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Caudate morphology and behavior of rats exposed to carbon monoxide in utero
EXPERIMENTAL
Caudate
NEUROLOGY
80,265-278
(1983)
Morphology and Behavior of Rats Exposed to Carbon Monoxide in Utero WAYNE
C. DAUGHTREY
AND STATA
NORTON’
Department of Pharmacology and Ralph L. Smith Mental Retardation Research Center, University of Kansas College of Health Sciences, Kansas City, Kansas 66103 Received June 24, 1982; revision received November 18, 1982 Postnatal morphologic damage was found in the caudate nucleus of rats exposed 2 or 3 h to carbon monoxide on gestational day 15. There were gross abnormalities in the form of ecotopic swellings of caudate tissue into the lateral ventricles. The incidence of caudate ectopias was about 20% in rats exposed 2 h as fetuses and 70% from 3 h of exposure. In addition, in the body of the caudate the number of dendritic branches was reduced in Golgi type II neurons. Postnatal behavior of the exposed rats was not significantly altered in a series of behavioral tests of motor function. Growth rate was not retarded. The failure to detect behavioral changes may be due to insensitivity ofthese tests detecting functional damage or to compensation by the developing brain, resulting in normal function. INTRODUCTION
The vulnerability of the central nervous system to hypoxic damage during development is well documented. In a study of hypoxic damage in human fetuses, Towbin ( 19) described two basic patterns of cerebral damage depending on fetal age. In the early fetus and premature newborn, the damage involved predominantly periventricular tissues, basal ganglia, and other deep neuronal aggregations. In the mature fetus and newborn the cerebral cortex became the primary target of injury. The functional consequences of hypoxic brain damage reflect the severity of the hypoxic event and the site of the lesion in the brain. Interest has generally focused on the easily recognizable, large destructive lesions encountered in cerebral palsy and mental retardation. Less severe hypoxic le’ Supported in part by U.S. Public Health Service grants MH 17279, ES 07079, and HD 02528. Please address correspondence to Stata Norton, Ph.D., Department of Pharmacology, University of Kansas Medical Center, 39th St. at Rainbow Blvd., Kansas City, KS 66103. 265 0014-4886/83 $3.00 Copyright All
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1983 reproductmn
by
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sions during the fetal period may contribute to more subtle forms of central nervous system disturbance, such as minimal brain dysfunction (20). However, the relationship between structural damage and function is complicated by the ability of the brain to compensate for damage through structural redundancy and plasticity. Therefore, functional deficits are not always observed to follow perinatal brain damage (5, 10, 11). It has been shown that exposure to carbon monoxide (CO) at a concentration causing severe hypoxia on gestational day 15 causes marked damage to the germinal matrix of the caudate nucleus of the fetal rat brain (6). The intent of the present investigation was to examine the consequences of COinduced caudate damage in parallel morphological and behavioral studies of the offspring of control and CO-exposed female rats, from birth through 7 months of age. The behavioral studies in preweaning offspring consisted of a series of developmental tests similar to those proposed for use in the evaluation of neurobehavioral status after prenatal exposure to toxic substances (3, 23). Behavioral testing of juvenile and adult animals was carried out to evaluate motor function and locomotor activity. The rationale behind the selection of these functional tests was the established involvement of the caudate nucleus in the regulation of motor behavior. In addition, behavioral testing with drug challenge was used in an attempt to unmask latent central nervous system damage. METHODS Breeding and Carbon Monoxide Exposure. Female, Sprague-Dawley-derived rats weighing approximately 150 grams were obtained from Charles River (Cambridge, Mass.). After 2 weeks of acclimation, female rats were placed with males of the same strain. Vaginal washings were examined daily until sperm positive (day 1 of gestation), then the female rat was removed from the breeding cage and housed individually for the duration of the experiment. On day 15 of gestation, the pregnant female rats were exposed to CO for either 2 or 3 h. Exposure to CO was carried out individually in a dynamic flow chamber (Bethlehem Model No. 6 15HP). The concentration of CO in the chamber was approximately 1000 ppm in filtered air; flow was 4 liters/ min. The exposure protocol produced maternal carboxyhemoglobin concentrations of about 50% at equilibrium, maintained for several hours (6). Control animals were exposed to air flow only. Exposure to CO at this concentration resulted in loss of righting reflex and coma after 2 h with recovery in less than 1 h. No deaths occurred from exposure with this protocol. At day 1 postpartum, the litters were culled to eight pups with equal distribution of gender. In instances where a litter contained fewer than eight
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pups at birth, additional ilarly treated dams.
