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Effect of cycloheximide administered to rats in early postnatal life: correlation of brain changes with behaviour in adulthood
200
Brain Research, 167 (1979)200-205 (t_~)Elsevier/North-Holland Biomedical Pre~s
Effect of cycloheximide administered to rats in early postnatal life:correlation of brain changes with behaviour in aduRhood
ALFRED PAVLIK and RICHARD JELiNEK* Institute of Physiology and Institute o]I Experimental Medicine, Czechoslovak Academy o~ Sciences. Prague ,rCzechoslovakia l
(Accepted January 4th, 1979)
Behavioural deviations have been observed after malnutrition, hypoxia, hormonal disturbances, drugs, irradiation, sensory deprivation, etc., provided the interventions coincided with the critical periods of brain development. One of the significant critical periods - - the brain growth spurt period 6 is in some species naturally exposed to the extrauterine environment providing a unique opportunity for studying the causes and mechanisms of brain maldevelopment. The morphogenetic processes 8 underlying the brain growth spurt period especially involve intensive proliferation and growth of individual brain cells 2,6 that are accompanied by a conspicuous net synthesis of macromolecules and their fast accumulation in the brainlL It may be expected that the interference with these biochemical processes will be reflected not only m impaired proliferation and growth but also in impaired cytodifferentiation resulting eventually in aberrant structural organization and abnormal function. In this study the relations between altered protein synthesis and structural and/or functional abnormalities were investigated in the developing rat brain. Male hooded rats of the Druckray strain were used. The date of birth was assigned as postnatal day 0. Size of the litter was standardized to 8 animals and littermates were divided randomly to experimental and control groups. On day 7 the experimental animals received 0.02 )~ cycloheximide (CHX) in saline subcutaneously in the suprapelvic region. The same volume of saline was administered to the littermate controls. At the time of injection the litter was weighed and the weighing repeated on days 14, 21, 35, 70, 150 and 300. The cerebrum, cerebellum and olfactory bulbs were dissected, weighed and frozen in liquid Nz. For DNA estimation the tissues were homogenized in 0.4 M perchloric acid and DNA separated from R N A by an alkaline hydrolysis 3. The DNA content was measured by the diphenylamine method 4. Inhibition o f protein synthesis following CHX treatment was estimated by means of incorporation of L-(U-14C)leucine. Having received the subcutaneous injection (200 #Ci/kg body weight) of * To whom correspondence should be addressed at: Institute of Experimental Medicine, Legerova 61,120 00 Prague 2, Czechoslovakia.
201 labelled leucine which followed 30 min, 210 min and 15.5 h after the injection of CHX, the animals were decapitated 30 min later. One half of the cerebrum was taken for the determination of acid soluble radioactivity (ASR) and specific radioactivity of protein (SRP) as described earlier 13. The relative specific activity (RSA) of protein was expressed as SRP/ASR ratio. Open-field behaviour, consisting of measures on lines crossed, rearing on hind limbs and defecation in the novel environment, was tested at 3 and 10 months. The open-field apparatus was fashioned so that its circular field (50 cm in diameter) was divided into a central area and 8 peripheral circle cuts with the same surface area. An animal was placed into the central circle and activity counts were recorded in a well-lit apparatus during a 2 min session. The animals were subjected to 5 consecutive sessions in the course of a week (one session a day). Conditioned avoidance behaviour was assayed by the shuttle-box procedurO t with the following modifications: a buzzer was presented for 10 sec before the electric shocks (90 V, 1.3 mA, AC) were applied to grid floor as the conditioned stimulus. Two electric shocks were delivered each second for a maximum of 10 sec if the rat had not yet escaped into the opposite compartment. Independently of the duration of individual trials, 30 sec intertrial intervals were maintained between two consecutive trials. The rats were subjected to 50 trials in a session and the conditioned avoidance reactions (CAR) were recorded. The percentage of CAR during the whole 50-trial session was calculated and the number of trials to criterion 3/4 (i.e., three CAR out of four consecutive trials) and 6/7 was determined. Data were statistically treated at the Prague Centre of Biomathematics using BMDP-Program Series made up at U.C.L.A., U.S.A. (BMDP 7D, 2R, 2V and 3S). A single systemic injection of CHX (0.6 mg/kg body weight) to 7-day-old rats transiently retarded the body growth with maximum to be seen in 14-day-old rats (reduction of body weight to 65 ~ , P < 0.01) and the catch-up recorded 9 weeks postinjection. Since higher doses of CHX were already toxic, this dose was chosen as effective sublethal treatment inducing short-term inhibition of protein synthesis at the time of maximum brain growth spurt. Following CHX treatment the feeding of CHXtreated rats was