Behavioral changes in preweaning and adult rats exposed prenatally to low lonizing radiation

TOXICOLOGY

AND

APPLIED

Behavioral

83, 240-249

PHARMA(‘OLCK;Y

( 1986)

Changes in Preweaning and Adult Rats Exposed Prenatally to Low Ionizing Radiation STATANORTON

Department

yf‘Pharmaco1og.v;. To.uicologJ, and Therapeutxs and Ralph L. Smith Mental Retardalion Cenfer. University cf Kansas Medical Center, Kansas City. Kansas 66103

Received

Augusi

I Y. I YKS.- accepled

November

Re.vearclr

19. I YX5

Behavioral Changes in Preweaning and Adult Rats Exposed Prenatally to Low Ionizing Radiation. NORTON, S. (1986). Tolicol. Appl. Pharmacol. 83,240-249. Seven behavioral tests were used to evaluate the postnatal behavior of rats after exposure on gestational Day 15 to 0, 25. 50, 75. or 125 r, whole body irradiation of the pregnant rat. Three tests were administered in the first 2 postnatal weeks (righting reflex, negative geotaxis. and reflex suspension): three tests were administered on postnatal Day 21 (modified open field. spatial maze, and continuous corridor). As adults, the rats were retested with the same tests as at 21 days and also in the running wheel. Dose-response decreases in body weight were greater in the younger rats. Some behavioral tests were not altered by irradiation, while others showed clear dose-response relationships, starting as low as 25 r. The early changes were characterized by light body weight, delays in behavioral development and hypoactivity. followed by recovery of some parameters with maturation. Eventually hyperactivity developed in adult rats after gestational irradiation. However, it cannot be concluded that either morphological or behavioral tests are more sensitive than neonatal body weight change for detection of damage from gestational irradiation. G> 1986 Academic press, hc.

The doses of ionizing radiation which are lethal to dividing cells have been identified in detail. It is axomatic that doses which damage a percentage of dividing cells may be more deleterious to an embryo or fetus than to the maternal organs. Doses of ionizing radiation below 200 r cause minimal deleterious effects to adult rats (Sipila, 1960) whereas 200 r may cause marked toxicity to the fetus, depending on the time of irradiation (Hicks and D’Amato, 1966). The effects of ionizing radiation on dividing cells are complex. Chromosomal damage may result in cell death but hypoploidic cells may continue to divide. Micronuclei formed from acentric chromosomal fragments may be found in embryos several days after X-ray doses as low as 60 r (Molls et al., 1982). In spite of extensive investigation of some of the anatomical consequencesto the CNS of fetal exposure to dosesof ionizing radiation 004 1-008X/86

$3.00

Copyright ic“ 1986 by Academic Press. Inc All rights of reproductmn in any form reserved.

240

below 200 r, behavioral effects have been less systematically examined. In addition, some studies which have included dose-effect data have not demonstrated dose-related changes. Using a small number of rats irradiated on Days 13, 15, 17, or 19 with 200 r, Hicks and co-workers found no consistent difficulty in the performance of adult rats on a fixed ratio scheduleand concluded that rats with bizarre visual cortices could make the same visual pattern discrimination as control rats (Falk and D’Amato, 1962: Hicks et al., 1962). In a study on 3- to 4-month-old rats, irradiated with 150 r on the 14th gestational day, the irradiated rats showed lessalternation in 12 test patterns in a maze and poorer visual discrimination in a operant reward test (Fowler et al., 1962). Furchtgott and Echols ( 1958) and Furchtgott and co-workers (1958) studied rats after irradiation with 50, 100, 200, or 300 r on ges-

