. Injections were given at three stages of post-emergence: immediately after emergence, just before visual experience; immediately after 5 h of visual experience; and 5 h after cessation of visual experience. When the inhibitors were injected immediately after emergence and immediately after visual experience, pattern discrimination did not develop regardless of the kind of inhibitor (Figs. 6 and 8). The injection of inhibitors immediately after visual experience was more effective than just before visual experience, because slight discrimination was seen with injections of low concentration (0.14 /~g) just before visual experience, while discrimination did not develop at all with injections immediately after visual experience except with 0.14/~g of anisomysin injection. In contrast to the above results, injections at 5 h after cessation of visual experience did not influence at all the
79 development of pattern discrimination (Fig. 10). These results support the findings of the chilling experiments. One more important finding involved the influence of inhibitors on phototactic behavior. Regardless of the stage of injection, the phototactic behavior was hardly influenced by the injection of inhibitors, although the number of arrivals decreased slightly (Figs. 7, 9 and 11). This suggests that the visual mechanisms for pattern discrimination and for phototaxis are two separate functions from the viewpoint of the developmental process. DISCUSSION It has been demonstrated in vertebrates that postnatal signals from sensory surfaces modulate neuronal activity and hence interfere with the self-organizing processes 3'2°. Even in invertebrates such as the fly, the same phenomenon has been demonstrated from the development of visual pattern discrimination 12. In the mammalian visual cortex, these experiencedependent modifications are restricted to a critical period of postnatal development 1'2°. As shown also in this study of the fly, visual experience had to be given within a very restricted period post-emergence for the normal development of pattern discrimination. Moreover, it was demonstrated that brief, normal neuronal activity immediately after visual experience plays a role in driving the development, and that the normal activity of the nervous system had to be maintained for at least 5 h for the normal development, suggesting identification of short-term and long-term memory, which may correspond to neuronal activity and molecular modification, respectively. In this experiment, the normal activity level was lowered in the fly anesthetized by chilling. The anesthetizing influence upon the development of pattern discrimination was evident when the fly was chilled in the suitable stage and period. Similar phenomena were seen in the development of cortical functions of the kittens, in which visual behavior failed to develop when they are paralyzed and/or anesthetized while exposed to light 1'6'19. Hence, for the normal development of higher functions such as pattern discrimination in the fly and such as the complicated visual behavior in the kitten, it is a prerequisite that the neuronal circuits are active2°. As one example, this was demonstrated from the effect of a REFERENCES 1 Buisseret, P., Gary-Bobo, E. and Imbert, M., Ocular motility and recovery of orientational properties of visual cortical neurones in dark-reared kittens, Nature (Lond.), 272 (1987)
certain level of norepinephrine in the extracellular space 7,8. The effect of several inhibitors of protein synthesis upon memory formation has been demonstrated: anisomycin influenced memory formation in miceS; retrograde amnesia for a passive avoidance response was produced in mice by cycloheximideXS; and puromycin caused loss of memory of avoidance discrimination learning in mice 4. These 3 kinds of inhibitors of protein synthesis impair 'long-term' but not 'short-term' memory 21. In fact, Castellucci et al. 2 detected, using 2-D gel analysis, 4 proteins, which changed in their rates of synthesis in long-term memory. The present experiment demonstrated the effect of 3 kinds of inhibitors of protein synthesis upon formation of pattern discrimination in flies. The effective time of injection was coincident with the period of normal activity, which was necessary for formation of pattern discrimination. It has been proposed that long-term memory depends on protein synthesis in synaptic membrane 17. Furthermore, it has been demonstrated that the formation of imprinting behavior accompanies protein synthesis at special locations of the brain and morphological changes of synapse z2. In flies, Kral and Meinertzhagen 9 observed electronmicroscopically that light experience relates to formation of retina-lamina synaptic connection. The development of pattern discrimination may imply formation of neuronal circuits after emergence including modification of synaptic connection, which accompanies molecular changes. The other important finding in the present experiment was the separation of the two visual functions for phototaxis and pattern discrimination. The former mechanism is already completed at emergence as a natural function and the latter develops after emergence as an acquired function. This has been previously suggested in the first experiment on experience-dependent development of pattern discrimination in the fly12. That is, pattern dicrimination did not develop when flies were kept in the dark for more than 5 days after emergence, while phototactic behavior was not influenced even under such condition. Acknowledgements. I wish to thank Dr. T. Nagao of Hokkaido University for helpful advice on injection of inhibitor of protein synthesis and also Dr. K. Yorichika for improving the English. This work was supported by Grants-in-Aid from the Ministry of Education, Science and Culture of Japan (60304011, 63540578 and 63304005).