apparently not changed but it cannot be excluded that they were relatively undernourished for some time. The inhibition of gastrointestinal functions by comparable doses of CHX has been described 9 and, indeed, diarrhoea was frequently seen in CHX-treated rats. In order to estimate the intensity and duration of CHX action, the inhibition of labelled leucine incorporation into brain protein was determined 1, 4 and 16 h after the injection of CHX. A dose-dependent inhibition of incorporation caused by a single injection of CHX was found and the dose 0.6 mg/kg body weight of CHX reduced SRP by about two-thirds both 1 and 4 h after the injection. Then protein synthesis was disinhibited and the reattainment of normal values of SRP occurred 16 h after the injection. As the inhibition of incorporation was accompanied by an elevated ASR, the calculated RSA of brain protein indicated that the rate of cerebral protein synthesis may have been even more inhibited than stated above. However, the increase of ASR was associated with a concomitant rise of endogeneous free leucine concen-
202 tration in the cerebral tissue which was similar in magnitude to that of radioactivit~ (unpublished results). The measured SRP was, therefore, accepted as a closer representation of the actual inhibition of protein synthesis than the calculated RSA value. Effect of CHX on the weight of the cerebrum, cerebellum and olfactory bulbs was determined in 7-, 14-, 21- and 35-day-old rats. Analysis of variance with two-way classification (experimental or control vs age) of log-transformed data was used ~2 CHX depressed their growth curves (S.E. of log-transformed data were: 0.009. 0.014 and 0.023, respectively, 'treatment': P < 0.01). The cerebral weight was decreased significantly only in 14-day-old rats (to 88 ~o, P < 0.05), the weight of the cerebellum and the olfactory bulbs was still reduced in 35-day-old rats (to 87~',~ and 82",, respectively, P < 0.05). Some parameters of body and brain growth were also determined in 5-month-old rats. Apparent atrophy of the olfactory bulbs (to 67'~,, P 0.02) predominated over the small reduction of body and brain (to 93'!,,, non-significant, and 95 ~ , P < 0.05, respectively). This finding initiated a deeper analysis of the changes in the olfactory bulbs. The determination of DNA content of the olfactory bulbs performed on days 7. 14, 2 l and 35 showed a sharp rise of D N A content in the controls which levelled offby the fifth week. CHX treatment reduced the D N A content indicating that the acquisition of the cells had been retarded (S.E. of log-transformed data was 0.0t8, 'treatment': P < 0.01). The olfactory bulbs of CHX-treated 5-month-old rats only slightly diminished the DNA content (to 90~o, non-significant). This resulted in an increased packing density (DNA concentration) of their cells (up to 137'~.,i, P "< 0.01 }. Since the renewal of olfactory bulb microneurons and neuroglial cells is a life-long process in the rat t,z, the acquisition of nearly-to-normal cell number at this age might indicate that compensatory proliferation had taken place in the meantime. Increased packing density supports the possibility that the complicated spatiotemporal pattern of morphogenetic processes s has been altered in the olfactory bulbs of CHX-treated rats. Open-field behavior was tested in 3- and 10-month-old rats. CHX-treated rats were hyperactive in both horizontal (crossing) and vertical (rearing) components of exploratory activity by the end of the third month (analysis of variance of repeated measures, F 345.83 and 313.30, respectively, P < 0.01). Inthis group the differences between CHX-treated and control rats varied from 0 to 35~,, and from 12 to 36",, respectively, daily. The hyperactivity seemed to increase with age as the differences were augmented from 22 to 99~,,, (crossing) and from 18 to 109% (rearing) in 10month-old rats (Fig. 1). Such hyperactivity corresponded well to that of rats which were exposed to carbon monoxide on day 5 and which persisted until adulthood in males only 1°. The postnatal administration of corticoids which had been shown to induce severe atrophy of the olfactory bulbs in the rat ~ was accompanied by hyperactivity and impaired conditioned avoidance learning 7. The aberrant signalization from maldeveloped olfactory bulbs may be an important factor as the exploratory or primitive search behaviour is a function of olfaction used for the detection of food. The rate of defecation was not found to be influenced by CHX treatment. Conditioned avoidance behaviour was tested by the shuttle-box procedure in 5-
203
Activity
omCROSSING
Oe REARING
•~ •
DEFECATION
units
30
20
10
tt
i
! st
2 nd
3 rd
4th
sth
session
Fig. I. Effect o f postnatal a d m i n i s t r a t i o n o f C H X on the exploratory behaviour in adulthood. A g r o u p of 28 rats was tested at the age o f 10 m o n t h s . The data were analyzed by a n o v a r o f repeated measures. T h e curves for C H X - t r e a t e d (full symbols) a n d control (open symbols) rats were different in crossing a n d rearing ( F = 346.81 a n d 258.20, respectively, P < 0.01). T h e arrows delimit the interval when differences between the m e a n values were significant (P < 0.01).