BEHAVIOR

AFTER

tational Days 14 to 18. They concluded that maze learning in adults was maximally affected by irradiation on gestational Days 14 to 15. Variability in response to irradiation was noted. When tested shortly after weaning, 50 r rats were hypoactive in the open field while 300 r rats were hyperactive and 100 and 200 r rats were like controls. The most consistent finding for rats irradiated on gestational Days 14 to 15 was a delay in home cage emergence, tested starting on postnatal Day 3 1. All doses, 50, 100, 200, and 300 r, showed this effect. The two tests. open field activity and home cage emergence, presumably measured different behavioral attributes, since control rats showed no correlation between responses on the two tests (Furchtgott and Echols, 1958). Very few studies have assessed behavior from birth to weaning. A study in many ways comparable to the present experiments has been carried out in the squirrel monkey. Ordy and co-workers (1982) have reported the development of 15 squirrel monkeys exposed to 0, 50, or 100 r (‘j*Co source) between 80 and 90 days of the approximately 150 days of gestation (7 controls, 3 receiving 50 r and 5 receiving 100 r were studied). Body righting was delayed at 2, 14, and 28 days in 100 r offspring. These monkeys also took longer to orient head-up (at 14 days) and longer to climb a 45’ incline (at 14 and 28 days). Visual discrimination in a Y-maze was lower in 100 r offspring, tested between 30 and 90 days. At 90 days plasma cortisol values were higher in 100 r monkeys than in controls. At birth the 50 and 100 r infants were smaller than controls. but at 3 months body weight was not different from controls. These investigations resulted in the suggestion that the hypothalamic-pituitary axis may represent a vulnerable point for the deficits observed in the study (Ordy et al., 1982). The reasons for failure to detect dose-related effects of ionizing radiation on behavior are not obvious. There may be a qualitative change in behavior with increasing degrees of CNS damage, instead of a quantitative change. Furthermore, the developing CNS has a po-

FETAL

241

IRRADIATION

tential for functional compensation in the presence of small amounts of damage. These considerations suggest that not all behavioral tests are equally likely to predict CNS damage in a dose-related study. The reliability of many behavioral tests for prediction of types CNS damage is unknown. Therefore a reasonable approach is to utilize a series of tests which may evaluate more than one aspect of CNS damage from gestational exposure to ionizing radiation and which cover a long period of postnatal development. Seven behavioral tests, beginning with neonates, were used in the following experiments. All of these tests monitor integrated motor function in one way or another, but the degree to which these tests also monitor more complex interpretations of the test environments is uncertain. Gestational Day 15 was chosen for irradiation since this time has been reported to be in the period when behavior is altered. It is past the time of major organogenesis and is in the period of development of the neocortex and other forebrain structures (Hicks, 1953) and diencephalic hypothalamic and preoptic nuclei (Altman and Bayer, 1978). METHODS Over a period of 4 months, 37 Charles River SpragueDawley derived (CD strain) female rats, weighing 175 to 200 g, were mated to male rats of the same strain. Gestational Day I was the day on which sperm were observed in the vagina. Rats were checked every morning by vaginal wash. On gestational Day 15 (between 10:00 AM and 12:OO noon), pregnant rats were exposed to ionizing radiation from a GE Maximar X-ray machine. Exposure parameters were: 15 mA 250 KV; filters of 0.25 mm Ca and 1 mm Al; delivered at about 40 r/min; distance to uterus approximately 50 cm: rats rotated at 3 rpm during exposure. Doses were 0, 25. 50. 75. or 125 r. Control rats were handled in the same way and were placed on the rotating platform in the plexiglass exposure box but with no radiation. Selection of rats for exposure conditions was randomized over the period of mating. Pregnant rats were maintained in plastic rat cages on crushed corn cob bedding with Purina rat chow and water ad libitum in a temperature controlled room (about 22°C) with 12/ 12 hr light/dark cycle starting at 6:OO AM. Within 24 hr after parturition, litters were reduced to 8 rat pups. as nearly as possible evenly divided between the sexes. In

242

STATA

both control and irradiated litters, the larger pups of each sex were retained. Of 37 litters, 2 contained less than 8 pups (one 125 r and one 75 r) and these litters were not supplemented. All pups were weaned on postnatal Day 22. Starting with postnatal Day 2. rats were weighed daily for the first 10 days and then on Days 14. 2 I, 28. and at 4 months of age. Seven behavioral tests were employed at different periods of postnatal development as outlined below, All tests were carried out between 8:00 AM and 1 1:oo AM.

