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Brightness discrimination learning in a Skinner box in prenatally X-irradiated rats
Physiology & Behavior, Vol. 16, pp. 343-348. Pergamon Press and Brain Research Publ., 1976. Printed in the U.S.A.
Brightness Discrimination Learning in a Skinner Box in Prenatally X-Irradiated Rats YOSHITAKA TAMAKI AND MINORU INOUYE l
Department o f Physiology and Department o f Embryology Institute for Developmental Research, Aichi Prefectural Colony, Aichi, Japan (Received 30 June 1975)
TAMAKI, Y. AND M. INOUYE. Brightness discrimination learning in a Skinner box in prenatally X-irradiated rats. PHYSIOL. BEHAV. 16(3) 343-348, 1976. - Male MP 1 albino rats were exposed to X-irradiation in utero at a single dose of 200 R on Day 17 of gestation. The light-dark discrimination training in a Skinner box was continued until the animals attained a learning criterion of 0.80 correct response ratio for 3 consecutive days. Although during the unreinforced baseline sessions the total number of bar pressings in the irradiated animals was superior to that in the controls, performance between the control and the irradiated animals did not differ significantly in (a) the number of training days required to attain the learning criterion, (b) the total number of days on which the animals produced a correct response ratio more than 0.80, and (c) the number of consecutive days during which the correct response ratio was more than 0.75. The results obtained suggest that the irradiated animals were able to discriminate in brightness cues as well, or nearly as well, as the controls. The cortical-subcortical system mediating brightness discrimination in the irradiated animals was discussed. Prenatal X-irradiation
Microcephalia
Brightness discrimination
IT has been known that pregnant rats exposed to Xirradiation subsequently develop in brain malformations such as exencephalia, hydrocephalia or microcephalia in the young [21]. In microcephalia manifested by X-irradiation at a single dose of 200 R on gestation Day 16 or 17, for example, severe malformation of the prosencephalon was observed, while the diencephalon was relatively well formed. Studies in animals with selective regional deficits due to X-irradiation during the prenatal period may contribute to the understanding of the dynamic function o f the brain, if supposed that each brain structure has a timetable of development and of a correlated differential radiosensitivity [8]. Kimeldorf and Hunt [14] have summarized a number of experiments dealed with the behavioral sequelae of animals exposed to ionizing radiation prenatally. There are certain amount of agreement concerning the behavioral characteristic after prenatal X-irradiation. For example, the prenatally X-irradiated animals showed an increased fearfulness or arousal in a open field [3,5], in escape-avoidance tasks [1, 4, 6], in a conditioned emotional response situation [20] and in examination of the EEG frequency analysis and the sleep-wakefulness cycle [15]. These findings served as the basis of our previous study in a 2 way avoidance situation [Tamaki, et al., unpublished observation]. That is, the irradiated rats treated in the same manner as in this study learned the avoidance response more rapidly than did the controls, and performed more
Irradiation and behavior
concurrent barrier crossings during the intertrial intervals (intertrial responses) than the controls. However, their performance could not be attributable to increased locomotive activities, because the number of spontaneous crossings during the habituation period was not correlated with the number of avoidance responses or of intertrial responses. This suggestion may be supported by an earlier finding [ 1 ] that the irradiated rats performed more passive avoidance behavior than the controls. Judging from the fact that concurrent intertrial responses were neither reinforced nor punished, the irradiated animals might be unable to inhibit responding. The present study using a l i g h t - d a r k discrimination task in a Skinner box was designed to investigate the inhibitory function in adult male rats treated with prenatal X-irradiation. METHOD
Animals Albino rats of the MP 1 strain were used. A nulliparous female of 13 weeks or older was kept overnight with 2 males, and females having spermatozoa in the vaginal smear examination were considered to be on Day 0 o f gestation. Three gravid females were exposed to a single whole-body X-irradiation at a dose of 200 R on Day 17 of gestation. A deep therapy 200 kVp, 25 mA X-ray machine (Toshiba, K X C - 1 8 - 3 ) was used with the following radiation factors: 200 kVp, 20 mA, TSD 70 cm, inherent filtration 0.5 mm
a Minoru Inouye is a member of the Department of Embryology, Institute for Developmental Research, Aichi Prefectural Colony, Aichi, Japan. 343
344 A1 + 0.5 mm Cu, HVL 12.7 mm A1 and a dose rate of 25.7 R/min. The dosimetry was made by a Victoreen integrating ratemeter, model 555, inserted in the plastic box to which the gravid female was confined during the irradiation. Ten male offspring of these irradiated females were designated as the irradiated group. Nine male offspring born from 3 dams without irradiation were taken as the control group. The body weight at the beginning of the experiment was 191.9 -+ 27.7 (mean +_ SD) g in the control and 195.0 +_ 32.6 g in the irradiated group, the difference being not significant (t = 0.305, df = 17). Experiment began when the animals were 77 days of age.
TAMAKI AND INOUYE number of responses emitted during S+ was divided by the total number of responses (S+ + S-).
Histological Preparation At the end of the experiment, the animals were sacrificed under ether anesthesia. Their brains, separated from the spinal cord at the cervico-medullary junction, were weighed immediately after removal from the skull. The sagittal, horizontal and dorsoventral lengths of the cerebral hemispheres were measured. The brains were fixed in 10% Formalin, embedded with paraffin, serially sectioned in 10 um and then stained with Klfiver-Barreara's cresyl violet and Luxol fast blue.