month-old rats using two measures of CAR acquisition. The percentage of CAR as well as the number of trials to criteria 3/4 and 6/7 were altered insignificantly (to 79 %, 116~/,, and 106~, non-significant, respectively, Mann-Whitney U-test). Following behavioural testing, the rats were sacrificed and the body, brain and olfactory bulbs were weighed and DNA content of the olfactory bulbs was determined. Linear correlation and regression analysis was performed between these data and those of conditioned avoidance. Close correlation was found between the weight of the olfactory bulbs and criterion 3/4 (r -- -0.6939, P < 0.01), and between their DNA content and this criterion (r ~ -0.5481, P < 0.05). Similar correlation was seen if the olfactory bulbs' weight was correlated with the percentage of CAR (r ~-- 0.6137, P < 0.05). It was of interest that the correlations between body and/or brain weight and any of the two measures of avoidance learning were not statistically significant. Step-wise regression analysis indicated that there is a very close relationship between the weight of the olfactory bulbs and the speed of CAR acquisition (criterion 3/4). In the scatter diagram (Fig. 2) such a relationship may be assumed by observing the data of CHX-treated rats but it is strengthened by adding the data of control
204
"/°I o •
CklX
•
•
SO •
qO~
•
•
•
0
0
30 20
10
0
10
20
30
40
50
60
70
80
90
100 m 9
Fig. 2. Relationship between the weight of the olfactory bulbs (independent variable) and the speed of conditioned avoidance acquisition-criterion 3/4:ATC (dependent variable) of both exleerimental and control rats at the age of 5 months. The anovar of the regression (ATC ~ 58.424-0.333 × weight of the olfactory bulbs) revealed that the null hypothesis (fl ~ O) may be rejected (P ~ 0.01).
rats. T h e association between the a t r o p h y o f the o l f a c t o r y bulbs and the deficit o f c o n d i t i o n e d a v o i d a n c e learning implies t h a t the sensitivity o f t h i s p a l e o c o r t i c a l s t r u c t u r e to s h o r t - t e r m i n h i b i t i o n o f p r o t e i n synthesis is similar to the susceptibility o f the processes which underlie the d e v e l o p m e n t o f n o r m a l learning capabilities, at least in the rat. The results o f this study show the i n h i b i t i o n o f p r o t e i n synthesis in b r a i n g r o w t h s p u r t period b r o u g h t a b o u t p e r m a n e n t changes o f the susceptible structures a n d functions. W e wish to t h a n k to Dr. T. Havr~inek for the statistical analysis and to Miss I r e n a Slavikov~i for the skilled technical assistance.
1 Altman, J.. Autoradiographic and histological studies of postnatal neurogenesis. IV. Cell proliferation and migration in the anterior forebrain with special reference to persisting neurogenesis in the olfactory bulb, J. comp. NeuroL, 137 (1969) 433--458. 2 Altman, J., Postnatal neurogenesis and the problem of neural plasticity. In W. A. Himwich (Ed.), Developmental Neurobiology, Ch. C. Thomas, Springfield, 1970, pp. 197-237. 3 Bal~lzs,R., Kov~ics, S., Cocks, W. A., Johnson, A. L. and Eayrs, J. T., Effect of thyroid hormone on the biochemical maturation of rat brain: postnatal cell formation, Brain Research, 25 (1971) 555-570. 4 Burton, K., A study of conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid, Biochem. J , 62 (1956) 315-323. 5 Cotterrell, M., Balfizs, R. and Johnson, A. L., Effects of corticosteroids on the biochemical maturation of rat brain: postnatal cell formation, J. Neurochem., 19 (1972) 2151-2 t 67.