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pups of the same age were cross-fostered from sim-
Morphology Toluidine Blue. Offspring of control rats and rats exposed 3 h to CO were randomly selected for morphological evaluation at 1 day, 7 days, 14 days, 2 months, or 7 months of age. After decapitation, the brains were rapidly removed and fixed in neutral, buffered Formalin. Paraffin sections were cut at 8 pm and stained with toluidine blue and Luxol fast blue. For evaluating architectural integrity, every 10th transverse section through the forebrain showing the caudate nucleus, lateral ventricles, septum, and cerebral cortex was examined from 6.9 to 8.9 mm anterior to the external auditory meatus as described by Konig and Klippel (12). Golgi Stain. Brains of control and CO-exposed offspring were taken for Golgi analysis at 1 day, 7 days, 14 days, or 7 months of age. Pieces of forebrain were fixed in an osmic acid-dichromate solution followed by silver nitrate as described by Valverde (22). Sections were cut at 100 pm. Golgi type II neurons in the caudate nucleus were chosen for analysis of dendritic branching patterns, excluding neurons in ectopic caudate. Under low magnification (100X) neurons appearing to have intact dendritic fields, not cut in a plane of section, were selected for study. The neurons were selected randomly throughout the caudate nucleus. When a neuron was selected under low power, the most branched dendrite of that neuron was counted, again to avoid truncation of branches by a plane of section. At 100 pm the sections were thick enough to encompass the entire dendritic field of the early postnatal neurons. For older neurons the method was increasingly important in order to study intact dendritic fields. Branching patterns of dendrites were drawn schematically from Golgi-stained neurons using oilimmersion optics (1250X) and were assigned orders using the centrifugal method of Uylings and co-workers (2 1). All slides were coded and the analysis was conducted in a blind fashion. Litter Size. At 24 h postpartum, the number of live offspring in each litter was recorded. Behavioral
Protocol
Weight Gain. All animals were weighed daily from day 1 after birth until 2 1 days of age, and at 28 days of age. Surface Righting. In tests of surface righting ability, a pup was placed on its back on a smooth surface. The time required for the pup to turn and place all 4 feet in contact with the surface was recorded. A maximum of 60 s per trial was allowed. The criterion for the mature righting response was
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righting in 1 s on each of 3 consecutive trials on a given day. Pups were tested daily, from postnatal day 1 until the criterion response was achieved. Auditory Startle. The criterion used for the appearance of auditory startle was a characteristic jump (startle) in response to a loud, brief noise. In testing for this response, an animal was placed in a glass beaker and a brief noise was produced by a metal rod being tripped to fall 3 cm onto an aluminum plate suspended above the beaker. The response was recorded as either present or absent. Animals were tested once daily from postnatal day 10 until the auditory startle response appeared. Eye Opening. The onset of eye opening was recorded as the first day on which both eyelids were well separated. Reflex Suspension. As an evaluation of grip strength and motor development, a test of reflex suspension was carried out daily between postnatal days 7 and 14. The forefeet of an animal were brought into contact with a horizontal metal rod 2 mm in diameter. The length of time that an animal remained suspended was recorded. Each animal was given three consecutive trials with a maximum of 60 s per trial. The time of the longest of the three suspension periods was recorded. Open Field. Spontaneous locomotor activity was measured in 21-day-old animals placed in an open area surrounded by a 30-cm high wall. The 60cm2 floor was marked off into 5-cm squares. The total number of squares entered with both front feet during a IO-min period was recorded. Residential Maze. Locomotor activity of control and CO-exposed rats was assessedin residential mazes at 1,4, and 6 months of age. The maze consisted of two continuous corridors shaped like a figure 8 with a blind corridor. An electronic counter provided cumulative counts for all eight photocells in the maze (16). The rats were housed in the mazes individually for 23-h starting at 1000 h on a 0600 to 1800 diurnal light cycle. Motor activity recorded during the initial 2-h period after a rat was placed in the maze was designated exploratory activity. Activity was recorded throughout the dark cycle beginning at 1800. The rats were removed from the mazes at 0900 the following morning. Swimming. At 6 weeks of age all rats were evaluated for swimming ability. The rats were placed individually at one end of a clear plastic tank (90 cm long X 18 cm wide X 30 cm deep) filled with water at 22 to 25°C and allowed several practice trials in swimming to the opposite end from which they could exit. Following this brief learning period, each animal swam the length of the tank. At the midway point, the rat was photographed. From the photograph the body angle was measured relative to the surface of the water. The body angle was the acute angle between the line from the base of the head to the base of the tail and the surface of the water. Exploratory Hole Board. The use of this apparatus has been described