Apparatus The apparatus consisted of an conventional operant chamber having inside dimensions of 30 × 20 × 30 cm, a single bar located in the center of the frontal panel 7 cm above the grid floor, a 20 mg food pellet dispenser and two 10 W light bulbs 20 cm above the grid floor. A daily session began when 2 light bulbs were turned on and ended when the lights were turned off. However, their brightness of the lights was interchanged by light controllers (National, LQ 20018), in order to serve as a discriminative stimulus. The brightness was 80 l x for the light condition and 10 lx for the dark condition. It was measured in the center of the frontal panel at the grid floor. The apparatus was located in a sound proof room where the white-noise level was approximately 85 dB sound pressure level (SPL) at the grid floor. Experimental events were controlled by electronics equipment (Unitec, UP 600) located in an adjacent room. Digital counters recorded the total number of bar pressings, and the number of bar pressings emitted during the light condition.
RESULTS
Histology The histological findings in the present study were comparable with those of Hicks, et al. [10]. The principal features were small cerebral hemispheres and abnormal cortical cytoarchitecture. The brains of the irradiated animals, tentatively called microcephalia, were approximately 35% lighter in mean wet weight than the control group. The control brains weighed 1.89 g in average (range: 1 . 8 3 - 2 . 0 7 g), while the irradiated brains 1.22 g (range: 1.06-1.45 g). The difference was highly significant with no overlap between the 2 groups. The cerebral hemispheres of the irradiated group were reduced by 27% in sagittal, 18% in horizontal and 26% in dorsoventral lengths, while the midbrain and cerebellum were scarcely reduced in size as compared with the controls (Fig. 1).
Procedure Ten days prior to the start of discrimination training, the animals were put on a 23 hr food deprivation schedule and were maintained on this schedule throughout the experiment. Water was available in home cage at all times. The procedure employed by Notterman and Block [ 18] was used in a modified form. Two brightness conditions (light or dark) were replicated with 5 durations (10, 20, 40, 80 or 160 sec). A daily session consisted of 20 min and 40 sec. In order to preclude any temporal conditioning, 2 brightness conditions were alternately presented with 1 of 3 irregular sequences (A, B or C) for these durations. Each of 3 sequences was presented every 3 days (A,B,C,A,B,C, --). The first 2 days were used solely to determine the animal's brightness preference and their operant level. Bar pressings in these baseline sessions were not reinforced. The light condition was taken as the positive stimulus (S*), since rats of both groups in the baseline sessions prefered slightly but not significantly the dark rather than the light condition (see Fig. 4). A CRF schedule was maintained during S+ with a 20 mg pellet as a reinforcer, but any responses emitted during the dark condition (negative stimulus; S-) were not reinforced. The discrimination training was continued until the animals attained a learning criterion of 0.80 correct response ratio for 3 consecutive days. But training was terminated when the animal failed to reach the learning criterion within 45 training days. The correct response ratio was calculated as follows: the
FIG. 1. Brain of an adult rat exposed to 200 R on gestation Day 17 (right) as compared with a control rat (left). Notice more severe retardation on prosencephalic development in the irradiated rat.
345
PRENATAL X-IRRADIATION AND DISCRIMINATION Irregularities of the folia in the anterior vermis of the cerebellum were observed, as shown in Figs. 1 and 3. Microscopically, the dorsal areas of the cerebral cortex were strikingly reduced in thickness, but the ventral regions (pyriform and olfactory lobes) below the rhinal fissure were spared. The outer layers of the neocortex were not discernible, but the inner pyramidal and polymorphous cell layers were apparently well preserved (Fig. 2). The trunculs corporis callosi was absent and the lateral ventricles were enlarged. There was marked underdevelopment of the caudate nucleus, putamen, hippocampus and dentate gyrus. The thalamus, hypothalamus, central tegmentum, red nucleus, substantia nigra and interpeduncular nucleus were apparently well developed (Fig. 3).