205 6 Dobbing, J., Effect of experimental undernutrition on development of the nervous system. In N. S. Scrimshaw and J. E. Gordon (Eds.), Malnutrition, Learning and Behavior, MIT Press, Boston, 1968, pp. 181-202. 7 Howard, E. and Benjamins, J. A., DNA, ganglioside and sulfatide in brains of rats given corticosterone in infancy, with an estimate of cell loss during development, Brain Research, 92 (1975) 73-87. 8 Jelinek, R. and Rychter, Z., Morphogenetic systems and the central phenomena of teratology. In T. V. N. Persaud (Ed.), Progress in the Study of Birth Defects, MTP Press, Lancaster, 1979, in press. 9 Korbov~i, L., Kohout, J., (~if~kovfi,J. and (~ih~ik, A., Inhibitory effect of cycloheximide on gastric secretion in rats, Biochem. Pharmacol., 26 (1977) 979-981. 10 Norton, S., Mullenix, P. and Culver, B., Comparison of the structure of hyperactive behavior in rats after brain damage from X-irradiated, carbon monoxide and pallidal lesions, Brain Research, 116 (1976) 49-67. 11 Nyakas, C., van Delft, A. M. L., Kaplanski, J. and Smelik, P. G., Exploratory activity and conditioned avoidance acquisition after early postnatal 6-OHDA administration, J. neural Transm., 34 (1973) 253-266. 12 Patel, A. J., Rabi6, A., Lewis, P. D. and Bal~izs, R., Effect of thyroid deficiency on postnatal cell formation in the rat brain: a biochemical investigation, Brain Research, 104 (1976) 33-48. 13 Pavlik, A. and Jakoubek, B., Developmental changes of protein-bound radioactivity distribution in rat brain slices incubated with labelled leucine, Brain Research, 154 (1978) 95-104.
Brain Research, 167 (1979)200-205 (t_~)Elsevier/North-Holland Biomedical Pre~s
Effect of cycloheximide administered to rats in early postnatal life:correlation of brain changes with behaviour in aduRhood
ALFRED PAVLIK and RICHARD JELiNEK* Institute of Physiology and Institute o]I Experimental Medicine, Czechoslovak Academy o~ Sciences. Prague ,rCzechoslovakia l
(Accepted January 4th, 1979)
Behavioural deviations have been observed after malnutrition, hypoxia, hormonal disturbances, drugs, irradiation, sensory deprivation, etc., provided the interventions coincided with the critical periods of brain development. One of the significant critical periods - - the brain growth spurt period 6 is in some species naturally exposed to the extrauterine environment providing a unique opportunity for studying the causes and mechanisms of brain maldevelopment. The morphogenetic processes 8 underlying the brain growth spurt period especially involve intensive proliferation and growth of individual brain cells 2,6 that are accompanied by a conspicuous net synthesis of macromolecules and their fast accumulation in the brainlL It may be expected that the interference with these biochemical processes will be reflected not only m impaired proliferation and growth but also in impaired cytodifferentiation resulting eventually in aberrant structural organization and abnormal function. In this study the relations between altered protein synthesis and structural and/or functional abnormalities were investigated in the developing rat brain. Male hooded rats of the Druckray strain were used. The date of birth was assigned as postnatal day 0. Size of the litter was standardized to 8 animals and littermates were divided randomly to experimental and control groups. On day 7 the experimental animals received 0.02 )~ cycloheximide (CHX) in saline subcutaneously in the suprapelvic region. The same volume of saline was administered to the littermate controls. At the time of injection the litter was weighed and the weighing repeated on days 14, 21, 35, 70, 150 and 300. The cerebrum, cerebellum and olfactory bulbs were dissected, weighed and frozen in liquid Nz. For DNA estimation the tissues were homogenized in 0.4 M perchloric acid and DNA separated from R N A by an alkaline hydrolysis 3. The DNA content was measured by the diphenylamine method 4. Inhibition o f protein synthesis following CHX treatment was estimated by means of incorporation of L-(U-14C)leucine. Having received the subcutaneous injection (200 #Ci/kg body weight) of * To whom correspondence should be addressed at: Institute of Experimental Medicine, Legerova 61,120 00 Prague 2, Czechoslovakia.