Behavior Tcm Three tests were employed in the very young rats prior to eye opening. These were surface righting reflex, negative geotaxis. and reflex suspension, performed in that sequence on days where testing overlapped. Three tests were used in 2 l-day-old rats. prior to weaning and after normal locomotion had developed. These were the continuous corridor, spatial maze, and modified openfield performed in that sequence. These tests were repeated in adult rats. In addition, adults were tested in the activity wheel at about 5 months of age. Testing was randomized among litters and the observer did not know the prenatal exposure condition of the animals during the testing, Righting re/le.~ From postnatal Day 2 to Day 10. rats were observed for speed with which they righted themselves when placed on their backs on a solid surface. The rats were timed to the nearest second with a stopwatch. when all 4 feet were in contact with the surface. The test was terminated at 60 set if the rat failed to turn. Negative grota.ris. On postnatal Days 5 through Y the rats were placed head down on a surface covered with screen at an angle of 25” from horizontal. At this age rats will attempt to turn 180” and orient with the head upward. The motor ability required to complete this turn develops in control rats during the second postnatal week and the time required to turn decreases. A maximum time of 2 min was allowed for each rat. R&Y .sn.sper~ror~. The grasp reflex when the forepaws are placed in contact with a 3 mm bar develops about the end of the first postnatal week. The length of time (up to 60 set) that the rats could suspend themselves from the bar was tested from Days 9 through 15. Modified open,held actbit): When the rats reached 2 I days of age, they were placed singly in a small open-field (40 X 40 cm) with 2.5-cm holes symmetrically placed Y cm apart in the floor. This apparatus was described by Makanjuola and co-workers (1977). The activity of the rat was recorded as the number of times the rat dipped its head into one of the holes in a 5-min period. This test was repeated when the rats were about 5 months old. Sputial maze. In a novel environment consisting of blind corridors from a central area, rats may enter part way along the corridors (partial entry) and return or may continue to the end of the corridor (complete entry). Use of

NORTON a 6-corridor maze with a central starting area has been described (Comer and Norton. 1985). Rats were tested singly for 3 min at 21 days of age and again as adults at 5 months of age. Continuous corridor. Because previous experience with the spatial maze showed that rats preferred corridors to open spaces and, following the research by Olton (1977) on spatial exploration of complex corridors by rats, a different corridor design was developed which allowed rats to explore back and forth continuously. reaching an end only in the center of the maze or at the starting point (Fig. I ). The total length of continuous corridor in one direction was about 5 m. The cross-sectional dimension of the corridor ( IO X IO cm) was based on the size of corridors dug by wild Norway rats (Norton et al., 1975b). It was predicted that the long corridor might discriminate between control exploratory behavior and the expected delay in exploration of irradiated rats, noted as delay in home cage emergence by Furchtgott and Echols (1958). For recording activity, each successive right-angle bend in the corridor was considered to delineate a separate “area.” dividing the corridor into 1 I areas. Movement in the corridor was scored as number of areas entered in 5 or IO min. The apparatus was covered with a clear plastic top. Statistical evuhration. Analyses of variance between groups, litters, and rats were performed on most of the behavioral data with post hoc tests for sources of difference when treatment groups were significantly different (p G 0.05). All rats from the litters were tested on each test

FIG. I. Diagram of the continuous corridor apparatus. Outside dimensions 60 x 70 cm, corridor 10 x 10 cm. Arrow at lower left indicates starting point where rat is placed. In recording activity, successive entry into one of the straight portions of the corridor is given a score of one when a rat completes a tight angle turn to enter the next area of the corridor (a complete entry required that all feet be in the next area). Activity either away from or returning toward the starting point is recorded.

BEHAVIOR

AFTER

with litters randomized as much as possible. Numbers of rats tested under each condition varied because testing was done 6 days a week instead of 7. and because, at intervals, rats were removed randomly from each litter for morphological assessment of CNS damage which is reported separately (Norton and Donoso, 1985). No spontaneous deaths occurred in any litters.