Discrimination Learning Figure 4 presents the customary S+-S- separation of the number of responses emitted during light-dark cycle in the 2 baseline sessions. An analysis of variance with mixed design [ 17] revealed that the total number of responses in the irradiated group was superior to that in the control group, F(1,17) = 5.69, p<0.05, but bar pressings between the light and dark conditions did not differ at the conventional significance level of 0.05, F(1,17) = 4.12, p<0.10. The Group x light-dark interaction was not significant (F< 1.00). Table 1 shows the acquisition data. There was no significant difference between these groups in the number of training days taken to reach the learning criterion (t = 0.709, d f = 17). One out of 10 irradiated rats did not attain the learning criterion within 45 days, therefore, it had an arbitrary score of 45 days. Furthermore, acquisition
TABLE 1 ACQUISTIONDATA IN BRIGHTNESSDISCRIMATIONLEARNING
Measure No. of days to criterion* No. of total days (ratio>0.80) No. of consecutive days (ratio>0.75)
Control Group
Irradiated Group
19.8±7.4 4.1±2.5 4.4±1.8
23.0±10.8 4.3± 2.5 4.6±1.7
*Number of training days took to reach the learning criterion. One irradiated rat which failed to reach the learning criterion within 45 days had an arbitrary score of 45 days. curves to evaluate the temporal course of improvement in performance. Here the indivdual curves are displaced horizontally, so that their final points coincide [7]. These learning curves were plotted at the case on which the animals consecutively performed the total number of responses more than 3 SD of the operant level, and omitted the results obtained from the 1 irradiated rat which failed to reach the learning criterion within 45 days. It was noted that the animals of both groups learned the brightness discrimination gradually, and that the pattern of learning curves in the irradiated group did not appear essentially different from those in the control group. Figure 6 presents the S*-S- separation of the number of bar pressings in representative correct response ratios of the total training days. Although the response differentiation in the both groups did develop as the training progressed, the number of responses emitted during S*-S- cycle with the correct response ratio less than the chance level (0.50) was approximately twice as high in the irradiated group as that in the control group. Accordingly, behavioral characteristics after prenatal X-irradiation might be reflected on the early training phase in a light-dark discrimination situation. DISCUSSION
FIG. 2. Dorsolateral cerebral cortex of an adult rat exposed to 200 R on gestation Day 17 (right) as compared with a control rat (left). The outer laminae are deficient in the irradiated rat. Cresyl violet and Luxol blue. performance between the 2 groups differed significantly neither in the total number of days on which the animals made the correct response ratio more than 0.80 (t = 0.159, d f = 17), nor in the number of consecutive days during which the correct response ratio was more than 0.75 (t = 0.190, d f = 17). Figure 5 presents the individual learning
The irradiated animals were able to discriminate in brightness cues as well, or nearly as well, as the control animals, despite of rather large distortions in neural organization. These results are consistent with those obtained by Falk and D'Amato [2] and Hicks, et al. [9], who used a visual pattern as a discriminative stimulus, and tended to confirm Lashley's hypothesis [16] that brightness discrimination habits are mediated by the striate cortex in normal rats and by subcortical mechanisms in rats after decortication. A further extension of this view was elabolated by Thompson [23,24], who found that the occipital cortex, posterior thalamus or ventral midbrain areas (red nucleus, substantia nigra, central tegmentum or interpeduncular nucleus) mediated a brightness discrimination. It might be proposed, therefore, that the prenatal X-irradiation had little effects on performance of the light-dark discrimination task. There is a good reason for suggesting this possibility, because the posterior thalamus and ventral mesencephalon displayed no selective regional deficits in rats treated with X-irradiation of 200 R on Day 17 of gestation, despite of severe malformation of the prosencephalon (see Fig. 3). Defective development of other regions (caudate nucleus, putamen, dentate gyrus and cerebellum) in the irradiated animals was observed in our histological study, but Thompson [24] pointed out that
346
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FIG. 3. Frontal sections of the brain of an adult rat exposed to 200 R on gestation Day 17 (right) as compared with a control rat (left). It is obvious that prosencephalic parts of the cerebrum in the irradiated rat are severely reduced in size, including myelinated pathways, and that truncus corporis callosi is absent. In contrast, diencephalon is relatively well developed. Normal appearance of the medulla oblongata but malformation of cerebellar cortex in the irradiated rat. Cresyl violet and Luxol blue.
PRENATAL X-IRRADIATION AND DISCRIMINATION
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FIG. 6. Frequency distribution of the number of responses in representative correct response ratios of the total training days for the control group (upper) and for the irradited group (lower). Responses during the light (reinforced) condition are symbolized by S* and responses during the dark (unreinforced) condition by S-. Percentage of the occurrence of each class to the total training days for both groups is shown in the inset. Vertical bars indicate + 1 SD. the damage of the above m e n t i o n e d areas did not affect brightness discrimination learning. F u r t h e r m o r e , underd e v e l o p m e n t of the h i p p o c a m p u s was also observed in the irradiated animals. A l t h o u g h it is postulated that the h i p p o c a m p u s normally has some kind of inhibitory f u n c t i o n o f behavior [ 1 3 ] , rats with damage to the h i p p o c a m p u s were not impaired b o t h in the acquisition of a brightness discrimination [12,22] and in the r e t e n t i o n o f brightness discrimination habits [25]. Hippoc a m p e c t o m i z e d animals were severely impaired in t h e learning w h e n the task required the inhibition of a p o t e n t competing responses [11]. Consequently, underd e v e l o p m e n t o f the h i p p o c a m p u s in the irradiated animals may show a severe deficit in the reversal learning of the discrimination. Initially, during the early training phase, the irradiated group showed an elevated n u m b e r of bar pressings as compared with the controls. Similar results were obtained by Flower, e t al. [ 3 ] , who found that the irradiated animals made more errors than the: ~ controls in a brightness discrimination learning within a training phase o f 7 days. A l t h o u g h this feature o f the irradiated animals might reflect the heightened operant level in the baseline sessions, a weakness of internal inhibition process [3,19] was not sufficient to account for the behavioral changes after prenatal X-irradiation. The reason for this was that in the
348
TAMAKI AND INOUYE
present s t u d y t h e irradiated animals were able to i n h i b i t b a r pressing d u r i n g d a r k ( u n r e i n f o r c e d ) c o n d i t i o n as discrim i n a t i o n h a b i t s established. Moreover, a possible exp l a n a t i o n of t h e h e i g h t e n e d o p e r a n t level in t h e irradiated animals c o u l d b e based o n t h e f i n d i n g o f a relative p r e d o m i n a n c e of t h e e x c i t a t i o n state as a result o f p r e n a t a l X-irradiation [ 1 5 ] .