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elsewhere as a measure of exploration (7, 13). At 7 to 8 weeks of age, the rats were tested in the hole board apparatus, which consisted of an open field (40 cm2) with 16 evenly spaced holes into which the animal was able to dip its head. Each animal was tested on 2 consecutive days following a S.C. injection of either saline or d-amphetamine sulfate (2.5 mg/kg). Thirty minutes elapsed between the time of injection and the start of testing. The total number of head-dips was recorded for 10 min. Half of the animals received saline and half received amphetamine on the first day of testing. On the next day, treatments were reversed. Response to amphetamine was determined for each rat by subtracting the number of head-dips after saline from the number of head-dips after amphetamine. Residential Maze with Drug Challenge. At 7 months of age, the locomotor response of rats to an amphetamine challenge was tested in a residential maze. The animals were placed in pairs in the mazes for several days prior to commencing drug challenges to assure that they were acclimated to the environment. Activity was recorded for 2 h following injection of either saline or d-amphetamine (1 mg/kg, s.c.) on separate days. Half received saline and half received amphetamine first. Response to amphetamine was determined in each pair of rats by subtracting the activity recorded after saline from the activity recorded after amphetamine. Statistics Comparison of the incidence of ectopic caudate in control and CO-exposed groups was made using a chi-square test of independence. In the Golgi study, comparison of neurons was made using a Mann Whitney U test on the mean number of branches per dendrite in control and CO-exposed animals. For each dendrite examined, the total number of branches present was tabulated. A mean value for the number of branches per dendrite was then calculated for each animal based on the evaluation of either 10 or 20 neurons as indicated for the rats of various ages. These mean values, each representative of a single animal, were the basis for comparison of control and exposed groups. Litter size in control and CO-exposed female rats were compared using a Mann Whitney U test. Student’s t test was used to compare the behavioral measures of control and CO-exposed animals. RESULTS Morphology. Toluidine blue-stained sections from forebrains of CO-exposed animals were examined for malformations in the structure of the caudate nucleus. In control animals (Fig. la), the caudate nucleus outlined the smooth external border of the lateral ventricle, seen as a triangular area in cross section at a level about 7 mm anterior to the external auditory meatus
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TABLE Incidence
of Ectopic
Caudate
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I
in Control
and CO-Exposed
Offspring
Incidence Control
Age 1 day 7 days 14 days 2 months I months
No. of animals” 017 015 O/5 014
O/6
Litter? 4 5 5 4 5
CO, 3 h
Percentage 0 0 0 0 0
No. of animals” 9/12* 6/8* 317 5/8+ 9/13*
Litters 6 8 I 8 6
Percentage 75 75 43 62 69
’ Values represent the number of brains showing the abnormality over the number of brains examined. * Values represent the number of different litters from which the animals of that group were obtained. * Significantly different from control, P < 0.0 I, chi-square test of independence.
(12). In the CO-exposed rats (Fig. lb), the margin of the caudate nucleus bordering the lateral ventricle was disrupted. An abnormal mass of tissue, or ectopic caudate, projected from the main body of the caudate into the ventricular lumen. Ectopic caudate was present in CO-exposed offspring at all ages examined (Table 1). Ectopic caudate was never observed in control animals. In animals exposed 2 h to CO the incidence was about 20%. In animals exposed 3 h to CO, two-thirds of the animals had ectopic caudate. Although the locus and appearance of the malformation was consistent, the severity of the damage varied. In some CO-exposed animals, ectopic caudate was present bilaterally whereas in other animals it was present only on one side. In most cases, the abnormal tissue at the maximum size projected into the ventricular lumen a considerable distance, but some ventricular space was retained. In a few instances, however, the ectopic caudate at the maximum point nearly obliterated the ventricular lumen. Bundles of fibers were seen occasionally within the masses of ectopic caudate. In most instances the ectopia was hemispherical and covered a distance of 1 to 2 mm in the anterior-posterior dimension. In Golgi-stained preparations, the ectopic tissue contained Golgi type II bipolar spiny neurons typical of the caudate nucleus. The ectopic tissue FIG. I. Photomicrographs of forebrain sections from 2-month-old control and CO-exposed rats. A-control. B-CO-exposed showing caudate ectopia. Corpus callosum at top, septum at right and caudate nucleus at left side of photomicrograph. Magnification X29.