FETAL

IRRADIATION

243

parametric tests could not be used. The difference between the number of control rats which took more than 60 set (23%) and the number of 75 r rats (74%) or 125 r rats (68%) on postnatal Day 2 was significant (x2 test, p < 0.05). The delay in surface righting at the highest dose was quite marked. About 40% of the rats still did not turn in 2 set by the 9th RESULTS postnatal day, at which time the number of rats reaching this criterion was much greater The number of offspring born to rats irrain all other groups (Table 3). diated on gestational Day 15 was not signifiNegative geotaxis. On postnatal Days 5 cantly different from control rats (Table 1). through 9, rats were tested for ability to turn However, body weight at birth was reduced in 180” from a head-down to a head-up position a dose-related way and the irradiated rats conon a screeninclined 25” from horizontal. Data tinued to be lighter in body weight up to were recorded for time taken up to 120 set weaning in litters receiving 125 r. The light and reported as average time and number birth weight of rats exposed to 50 or 75 r disachieving criterion (turning in 30 set or less) appeared by the second postnatal week (Table on each day (Table 4). Although rats receiving 2). Thus the postnatal growth rate of rats exwith 75 and 125 r were slower to turn than posed to these dosesduring gestation was as control rats, the variability was large. No sighigh or higher than the postnatal rate of growth nificant dose-related effect of irradiation was of control rats. Surface righting r&x. Shortly after birth, demonstrated with this test. Rclflex suspension. lrradiated rats, tested on rats are able to turn from their backs to lie postnatal Days 10 to 15 for ability to suspend with feet down, the position in which they themselves by their forepaws, had generally usually nurse. Although this reflex is present shorter times than control rats. The developin the neonate, the speedwith which it is permental delay in rats irradiated with 125 r was formed develops over the first 10 days after marked both in the number of secondseach birth until the rats turn over in lessthan 2 set rat was able to stay suspended and days to when placed on their backs. Although all irreach the point at which 50% of the rats susradiated rats eventually achieved this criterion pended themselvesfor 5 set or more (Table 5). of performance. there wasa dose-relateddelay C’ontinuolls corridor. At 2 1 days of age the in surface righting. Becausea large number of rats’ activity was recorded for 10 min in the rats in all groups did not right themselves continuous corridor. The rats showed a dosewithin 60 set in the first 4 postnatal days, related decreasein activity in the starting area (Table 6). The activity in the remainder of the TABLE I corridor was also decreasedin irradiated rats and significant reductions were present in rats NLJMBEROF~FFSPRINGFOLLOWING MATERNAL receiving 25, 50, or 125 r. The 125 r were reIRRADIATIONONGESTATIONAL DAY 15 tested at about 5 months of agefor 5 min each Number of Average pups/litter in the corridor and the slightly greater activity Treatment litters (range) of irradiated rats was not significantly different from controls at that age (Table 6). ApproxiI1 12.7 (8-18) Control mately equal numbers of male and female rats 25 r 6 11.8 (8-14) 50 r 6 11.8 (9-14) were studied. Their activity levels were not 75 r 7 12.1 (4-16) significantly different and were combined. 125 r I 12.6 (7-16) Sputial maze. At 2 1days of ageand at about

244

STATA

NORTON

TABLE BODY

WEIGHT

2

OF IRRADIATED

RATS

G + SE (number At%

Control

24 hr

25 r

6.23 IT 0.05

6.55 +

(88) 3 days

7.64 +

7 days

50 r 0.07

582

(63) 0.1 I

7.68 +

(63)

of rats)

0.1 I

t 0.05” (71)

7.12 +

(40)

75 r

125 r

5.36 +- 0.14” (72)

5.14 i

0.09”

6.46 t 0.22’ (38)

6.12 2 0.07” (55)

(48)

0.06”

(88)

13.28 f (63)

0.18

13.45

+ (48)

0.23

12.43

2 0.15” (48)

12.20 + 0.31” 150)

10.55

i 0.23” (55)

I4 days

27.57

+ (62)

0.37

28.19

+ (46)

0.50

26.71

+ 0.37 (47)

27.1 I k 0.60 (49)

23.50

‘+ 0.40” (54)

21 days

49.95

*

0.71

48.13

*

1.00

46.02

46.81

0.97

38.58

i- 0.7 I a (41)

k 2.39 (14)

65.54

+ 2.60” (16)

(50) 28 days

77.73

i

(38) I.96

78.13

(31) 4 months Male

535.8

Female

295.7

f 34.5

553.0

from control.