201 labelled leucine which followed 30 min, 210 min and 15.5 h after the injection of CHX, the animals were decapitated 30 min later. One half of the cerebrum was taken for the determination of acid soluble radioactivity (ASR) and specific radioactivity of protein (SRP) as described earlier 13. The relative specific activity (RSA) of protein was expressed as SRP/ASR ratio. Open-field behaviour, consisting of measures on lines crossed, rearing on hind limbs and defecation in the novel environment, was tested at 3 and 10 months. The open-field apparatus was fashioned so that its circular field (50 cm in diameter) was divided into a central area and 8 peripheral circle cuts with the same surface area. An animal was placed into the central circle and activity counts were recorded in a well-lit apparatus during a 2 min session. The animals were subjected to 5 consecutive sessions in the course of a week (one session a day). Conditioned avoidance behaviour was assayed by the shuttle-box procedurO t with the following modifications: a buzzer was presented for 10 sec before the electric shocks (90 V, 1.3 mA, AC) were applied to grid floor as the conditioned stimulus. Two electric shocks were delivered each second for a maximum of 10 sec if the rat had not yet escaped into the opposite compartment. Independently of the duration of individual trials, 30 sec intertrial intervals were maintained between two consecutive trials. The rats were subjected to 50 trials in a session and the conditioned avoidance reactions (CAR) were recorded. The percentage of CAR during the whole 50-trial session was calculated and the number of trials to criterion 3/4 (i.e., three CAR out of four consecutive trials) and 6/7 was determined. Data were statistically treated at the Prague Centre of Biomathematics using BMDP-Program Series made up at U.C.L.A., U.S.A. (BMDP 7D, 2R, 2V and 3S). A single systemic injection of CHX (0.6 mg/kg body weight) to 7-day-old rats transiently retarded the body growth with maximum to be seen in 14-day-old rats (reduction of body weight to 65 ~ , P < 0.01) and the catch-up recorded 9 weeks postinjection. Since higher doses of CHX were already toxic, this dose was chosen as effective sublethal treatment inducing short-term inhibition of protein synthesis at the time of maximum brain growth spurt. Following CHX treatment the feeding of CHXtreated rats was apparently not changed but it cannot be excluded that they were relatively undernourished for some time. The inhibition of gastrointestinal functions by comparable doses of CHX has been described 9 and, indeed, diarrhoea was frequently seen in CHX-treated rats. In order to estimate the intensity and duration of CHX action, the inhibition of labelled leucine incorporation into brain protein was determined 1, 4 and 16 h after the injection of CHX. A dose-dependent inhibition of incorporation caused by a single injection of CHX was found and the dose 0.6 mg/kg body weight of CHX reduced SRP by about two-thirds both 1 and 4 h after the injection. Then protein synthesis was disinhibited and the reattainment of normal values of SRP occurred 16 h after the injection. As the inhibition of incorporation was accompanied by an elevated ASR, the calculated RSA of brain protein indicated that the rate of cerebral protein synthesis may have been even more inhibited than stated above. However, the increase of ASR was associated with a concomitant rise of endogeneous free leucine concen-
202 tration in the cerebral tissue which was similar in magnitude to that of radioactivit~ (unpublished results). The measured SRP was, therefore, accepted as a closer representation of the actual inhibition of protein synthesis than the calculated RSA value. Effect of CHX on the weight of the cerebrum, cerebellum and olfactory bulbs was determined in 7-, 14-, 21- and 35-day-old rats. Analysis of variance with two-way classification (experimental or control vs age) of log-transformed data was used ~2 CHX depressed their growth curves (S.E. of log-transformed data were: 0.009. 0.014 and 0.023, respectively, 'treatment': P < 0.01). The cerebral weight was decreased significantly only in 14-day-old rats (to 88 ~o, P < 0.05), the weight of the cerebellum and the olfactory bulbs was still reduced in 35-day-old rats (to 87~',~ and 82",, respectively, P < 0.05). Some parameters of body and brain growth were also determined in 5-month-old rats. Apparent atrophy of the olfactory bulbs (to 67'~,, P 0.02) predominated over the small reduction of body and brain (to 93'!,,, non-significant, and 95 ~ , P < 0.05, respectively). This finding initiated a deeper analysis of the changes in the olfactory bulbs. The determination of DNA content of the olfactory bulbs performed on days 7. 