ACKNOWLEDGMENT The authors greatly acknowledge to Dr. R. Shoji, M. Sc. I. K. Takeuchi and Mr. M. Kawabata for providing animals, to Miss N. Nakayama for skillful histological preparation, to Miss M. Seo for technical assistance, and to Dr. S. Kiyono for kindly reading the manuscript.
REFERENCES 1. Deagle, J. and E. Furchtgott. Passive avoidance in prenatally X-irradiated rats. DevlPsychobiol. 1: 9 0 - 9 2 , 1968. 2. Falk, J. L. and C. J. D'Amato. Automation of pattern discrimination in the rat. Psychol. Rep. 10: 24, 1962. 3. Flower, G., S. P. Hicks, C. J. D'Amato and F. A. Beach. Effects of fetal irradiation on behavior in the adult rat. J. comp. physiol. Psychol. 55: 3 0 9 - 3 1 4 , 1962. 4. Furchtgott, E., J. R. Jones, R. S. Tacker and J. Deagle. Aversive conditioning in prenatally X-irradiated rats. Physiol. Behav. 5: 5 7 1 - 5 7 6 , 1970. 5. Furchtgott, E., R. S. Tacker and D. O. Draper. Open-field behavior and heart rate in prenatally X-irradiated rats. Teratology 1: 201-206, 1968. 6. Furchtgott, E. and S. Wechkin. Avoidance conditioning as a function of prenatal X-irradiation and age. J. comp. physiol. Psychol. 55: 6 9 - 7 2 , 1962. 7. Hayes, K. J. The backward curve: A method for the study of learning. Psychol. Rev. 60: 2 6 9 - 2 7 5 , 1953. 8. Hicks, S. P. and C. J. D'Amato. How to design and build abnormal brains using radiation during development. In: Disorders o f the Developing Nervous System, edited by W. S. Field and M. M. Desmond. Springfield: C. C. Thomas, 1961, pp. 6 0 - 9 7 . 9. Hicks, S. P., C. J. D'Amato and J. L. Falk. Some effects of radiation on structure and behavioral development. Int. J. Neurol. 3: 5 3 5 - 5 4 8 , 1962. 10. Hicks, S. P., C. J. D'Amato and M. J. Love. The development of the mammalian nervous system. I. Malformations of the brain, especially the cerebral cortex, induced in rats by radiation. II. Some mechanisms of the malformations of the cortex. J. comp. Neurol. 113: 4 3 5 - 4 6 9 , 1959. 11. Issacsson, R. L. The Limbic System. New York: Plenum Press, 1974. 12. Kimble, D. P. The effects of bilateral hippocampus lesions in rats. J. comp. physiol. PsychoL 56: 2 7 3 - 2 8 3 , 1963. 13. Kimble, D. P. Hippocampus and internal inhibition. Psychol. Bull. 70: 285-295, 1968. 14. Kimeldorf, D. J. and E. L. Hunt. lonizing Radiation, Neural Function, and Behavior. New York: Academic Press, 1965.
15. Kiyono, S., M. Seo, K. Takasu, R. Shoji, I. K. Takeuchi and U. Murakami. Spontaneous motility, EEG and sleep-wakefulness cycle disturbance in prenatally X-irradiated rats. J. Physiol. Soc. Japan 37: 4 - 6 , 1975. 16. Lashley, K. S. The mechanisms of vision: XII. Nervous structures concerned in the acquisition and retention of habits based on reactions to light. Comp. Psychol. Monogr. 11: 4 2 - 7 9 , 1935. 17. Lindquist, E. F. Design and Analysis of Experiments in Psychology and Education. Boston: Houghton Mifflin, 1953. 18. Notterman, J. M. and A. H. Block. Note on response differentiation during a simple discrimination. J. exp. Analysis Behav. 3: 289-291, 1960. 19. Piontkovsky, I. A. Features specific to the functions of the higher divisions of the central nervous system in animals subjected to the effect of ionizing radiation at various periods of antenatal development. J. comp. physiol. Psychol. 54: 314-318, 1961. 20. Sharp, J. C. The effects of prenatal X-irradiation on acquisition, retention and extinction of a conditioned emotional response. Radiat. Res. 24: 154-157, 1965. 21. Shoji, R., I. K. Takeuchi, U. Murakami and J. Tsuge. Experimental studies on maldevelopment in the rat and hamster brain following maternal X-irradiation (Abstract). Teratology 10: 98, 1974. 22. Silveira, J. M. and D. P. Kimble. Brightness discrimination and reversal in hippocampally-lesioned rats. Physiol. Behav. 3: 6 2 5 - 6 3 0 , 1968. 23. Thompson, R. Retention of a brightness discrimination following neocortical damage in the rat. J. comp. physiol. Psychol. 53: 2 1 3 - 2 1 5 , 1960. 24. Thompson, R. Localization of the "visual memory system" in the white rat. J. comp. physiol. Psychol. Monogr. 6 9 : 1 - 2 9 , 1969. 25. Trauax, T. and R. Thompson. Role of the hippocampus in performance of easy and difficult visual discrimination tasks. J. comp. physiol. Psychol. 67: 228-234, 1969.