2.69

79.04

i 40.4

272.8

0.72

+

i 55.0

5 16.0

3.9

2 25.0

? 72.4

297.3

47 I .O + 46.9

394.0

k 3 I .7” (101

236.4

i 22.0” 18)

(6)

f 20.0

262.7

18)

+ 33.9 (IO)

t test. p < 0.05

TABLE SURFACE

3

RIGHTING

REFLEX Postnatal

Treatment

72.69

(61

(8)

k (39)

(9)

(8)

(10) a Different

i (12)

(13)

+ (37)

days

5

6

7

8

9

IO

Control xc k SE % at criterion

(IV)~

15.2 i 2.7 29 (55)

6.1 + 1.6 58 (55)

2.8 + 0.42 69 (55)

2.3 2 0.30 84 (55)

1.6 f 0.18 84 (55)

1.5 2 0.26 96 (47)

25 r xc t SE % at criterion

(N)

12.3 2 3.0 38 (40)

3.7 + I.2 73 (48)

2.8 2 I.2 83 (48)

I .6 + 0.26 90 (48)

I.4 AO.13 93 (40)

1.2 + 0.17 96 (24)

50 Iset + SE W at criterion

(N)

16.9 k 3.4 33 (40)

12.7 k 2.6 48 (48)

3.1 + 0.54 69 (48)

4.0 + I.4 77 (48)

1.6kO.31 87 (31)

1.5 + 0.25 87 (31)

75 r set + SE % at criterion

(N)

18.7 f 3.4 41 (46)

8.7 + 2.1 50 (46)

6.2 + 2. I 74 (46)

4.6 k 1.1 65 (46)

I .2 + 0.09 95 (38)

I .o t 0.03 100 (29)

I25 r set i SE B at criterion

(IV)

22.9 2 3.7 36 (47)

9.9 i 2.2 45 (47)

8.0 2 1.6” 42 (55)

7.2 2 1.7” 65 (55)

2.9 t 0.66” 59 (39)

I .6 f 0.23 85 (39)

’ Significantly different ‘Criterion: % turning

from control. analysis in G2 set (total number

of variance with Dunnett’s of rats tested).

test. p < 0.05

BEHAVIOR

AFTER

FETAL

TABLE NEGATIVE

4

GEOTAXIS

TEST

Postnatal Treatment

245

IRRADIATION

day

5

6

7

8

9

Control set f SE” % at criterion

(N)b

112 f 3 I(71)

104*4 3 (79)

76 + 6 18 (79)

56 f 5 29 (79)

42 f 4 44 (79)

25 r set k SE 97 at criterion

(N)

117k2 0 (32)

91 k6 10 (40)

67 k 6 20 (40)

58 f 7 35 (40)

40 + 5 51 (39)

50 r set + SE % at criterion

(N)

115k3 0 (40)

103 f 5 3 (40)

77 i- 7 18 (40)

65 +6 18 (40)

37 iz 5 68 (40)

75 r set k SE 7% at criterion

(N)

115 * 3 0 (38)

112 f 3 2 (46)

97 Ik 5 7 (46)

66 f 6 26 (46)

50 * 5 38 (45)

125 r set 2 SE % at criterion

(Iv)

116+_2 0 (47)

103 rt4 2 (47)

85 t6 13 (47)

66 f 6 30 (47)

52 t 6 37 (46)

’ Average set before turning (maximum b Criterion: % turning in ~30 set (total

= 120 set). number of rats tested).