14, 2 l and 35 showed a sharp rise of D N A content in the controls which levelled offby the fifth week. CHX treatment reduced the D N A content indicating that the acquisition of the cells had been retarded (S.E. of log-transformed data was 0.0t8, 'treatment': P < 0.01). The olfactory bulbs of CHX-treated 5-month-old rats only slightly diminished the DNA content (to 90~o, non-significant). This resulted in an increased packing density (DNA concentration) of their cells (up to 137'~.,i, P "< 0.01 }. Since the renewal of olfactory bulb microneurons and neuroglial cells is a life-long process in the rat t,z, the acquisition of nearly-to-normal cell number at this age might indicate that compensatory proliferation had taken place in the meantime. Increased packing density supports the possibility that the complicated spatiotemporal pattern of morphogenetic processes s has been altered in the olfactory bulbs of CHX-treated rats. Open-field behavior was tested in 3- and 10-month-old rats. CHX-treated rats were hyperactive in both horizontal (crossing) and vertical (rearing) components of exploratory activity by the end of the third month (analysis of variance of repeated measures, F 345.83 and 313.30, respectively, P < 0.01). Inthis group the differences between CHX-treated and control rats varied from 0 to 35~,, and from 12 to 36",, respectively, daily. The hyperactivity seemed to increase with age as the differences were augmented from 22 to 99~,,, (crossing) and from 18 to 109% (rearing) in 10month-old rats (Fig. 1). Such hyperactivity corresponded well to that of rats which were exposed to carbon monoxide on day 5 and which persisted until adulthood in males only 1°. The postnatal administration of corticoids which had been shown to induce severe atrophy of the olfactory bulbs in the rat ~ was accompanied by hyperactivity and impaired conditioned avoidance learning 7. The aberrant signalization from maldeveloped olfactory bulbs may be an important factor as the exploratory or primitive search behaviour is a function of olfaction used for the detection of food. The rate of defecation was not found to be influenced by CHX treatment. Conditioned avoidance behaviour was tested by the shuttle-box procedure in 5-
203
Activity
omCROSSING
Oe REARING
•~ •
DEFECATION
units
30
20
10
tt
i
! st
2 nd
3 rd
4th
sth
session
Fig. I. Effect o f postnatal a d m i n i s t r a t i o n o f C H X on the exploratory behaviour in adulthood. A g r o u p of 28 rats was tested at the age o f 10 m o n t h s . The data were analyzed by a n o v a r o f repeated measures. T h e curves for C H X - t r e a t e d (full symbols) a n d control (open symbols) rats were different in crossing a n d rearing ( F = 346.81 a n d 258.20, respectively, P < 0.01). T h e arrows delimit the interval when differences between the m e a n values were significant (P < 0.01).
month-old rats using two measures of CAR acquisition. The percentage of CAR as well as the number of trials to criteria 3/4 and 6/7 were altered insignificantly (to 79 %, 116~/,, and 106~, non-significant, respectively, Mann-Whitney U-test). Following behavioural testing, the rats were sacrificed and the body, brain and olfactory bulbs were weighed and DNA content of the olfactory bulbs was determined. Linear correlation and regression analysis was performed between these data and those of conditioned avoidance. Close correlation was found between the weight of the olfactory bulbs and criterion 3/4 (r -- -0.6939, P < 0.01), and between their DNA content and this criterion (r ~ -0.5481, P < 0.05). Similar correlation was seen if the olfactory bulbs' weight was correlated with the percentage of CAR (r ~-- 0.6137, P < 0.05). It was of interest that the correlations between body and/or brain weight and any of the two measures of avoidance learning were not statistically significant. Step-wise regression analysis indicated that there is a very close relationship between the weight of the olfactory bulbs and the speed of CAR acquisition (criterion 3/4). In the scatter diagram (Fig. 2) such a relationship may be assumed by observing the data of CHX-treated rats but it is strengthened by adding the data of control
204
"/°I o •
CklX
•
•
SO •
qO~
•
•
•
0
0
30 20
10
0
10
20
30
40
50
60
70
80
90
100 m 9
Fig. 2. Relationship between the weight of the olfactory bulbs (independent variable) and the speed of conditioned avoidance acquisition-criterion 3/4:ATC (dependent variable) of both exleerimental and control rats at the age of 5 months. The anovar of the regression (ATC ~ 58.424-0.333 × weight of the olfactory bulbs) revealed that the null hypothesis (fl ~ O) may be rejected (P ~ 0.01).