816-817. 2 Castellucci, V.E, Kennedy, T.E., Kandel, E.R. and Goelet, P., A quantitative analysis of 2-D gels identifies proteins in which labeling is increased followinglong-term sensitization in aplysia, Neuron, 1 (1988) 321-328.
80 3 Chow, K.L., Neuronal changes in the visual system following visual deprivation. In R. Jung (Ed.), Central Processing of Visual
Information A: Intergrative Functions and Comparative Data, Handbook of Sensory Physiology, Vol. VII/3A, Springer, Berlin, pp. 599-627. 4 Flexner, J.B., Flexner, L.B. and Stellar, E., Memory in mice as affected by intracerebral puromycin, Science, 141 (1963) 57-59. 5 Flood, J.F., Bennett, E.L. and Orme, A.E., Relation of memory formation to controlled amounts of brain protein synthesis, Physiol. Behav., 15 (1975) 97-102. 6 Freeman, R.D. and Bonds, A.B., Cortical plasticity in monocularly deprived immobilized kittens depends on eye movement, Science, 206 (1979) 1093-1095. 7 Kasamatsu, T. and Pettigrew, J.D., Preservation of binocularity after monocular deprivation in the striate cortex of kittens treated with 6-hydroxydopamine, J. Comp. Neurol., 185 (1979) 139-162. 8 Kasamatsu, T., Pettigrew, J.D. and Ary, M., Restoration of visual cortical plasticity by local microperfusion of norepinephrine, J. Comp. NeuroL, 185 (1979) 163-181. 9 Kral, D. and Meinertzhagen, I.A., Functional plasticity in the fly's visual system, Soc. Neurosci. Abstr., 12 (1987) 930. 10 Mimura, K., Phototactic behaviour of walking flies in response to some visual patterns: correlation with receptive field patterns of photoreceptors, J. Comp. Physiol., 144 (1981) 75-82. 11 Mimura, K., Discrimination of some visual patterns in Drosophila melanogaster, J. Comp. Physiol., 146 (1982) 229-233. 12 Mimura, K., Development of visual pattern discrimination in the
fly depends on light experience, Science. 232 (1986) 83-85. 13 Mimura, K., Persistence and extinction of the effect of visual pattern deprivation in the fly, Exp. Biol., 46 (1987) 155-162. 14 Mimura, K., The effect of partial covering of the eye on the results of selective deprivation of visual pattern in the fly, Brain Research, 437 (1987) 97-102. 15 Mumura, K., Cytochrome oxidase histochemistry in the effect of light deprivation on the fly visual system, Brain Research, 445 (1988) 228-233. 16 Nagao, T., personal communication. 17 Nestler, E.J. and Greengard, P., Protein phosphorylation in the brain, Nature (Lond.), 305 (1983) 583-588. 18 Quartermain, D., McEwen, B.S. and Azmitia, Jr., E.C., Amnesia produced by electroconvulsive shock or cycloheximide: conditions for recovery, Science, 169 (1970) 683-686. 19 Singer, W., Central core control of developmental plasticity in the kitten visual cortex: I. Diencephalic lesions, Exp. Brain Res., 47 (1982) 209-222. 211 Singer, W., Learning to see: mechanisms in experience-dependent development. In P. Marler and H.S. Terrace (Eds.), The Biology of Learning, Springer, Berlin, pp. 461-477. 21 Squire, L.R. and Barondes, S.H., Anisomycin, like other inhibitors of cerebral protein synthesis, impairs "long-term' memory of a discrimination task, Brain Research, 66 (1974) 301-308. 22 Takamatsu, K. and Tsukada, Y., Neurochemical studies on imprinting behavior in chick and duckling, Neurochem. Res., 10 (1985) 1371-1391.