TABLE REFLEX

5

SUSPENSION

TEST

Postnatal

Treatment

IO

I1

I2

days tested

13

14

15

Control SW k SEY o/ at c!itenon

(N)*

7.7 + 1.1 45 (55)

7.6 5 0.87 62 (63)

7.5 t 0.94 60 (55)

11.1 * I.2 78 (55)

18.8 + 2.0 86 (63)

30.8 + 2.9 100 (39)

2s r set L SE % at criterion

(N:)

4.5 t 0.88 31 (39)

6.01 + 0.85 49 (39)

9.2 + 0.84 76 (46)

II.42 1.7 72 (46)

17.7 t 2.1 83 (46)

28.2 + 3.4 97 (30)

6.1 + 0.95 45 (47)

5.9

96 at criterion (N)

2 0.83 49 (40)

6.1 t 0.57 58 (40)

8.4 + 1.1 62 (47)

15.8 + 1.5 85 (47)

22.1 + 2.3 100 (31)

12.8 k 1.8 76 (45)

17.0 + 2.0 87 (45)

28.6 + 3.7 97 (31)

50 r SW + SE

75 r set + SE %’ at criterion

6.5 k 0.79 51 (45)

7.9

(N)

* 1.4 53 (45)

9.2 i 1.0 62 (45)

125 rc set t SE 7% at criterion

3.4 5 0.52 24 (54)

3.7

(N)

* 0.38 28 (54)

5.8 T 0.6 I 6 I (46)

’ Average set remaining suspended. b Criterion > 5 set (total number of rats tested). ’ Sigmficantly different from control values, analysis

of variance

with Dunnett’s

7.8 f 0.88 61 (46)

test. y = ~0.05

7.7 + 0.81 72 (54)

for all days at 125 r.

16.5 + 2.3 90 (39)

246

STATA TABLE CONTINUOUS No. rats (No. litters)

Control 25 r 50 I75 r 125 r

17 (3) 6 (1) 6 (I) I2 (2) I I (2)

retreat from an arm of the maze before the end. Therefore, the ratio of complete/partial entries (C/P ratio) is a measure of perseveration: the larger the ratio, the greater the degree of perseveration. At 2 1 days of age, rats which received 125 r were less active in the spatial maze: both complete and partial entries were significantly lower, There was no evidence for increased perseveration in these rats since complete entries were diminished more than partial entries and the C/P ratio dropped. The 75 r rats were intermediate between controls and 125 r rats in number of complete entries. The C/P ratio was lower than in controls. As adults, the depressionof activity seenat 2 1 days disappeared and the 125 r rats were more active than controls but the difference was not significant. The C/P ratio for both 75 and 125 r rats was larger than the ratio of control and lower dose groups, primarily due to a drop in partial entries, suggestingsomeperseveration in the adult high-doserats (Table 7). Modified open jield. This modified open field test was used asan additional measureof responseto a novel environment. The addition of openings in the floor into which rats dip

6

CORRIDOR

Treatment

NORTON

ACTIVITY

Actiwty in starting area’

Total activity*

2 1 days of age 4.41 2.83 2.17 2.00 1.36

ir t 2 t ?

0.47 0.40’ 0.51’ 0.32’ 0.20’

19.23 Il.84 6.18 15.08 5.46

i: 1.1 I + 1.80’ + 2.09’ + 2.54 _t 1.65

5 months of age Control I25 T

22 (6) 16 (4)

2.41 t 0.17 2.44 +- 0.46

10.86 2 0.63 12.75 ?I 1.34

’ Average number of times starting area was left or reentered (GE). ’ Average number of corridor areas ( I to I I) entered (+SE). ‘p G 0.05. analysis of variance with Dunnett’s test.

5 months of age, rats were tested for activity and perseveration in the spatial maze. Activity for each of the irradiated and control groups was recorded as the number of partial or complete entries into any arm of the maze. Perseverating animals are considered less likely to perform partial entries, i.e., to enter and TABLE EXPLORATION

Treatment

group

OF SPATIAL

7

MAZE BY WEANLING

No. rats (No. litters)

Complete (C) entries t SE

AND ADULT

RATS

Partial (P) entries f SE

c/p Ratio

2 I days of age Control 25 r 50 r 75 r 125 r

49 (8) 37 (6) 36(6) 39 (7) 40 (7)

6.7 6.9 7.1 5.4 3.9 5 months

Control 25 r 50 r 75 r 125 r ’ Significantly

22 (6) 14 (4) 14 (4) 18(5) 16 (4) less than control,

analysis

9.1 10.4 8.9 9.2 11.1 of variance.