rats. T h e association between the a t r o p h y o f the o l f a c t o r y bulbs and the deficit o f c o n d i t i o n e d a v o i d a n c e learning implies t h a t the sensitivity o f t h i s p a l e o c o r t i c a l s t r u c t u r e to s h o r t - t e r m i n h i b i t i o n o f p r o t e i n synthesis is similar to the susceptibility o f the processes which underlie the d e v e l o p m e n t o f n o r m a l learning capabilities, at least in the rat. The results o f this study show the i n h i b i t i o n o f p r o t e i n synthesis in b r a i n g r o w t h s p u r t period b r o u g h t a b o u t p e r m a n e n t changes o f the susceptible structures a n d functions. W e wish to t h a n k to Dr. T. Havr~inek for the statistical analysis and to Miss I r e n a Slavikov~i for the skilled technical assistance.
1 Altman, J.. Autoradiographic and histological studies of postnatal neurogenesis. IV. Cell proliferation and migration in the anterior forebrain with special reference to persisting neurogenesis in the olfactory bulb, J. comp. NeuroL, 137 (1969) 433--458. 2 Altman, J., Postnatal neurogenesis and the problem of neural plasticity. In W. A. Himwich (Ed.), Developmental Neurobiology, Ch. C. Thomas, Springfield, 1970, pp. 197-237. 3 Bal~lzs,R., Kov~ics, S., Cocks, W. A., Johnson, A. L. and Eayrs, J. T., Effect of thyroid hormone on the biochemical maturation of rat brain: postnatal cell formation, Brain Research, 25 (1971) 555-570. 4 Burton, K., A study of conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid, Biochem. J , 62 (1956) 315-323. 5 Cotterrell, M., Balfizs, R. and Johnson, A. L., Effects of corticosteroids on the biochemical maturation of rat brain: postnatal cell formation, J. Neurochem., 19 (1972) 2151-2 t 67.
205 6 Dobbing, J., Effect of experimental undernutrition on development of the nervous system. In N. S. Scrimshaw and J. E. Gordon (Eds.), Malnutrition, Learning and Behavior, MIT Press, Boston, 1968, pp. 181-202. 7 Howard, E. and Benjamins, J. A., DNA, ganglioside and sulfatide in brains of rats given corticosterone in infancy, with an estimate of cell loss during development, Brain Research, 92 (1975) 73-87. 8 Jelinek, R. and Rychter, Z., Morphogenetic systems and the central phenomena of teratology. In T. V. N. Persaud (Ed.), Progress in the Study of Birth Defects, MTP Press, Lancaster, 1979, in press. 9 Korbov~i, L., Kohout, J., (~if~kovfi,J. and (~ih~ik, A., Inhibitory effect of cycloheximide on gastric secretion in rats, Biochem. Pharmacol., 26 (1977) 979-981. 10 Norton, S., Mullenix, P. and Culver, B., Comparison of the structure of hyperactive behavior in rats after brain damage from X-irradiated, carbon monoxide and pallidal lesions, Brain Research, 116 (1976) 49-67. 11 Nyakas, C., van Delft, A. M. L., Kaplanski, J. and Smelik, P. G., Exploratory activity and conditioned avoidance acquisition after early postnatal 6-OHDA administration, J. neural Transm., 34 (1973) 253-266. 12 Patel, A. J., Rabi6, A., Lewis, P. D. and Bal~izs, R., Effect of thyroid deficiency on postnatal cell formation in the rat brain: a biochemical investigation, Brain Research, 104 (1976) 33-48. 13 Pavlik, A. and Jakoubek, B., Developmental changes of protein-bound radioactivity distribution in rat brain slices incubated with labelled leucine, Brain Research, 154 (1978) 95-104.