Brightness Discrimination Learning in a Skinner Box in Prenatally X-Irradiated Rats YOSHITAKA TAMAKI AND MINORU INOUYE l
Department o f Physiology and Department o f Embryology Institute for Developmental Research, Aichi Prefectural Colony, Aichi, Japan (Received 30 June 1975)
TAMAKI, Y. AND M. INOUYE. Brightness discrimination learning in a Skinner box in prenatally X-irradiated rats. PHYSIOL. BEHAV. 16(3) 343-348, 1976. - Male MP 1 albino rats were exposed to X-irradiation in utero at a single dose of 200 R on Day 17 of gestation. The light-dark discrimination training in a Skinner box was continued until the animals attained a learning criterion of 0.80 correct response ratio for 3 consecutive days. Although during the unreinforced baseline sessions the total number of bar pressings in the irradiated animals was superior to that in the controls, performance between the control and the irradiated animals did not differ significantly in (a) the number of training days required to attain the learning criterion, (b) the total number of days on which the animals produced a correct response ratio more than 0.80, and (c) the number of consecutive days during which the correct response ratio was more than 0.75. The results obtained suggest that the irradiated animals were able to discriminate in brightness cues as well, or nearly as well, as the controls. The cortical-subcortical system mediating brightness discrimination in the irradiated animals was discussed. Prenatal X-irradiation
Microcephalia
Brightness discrimination
IT has been known that pregnant rats exposed to Xirradiation subsequently develop in brain malformations such as exencephalia, hydrocephalia or microcephalia in the young [21]. In microcephalia manifested by X-irradiation at a single dose of 200 R on gestation Day 16 or 17, for example, severe malformation of the prosencephalon was observed, while the diencephalon was relatively well formed. Studies in animals with selective regional deficits due to X-irradiation during the prenatal period may contribute to the understanding of the dynamic function o f the brain, if supposed that each brain structure has a timetable of development and of a correlated differential radiosensitivity [8]. Kimeldorf and Hunt [14] have summarized a number of experiments dealed with the behavioral sequelae of animals exposed to ionizing radiation prenatally. There are certain amount of agreement concerning the behavioral characteristic after prenatal X-irradiation. For example, the prenatally X-irradiated animals showed an increased fearfulness or arousal in a open field [3,5], in escape-avoidance tasks [1, 4, 6], in a conditioned emotional response situation [20] and in examination of the EEG frequency analysis and the sleep-wakefulness cycle [15]. These findings served as the basis of our previous study in a 2 way avoidance situation [Tamaki, et al., unpublished observation]. That is, the irradiated rats treated in the same manner as in this study learned the avoidance response more rapidly than did the controls, and performed more
Irradiation and behavior
concurrent barrier crossings during the intertrial intervals (intertrial responses) than the controls. However, their performance could not be attributable to increased locomotive activities, because the number of spontaneous crossings during the habituation period was not correlated with the number of avoidance responses or of intertrial responses. This suggestion may be supported by an earlier finding [ 1 ] that the irradiated rats performed more passive avoidance behavior than the controls. Judging from the fact that concurrent intertrial responses were neither reinforced nor punished, the irradiated animals might be unable to inhibit responding. The present study using a l i g h t - d a r k discrimination task in a Skinner box was designed to investigate the inhibitory function in adult male rats treated with prenatal X-irradiation. METHOD
Animals Albino rats of the MP 1 strain were used. A nulliparous female of 13 weeks or older was kept overnight with 2 males, and females having spermatozoa in the vaginal smear examination were considered to be on Day 0 o f gestation. Three gravid females were exposed to a single whole-body X-irradiation at a dose of 200 R on Day 17 of gestation. A deep therapy 200 kVp, 25 mA X-ray machine (Toshiba, K X C - 1 8 - 3 ) was used with the following radiation factors: 200 kVp, 20 mA, TSD 70 cm, inherent filtration 0.5 mm
a Minoru Inouye is a member of the Department of Embryology, Institute for Developmental Research, Aichi Prefectural Colony, Aichi, Japan. 343
344 A1 + 0.5 mm Cu, HVL 12.7 mm A1 and a dose rate of 25.7 R/min. The dosimetry was made by a Victoreen integrating ratemeter, model 555, inserted in the plastic box to which the gravid female was confined during the irradiation. Ten male offspring of these irradiated females were designated as the irradiated group. Nine male offspring born from 3 dams without irradiation were taken as the control group. The body weight at the beginning of the experiment was 191.9 -+ 27.7 (mean +_ SD) g in the control and 195.0 +_ 32.6 g in the irradiated group, the difference being not significant (t = 0.305, df = 17). Experiment began when the animals were 77 days of age.
TAMAKI AND INOUYE number of responses emitted during S+ was divided by the total number of responses (S+ + S-).
Histological Preparation At the end of the experiment, the animals were sacrificed under ether anesthesia. Their brains, separated from the spinal cord at the cervico-medullary junction, were weighed immediately after removal from the skull. The sagittal, horizontal and dorsoventral lengths of the cerebral hemispheres were measured. The brains were fixed in 10% Formalin, embedded with paraffin, serially sectioned in 10 um and then stained with Klfiver-Barreara's cresyl violet and Luxol fast blue.