+ t i+ i

0.69 0.62 0.82 0.66 0.56”

0.91 0.86 0.97 1.30 0.56

* k + i +

0.20 0.20 0.18 0.28 0.12”

7.3 8.0 7.3 4.1 5.9

0.81 1.14 0.92 0.44 0.62

F i + +c

0.20 0.31 0.43 0. I6 0.38

Il.2 9.1 9.6 20.9 17.9

of age 1+ 0.75 * I.1 f 0.95 t 0.76 ri- I.1

p < 0.05.

BEHAVIOR

AFTER

their heads may encourage exploration of the open area, since some rats show prolonged hesitancy in exploring open areas, which accounts for some of the variability of the standard open field test. In the modified open field the control 21-day-old rats were somewhat more active than when they were about 5 months old. The irradiated rats showed a doserelated lower activity at 21 days which, like the continuous corridor and spatial maze tests, reversed in adult rats, with irradiated rats showing greater than control activity at 5 months of age (Table 8). Running wheel. At about 5 months of age, rats were placed in cages with access to an activity wheel of conventional design for 24 hr. As expected, females were more active than males. Because of the variability of the results, the slightly greater activity of irradiated than control rats was not significant for any dose. Total activity counts (wheel revolutions) for control males were 41 f 16 (N = 13) and 99 F 47 for 125 r males (N = IO); for females the values were 660 f 140 (N = 12) and 674 + 140

TABLE MODIFIED

Treatment

group

OPEN FIELD TEST No. rats (No. litters)

2 1-day-old Control 25 r 50 r 75 r 125 r

Activity

36 (6) 39 (7) 40 (7)

20 12 12 16 16

k SE”

rats

67(11) 37 (6)

5-month-old Control 25 r 50 r 15 r 125 r

8

5.75 6.27 6.08 4.67 4.73

2 f k + f

0.55 0.76 0.64 0.5 1 0.64

2.35 2.58 4.91 3.56 6.93

2 2 f 2 f

0.47 0.76 0.98’ 0.77 1.68’

rats (5) (3) (3) (4) (4)

a Activity measured as number of dips of head into floor openings. b Significantly greater than control activity, analysis of variance. p < 0.05.

FETAL

247

IRRADIATION

(N = 12) for control and 125 r rats, respectively. DISCUSSION The damaging action of ionizing radiation on dividing cells is well known. The extensive background information make this an interesting agent with which to examine toxic affects on the fetus. The dose to the fetus can be carefully controlled; maternal metabolism and placental distribution is not a factor as it is with chemicals; the onset and duration of effect are precisely timed by the exposure period; and doses below 200 r are relatively innocuous to the mother. Given these advantages, it is surprising how variable and incomplete the data are which document the behavioral consequences of damage to fetal central nervous system from exposure to doses below 200 r. It has been recognized for some time that gross morphological CNS damage is present after irradiation of fetal rats on gestational Days 13 to 17 with 150 to 200 r (Hicks, 1953; Hicks et al., 1959). The ventricles are enlarged, the cerebral cortex is considerably reduced in size, as are other forebrain structures, ectopic cortex is present, the corpus callosum is absent, and the anterior commissure is reduced in size. Several of these changes show graded effects in the lower dose ranges studied here from 25 to 75 r (Mullenix et al., 1975; Norton and Donoso, 1985). Nevertheless, the degree of behavioral involvement found in these studies has been minimal in view of the distinct, and even dramatic, morphological alterations. Perhaps because the behavioral effects have not been marked, there has been little attempt to develop a detailed analysis of behavioral changes over time in irradiated rats. The present study was intended to fill in some of these data, using a series of tests which complement existing information. In particular. previous studies in rats have not examined preweaning behavior. The day chosen for irradiation, gestational Day 15, has been used for detailed

248

STATA

morphological examination of CNS damage (Norton and Donoso, 1985). The body weight of rats was reduced at birth by doses as low as 50 r but growth rate was equal to or slightly greater than control rate, so that 50 and 75 r rats caught up with controls by 2 weeks, postnatally. The decreased body weight of 125 r rats at birth was retained throughout the study, in agreement with previous studies. In the first 2 postnatal weeks, three tests were used. Two of these, surface righting and reflex suspension, showed significant developmental delays in 125 r rats. Rats receiving 75 r showed early delay in surface righting with recovery to control performance by the 9th postnatal day. These developmental delays were very similar to the delays reported for rats made hypothyroid by perinatal exposure to an anti-thyroid drug (Comer and Norton, 1982). The negative geotaxis test was reported by Butcher and Vorhees ( 1979) to show delays in rats exposed to several different chemicals in uteru. However, the test results in the present experiments did not distinguish irradiated from control rats. Around the time of weaning (2 1 to 28 days), three activity tests were used and the same tests were applied when the rats were about 5 months old. Until rats are 5 weeks old, no sexlinked differences in activity are present in control rats (Norton et al., 1975a) and none were detected in this study of weanling rats. At the time of weaning, rats irradiated with 75 or 125 r were less active than controls. This difference was significant in the spatial maze and continuous corridor tests. The detection of effects as low as 25 r in continuous corridor activity was unexpected, although dose-response considerations make this effect of 25 r a possible result with a sensitive behavioral test. However, the continuous corridor is a new test, requiring additional information regarding its usefulness in detecting fetal CNS damage. The modified open field test did not show a difference between control and irradiated rats at this age. Previous studies with different tests have not reported dose-related depression of

NORTON

activity in rats 6 weeks old after in utero irradiation (Furchtgott et al., 1958; Norton, 1977; Schneider and Norton, 1979) except one report of decreased activity in the open field following 50 r but not higher doses on gestational Day 15 (Furchtgott and Echols, 1958). When the rats were tested as adults in all three tests, the activity measured was greater in irradiated rats than in controls. The increase in activity was significant only in the modified open field. Previous reports using different tests of adult rats after prenatal irradiation have shown that hyperactivity develops by 5 months of age, confirming the present results with the modified open field (Norton et al., 1976; Norton, 1977). A comparable picture of early hypoactivity followed by hyperactivity as adults was seen in rats made hypothyroid during the perinatal period by maternal administration of methimazole (Comer and Norton, 1983). In selecting the tests reported here, it was predicted that the use of a series of tests would reduce the likelihood of failure to detect a behavioral effect of gestational irradiation and to some extent this was the result of the experiments. Not all tests were equally affected by irradiation but a pattern of early developmental delay and adult hyperactivity emerged. Interpretation of the reasons for differences in the tests is difficult. Variability and number of rats account for some of this. For example, the negative geotaxis test had the largest variability of any of the tests which may account for the failure to detect significant changes from 75 and 125 r. Overall in these experiments, consistent behavioral changes were produced by gestational exposure to 125 r. With the exception of the continuous corridor test, no significant changes were produced by 25 r. This cut-off at 50 r agrees with morphological data on cortical thickness and cross-sectional area of the caudate nucleus in the forebrain (Norton and Donoso, 1985). However, in that study doseresponse considerations suggested that 25 r may be above control values in production of morphological damage. It cannot be con-

BEHAVIOR

AFTER FETAL

eluded that 25 r is without effect on the offspring. In other experiments with low-dose radiation, it has been shown that certain drug challenges to the low-dose irradiated animal resulted in differences in performance from controls, when performance in the test was similar without drug challenge (Schneider and Norton, 1979). Finally, body weight changes in pups were detected at birth at doses as low as 50 r. Therefore, it cannot be concluded that either morphological or behavioral tests are more sensitive than neonatal body weight changes for detection of damage from gestational irradiation. ACKNOWLEDGMENTS This research was supported in part by USPHS Grants NS 16694 and HD 02528. The expert technical assistance of Ms. Linda Landon is gratefully acknowledged.

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