Apparatus The apparatus consisted of an conventional operant chamber having inside dimensions of 30 × 20 × 30 cm, a single bar located in the center of the frontal panel 7 cm above the grid floor, a 20 mg food pellet dispenser and two 10 W light bulbs 20 cm above the grid floor. A daily session began when 2 light bulbs were turned on and ended when the lights were turned off. However, their brightness of the lights was interchanged by light controllers (National, LQ 20018), in order to serve as a discriminative stimulus. The brightness was 80 l x for the light condition and 10 lx for the dark condition. It was measured in the center of the frontal panel at the grid floor. The apparatus was located in a sound proof room where the white-noise level was approximately 85 dB sound pressure level (SPL) at the grid floor. Experimental events were controlled by electronics equipment (Unitec, UP 600) located in an adjacent room. Digital counters recorded the total number of bar pressings, and the number of bar pressings emitted during the light condition.
RESULTS
Histology The histological findings in the present study were comparable with those of Hicks, et al. [10]. The principal features were small cerebral hemispheres and abnormal cortical cytoarchitecture. The brains of the irradiated animals, tentatively called microcephalia, were approximately 35% lighter in mean wet weight than the control group. The control brains weighed 1.89 g in average (range: 1 . 8 3 - 2 . 0 7 g), while the irradiated brains 1.22 g (range: 1.06-1.45 g). The difference was highly significant with no overlap between the 2 groups. The cerebral hemispheres of the irradiated group were reduced by 27% in sagittal, 18% in horizontal and 26% in dorsoventral lengths, while the midbrain and cerebellum were scarcely reduced in size as compared with the controls (Fig. 1).
Procedure Ten days prior to the start of discrimination training, the animals were put on a 23 hr food deprivation schedule and were maintained on this schedule throughout the experiment. Water was available in home cage at all times. The procedure employed by Notterman and Block [ 18] was used in a modified form. Two brightness conditions (light or dark) were replicated with 5 durations (10, 20, 40, 80 or 160 sec). A daily session consisted of 20 min and 40 sec. In order to preclude any temporal conditioning, 2 brightness conditions were alternately presented with 1 of 3 irregular sequences (A, B or C) for these durations. Each of 3 sequences was presented every 3 days (A,B,C,A,B,C, --). The first 2 days were used solely to determine the animal's brightness preference and their operant level. Bar pressings in these baseline sessions were not reinforced. The light condition was taken as the positive stimulus (S*), since rats of both groups in the baseline sessions prefered slightly but not significantly the dark rather than the light condition (see Fig. 4). A CRF schedule was maintained during S+ with a 20 mg pellet as a reinforcer, but any responses emitted during the dark condition (negative stimulus; S-) were not reinforced. The discrimination training was continued until the animals attained a learning criterion of 0.80 correct response ratio for 3 consecutive days. But training was terminated when the animal failed to reach the learning criterion within 45 training days. The correct response ratio was calculated as follows: the
FIG. 1. Brain of an adult rat exposed to 200 R on gestation Day 17 (right) as compared with a control rat (left). Notice more severe retardation on prosencephalic development in the irradiated rat.
345
PRENATAL X-IRRADIATION AND DISCRIMINATION Irregularities of the folia in the anterior vermis of the cerebellum were observed, as shown in Figs. 1 and 3. Microscopically, the dorsal areas of the cerebral cortex were strikingly reduced in thickness, but the ventral regions (pyriform and olfactory lobes) below the rhinal fissure were spared. The outer layers of the neocortex were not discernible, but the inner pyramidal and polymorphous cell layers were apparently well preserved (Fig. 2). The trunculs corporis callosi was absent and the lateral ventricles were enlarged. There was marked underdevelopment of the caudate nucleus, putamen, hippocampus and dentate gyrus. The thalamus, hypothalamus, central tegmentum, red nucleus, substantia nigra and interpeduncular nucleus were apparently well developed (Fig. 3).
Discrimination Learning Figure 4 presents the customary S+-S- separation of the number of responses emitted during light-dark cycle in the 2 baseline sessions. An analysis of variance with mixed design [ 17] revealed that the total number of responses in the irradiated group was superior to that in the control group, F(1,17) = 5.69, p<0.05, but bar pressings between the light and dark conditions did not differ at the conventional significance level of 0.05, F(1,17) = 4.12, p<0.10. The Group x light-dark interaction was not significant (F< 1.00). Table 1 shows the acquisition data. There was no significant difference between these groups in the number of training days taken to reach the learning criterion (t = 0.709, d f = 17). One out of 10 irradiated rats did not attain the learning criterion within 45 days, therefore, it had an arbitrary score of 45 days. Furthermore, acquisition
TABLE 1 ACQUISTIONDATA IN BRIGHTNESSDISCRIMATIONLEARNING
Measure No. of days to criterion* No. of total days (ratio>0.80) No. of consecutive days (ratio>0.75)
Control Group
Irradiated Group
19.8±7.4 4.1±2.5 4.4±1.8
23.0±10.8 4.3± 2.5 4.6±1.7
*Number of training days took to reach the learning criterion. One irradiated rat which failed to reach the learning criterion within 45 days had an arbitrary score of 45 days. curves to evaluate the temporal course of improvement in performance. Here the indivdual curves are displaced horizontally, so that their final points coincide [7]. These learning curves were plotted at the case on which the animals consecutively performed the total number of responses more than 3 SD of the operant level, and omitted the results obtained from the 1 irradiated rat which failed to reach the learning criterion within 45 days. It was noted that the animals of both groups learned the brightness discrimination gradually, and that the pattern of learning curves in the irradiated group did not appear essentially different from those in the control group. Figure 6 presents the S*-S- separation of the number of bar pressings in representative correct response ratios of the total training days. Although the response differentiation in the both groups did develop as the training progressed, the number of responses emitted during S*-S- cycle with the correct response ratio less than the chance level (0.50) was approximately twice as high in the irradiated group as that in the control group. Accordingly, behavioral characteristics after prenatal X-irradiation might be reflected on the early training phase in a light-dark discrimination situation. DISCUSSION
FIG. 2. Dorsolateral cerebral cortex of an adult rat exposed to 200 R on gestation Day 17 (right) as compared with a control rat (left). The outer laminae are deficient in the irradiated rat. Cresyl violet and Luxol blue. performance between the 2 groups differed significantly neither in the total number of days on which the animals made the correct response ratio more than 0.80 (t = 0.159, d f = 17), nor in the number of consecutive days during which the correct response ratio was more than 0.75 (t = 0.190, d f = 17). Figure 5 presents the individual learning
The irradiated animals were able to discriminate in brightness cues as well, or nearly as well, as the control animals, despite of rather large distortions in neural organization. These results are consistent with those obtained by Falk and D'Amato [2] and Hicks, et al. [9], who used a visual pattern as a discriminative stimulus, and tended to confirm Lashley's hypothesis [16] that brightness discrimination habits are mediated by the striate cortex in normal rats and by subcortical mechanisms in rats after decortication. A further extension of this view was elabolated by Thompson [23,24], who found that the occipital cortex, posterior thalamus or ventral midbrain areas (red nucleus, substantia nigra, central tegmentum or interpeduncular nucleus) mediated a brightness discrimination. It might be proposed, therefore, that the prenatal X-irradiation had little effects on performance of the light-dark discrimination task. There is a good reason for suggesting this possibility, because the posterior thalamus and ventral mesencephalon displayed no selective regional deficits in rats treated with X-irradiation of 200 R on Day 17 of gestation, despite of severe malformation of the prosencephalon (see Fig. 3). Defective development of other regions (caudate nucleus, putamen, dentate gyrus and cerebellum) in the irradiated animals was observed in our histological study, but Thompson [24] pointed out that
346
TAMAKI
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FIG. 3. Frontal sections of the brain of an adult rat exposed to 200 R on gestation Day 17 (right) as compared with a control rat (left). It is obvious that prosencephalic parts of the cerebrum in the irradiated rat are severely reduced in size, including myelinated pathways, and that truncus corporis callosi is absent. In contrast, diencephalon is relatively well developed. Normal appearance of the medulla oblongata but malformation of cerebellar cortex in the irradiated rat. Cresyl violet and Luxol blue.
PRENATAL X-IRRADIATION AND DISCRIMINATION
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FIG. 6. Frequency distribution of the number of responses in representative correct response ratios of the total training days for the control group (upper) and for the irradited group (lower). Responses during the light (reinforced) condition are symbolized by S* and responses during the dark (unreinforced) condition by S-. Percentage of the occurrence of each class to the total training days for both groups is shown in the inset. Vertical bars indicate + 1 SD. the damage of the above m e n t i o n e d areas did not affect brightness discrimination learning. F u r t h e r m o r e , underd e v e l o p m e n t of the h i p p o c a m p u s was also observed in the irradiated animals. A l t h o u g h it is postulated that the h i p p o c a m p u s normally has some kind of inhibitory f u n c t i o n o f behavior [ 1 3 ] , rats with damage to the h i p p o c a m p u s were not impaired b o t h in the acquisition of a brightness discrimination [12,22] and in the r e t e n t i o n o f brightness discrimination habits [25]. Hippoc a m p e c t o m i z e d animals were severely impaired in t h e learning w h e n the task required the inhibition of a p o t e n t competing responses [11]. Consequently, underd e v e l o p m e n t o f the h i p p o c a m p u s in the irradiated animals may show a severe deficit in the reversal learning of the discrimination. Initially, during the early training phase, the irradiated group showed an elevated n u m b e r of bar pressings as compared with the controls. Similar results were obtained by Flower, e t al. [ 3 ] , who found that the irradiated animals made more errors than the: ~ controls in a brightness discrimination learning within a training phase o f 7 days. A l t h o u g h this feature o f the irradiated animals might reflect the heightened operant level in the baseline sessions, a weakness of internal inhibition process [3,19] was not sufficient to account for the behavioral changes after prenatal X-irradiation. The reason for this was that in the
348
TAMAKI AND INOUYE
present s t u d y t h e irradiated animals were able to i n h i b i t b a r pressing d u r i n g d a r k ( u n r e i n f o r c e d ) c o n d i t i o n as discrim i n a t i o n h a b i t s established. Moreover, a possible exp l a n a t i o n of t h e h e i g h t e n e d o p e r a n t level in t h e irradiated animals c o u l d b e based o n t h e f i n d i n g o f a relative p r e d o m i n a n c e of t h e e x c i t a t i o n state as a result o f p r e n a t a l X-irradiation [ 1 5 ] .
ACKNOWLEDGMENT The authors greatly acknowledge to Dr. R. Shoji, M. Sc. I. K. Takeuchi and Mr. M. Kawabata for providing animals, to Miss N. Nakayama for skillful histological preparation, to Miss M. Seo for technical assistance, and to Dr. S. Kiyono for kindly reading the manuscript.
REFERENCES 1. Deagle, J. and E. Furchtgott. Passive avoidance in prenatally X-irradiated rats. DevlPsychobiol. 1: 9 0 - 9 2 , 1968. 2. Falk, J. L. and C. J. D'Amato. Automation of pattern discrimination in the rat. Psychol. Rep. 10: 24, 1962. 3. Flower, G., S. P. Hicks, C. J. D'Amato and F. A. Beach. Effects of fetal irradiation on behavior in the adult rat. J. comp. physiol. Psychol. 55: 3 0 9 - 3 1 4 , 1962. 4. Furchtgott, E., J. R. Jones, R. S. Tacker and J. Deagle. Aversive conditioning in prenatally X-irradiated rats. Physiol. Behav. 5: 5 7 1 - 5 7 6 , 1970. 5. Furchtgott, E., R. S. Tacker and D. O. Draper. Open-field behavior and heart rate in prenatally X-irradiated rats. Teratology 1: 201-206, 1968. 6. Furchtgott, E. and S. Wechkin. Avoidance conditioning as a function of prenatal X-irradiation and age. J. comp. physiol. Psychol. 55: 6 9 - 7 2 , 1962. 7. Hayes, K. J. The backward curve: A method for the study of learning. Psychol. Rev. 60: 2 6 9 - 2 7 5 , 1953. 8. Hicks, S. P. and C. J. D'Amato. How to design and build abnormal brains using radiation during development. In: Disorders o f the Developing Nervous System, edited by W. S. Field and M. M. Desmond. Springfield: C. C. Thomas, 1961, pp. 6 0 - 9 7 . 9. Hicks, S. P., C. J. D'Amato and J. L. Falk. Some effects of radiation on structure and behavioral development. Int. J. Neurol. 3: 5 3 5 - 5 4 8 , 1962. 10. Hicks, S. P., C. J. D'Amato and M. J. Love. The development of the mammalian nervous system. I. Malformations of the brain, especially the cerebral cortex, induced in rats by radiation. II. Some mechanisms of the malformations of the cortex. J. comp. Neurol. 113: 4 3 5 - 4 6 9 , 1959. 11. Issacsson, R. L. The Limbic System. New York: Plenum Press, 1974. 12. Kimble, D. P. The effects of bilateral hippocampus lesions in rats. J. comp. physiol. PsychoL 56: 2 7 3 - 2 8 3 , 1963. 13. Kimble, D. P. Hippocampus and internal inhibition. Psychol. Bull. 70: 285-295, 1968. 14. Kimeldorf, D. J. and E. L. Hunt. lonizing Radiation, Neural Function, and Behavior. New York: Academic Press, 1965.
15. Kiyono, S., M. Seo, K. Takasu, R. Shoji, I. K. Takeuchi and U. Murakami. Spontaneous motility, EEG and sleep-wakefulness cycle disturbance in prenatally X-irradiated rats. J. Physiol. Soc. Japan 37: 4 - 6 , 1975. 16. Lashley, K. S. The mechanisms of vision: XII. Nervous structures concerned in the acquisition and retention of habits based on reactions to light. Comp. Psychol. Monogr. 11: 4 2 - 7 9 , 1935. 17. Lindquist, E. F. Design and Analysis of Experiments in Psychology and Education. Boston: Houghton Mifflin, 1953. 18. Notterman, J. M. and A. H. Block. Note on response differentiation during a simple discrimination. J. exp. Analysis Behav. 3: 289-291, 1960. 19. Piontkovsky, I. A. Features specific to the functions of the higher divisions of the central nervous system in animals subjected to the effect of ionizing radiation at various periods of antenatal development. J. comp. physiol. Psychol. 54: 314-318, 1961. 20. Sharp, J. C. The effects of prenatal X-irradiation on acquisition, retention and extinction of a conditioned emotional response. Radiat. Res. 24: 154-157, 1965. 21. Shoji, R., I. K. Takeuchi, U. Murakami and J. Tsuge. Experimental studies on maldevelopment in the rat and hamster brain following maternal X-irradiation (Abstract). Teratology 10: 98, 1974. 22. Silveira, J. M. and D. P. Kimble. Brightness discrimination and reversal in hippocampally-lesioned rats. Physiol. Behav. 3: 6 2 5 - 6 3 0 , 1968. 23. Thompson, R. Retention of a brightness discrimination following neocortical damage in the rat. J. comp. physiol. Psychol. 53: 2 1 3 - 2 1 5 , 1960. 24. Thompson, R. Localization of the "visual memory system" in the white rat. J. comp. physiol. Psychol. Monogr. 6 9 : 1 - 2 9 , 1969. 25. Trauax, T. and R. Thompson. Role of the hippocampus in performance of easy and difficult visual discrimination tasks. J. comp. physiol. Psychol. 67: 228-234, 1969.