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TABLE 2 Dendritic Branching of Caudate Golgi Type II Neurons”
Age 1 day 7 days 14 days 7 months
Control
CO, 2 h
Control
CO, 3 h
12.8 f 0.8 14.4 f 0.7 11.9 + 0.6 -
10.5 k 0.78 13.7 f 0.9 12.2 + 0.8 -
14.6 f 1.0 13.9 + 0.7
10.3 f 0.5* 12.7 f 0.6
LIValues represent the mean number of branches per dendrite. For l-day-old, 7-day-old, and 7-month-old animals, counts were averaged from 10 neurons per brain. For 14-day-old animals, counts were averaged from 20 neurons per brain. Each value shown in the table is a mean + SE determined from at least four different brains. * Significantly different from control value, P i 0.05. Mann Whitney .!I test.
represented a small portion of the total caudate nucleus projecting from the dorsolateral surface and no differences in neuronal orientation within the body of the caudate was observed in those caudates with ectopias. However, the number of branches per dendrite on the bipolar spiny neurons in the body of the caudate was decreasedin the rats exposed either 2 or 3 h to CO (Table 2). This reduced differentiation in neurons of CO-exposed rats was found in neonatal rats only. In the rats of later postnatal ages,the branching in control and CO-exposed neurons was not significantly different. Litter Size. Exposure of pregnant female rats to CO for 2 or 3 h did not significantly reduce litter size. In 10 control litters, the median number of pups was 12 (range, 6 to 15). In 7 litters exposed 2 h to CO, the median number of pups was also 12 (range, 5 to 16). In 11 litters exposed 3 h to CO, the median number of pups was 10 (range, 3 to 15). The smallest litter with only 3 pups suggeststhat CO exposure wascausing death of somefetuses. Growth Rate. The rate of weight gain during the preweaning period was not significantly affected by prenatal CO exposure. The growth rate of control offspring and offspring exposed 3 h to CO is shown in Fig. 2. The mean weight of CO-exposed animals was approximately 5% lessthan that of controls at most ages.However, statistical comparisons using the litter mean as the unit of analysis did not indicate that these differences were significant. Physical and Rejlex Development. In tests of physical and reflex development there were no significant differences between control and CO-exposed animals (Fig. 3). The surface righting response matured about the eighth postnatal day in control animals and in animals exposed either 2 or 3 h to CO. The auditory startle responsedeveloped in all groups of animals around the 12th postnatal day. The average age at which the eyes opened in all three groups was approximately 15 days.
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FIG. 2. Growth of control rats (solid line) and CO-exposed for 3 h (dashed line). Points are mean weights f SE of pups from 5 control and 6 CO-exposed litters.
Reflex Suspension. In agreement with previous studies (5) there was a general improvement in the performance of this test in all groups of animals as they matured. On postnatal day 7, the average time for four control litters was 6.4 * 1.2 s and that for five litters of offspring exposed 2 h to CO was 7.7 + 1.0 s. Animals exposed 2 h to CO lagged behind controls on days 8, 9, and 10 postnatally. By postnatal day 11, they were performing somewhat better than control animals. The difference in performance was not significant. When these tests were repeated in animals exposed 3 h to CO, a similar trend was not observed. Six litters of CO-exposed animals did not lag behind animals in five control litters during the early stages of testing and performed significantly better than controls on day 13. On days 7, 8, 10, and 11, COexposed animals lagged behind controls and on days 9, 12, and 14 performed slightly better. The lack of a consistent pattern or dose response suggests that any of the differences observed between control and CO-exposed animals were not treatment-related effects. Open Field. Spontaneous locomotor activity recorded in an open field at 2 1 days of age was not significantly different in 8 control male rats from four
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16-
SURFKE RIRHTlR6
AIR
CO 3HR
AIR
CO 31
FIG. 3. Physical and reflex development in control rats (open bars) and CO-exposed for 3 h (dashed bars). Bars represent the age of the pups at the time the criterion was met (mean age f SE for 5 control and 6 CO-exposed litters).
litters and 9 CO-exposed male rats from five litters. Average activity in animals exposed 2 h to CO (329 + 26 squares crossed) was somewhat higher than that in corresponding controls (248 + 48 squares), although the difference was not statistically significant. Comparable data were obtained with female rats. Locomotor activity in 18 rats from six litters exposed 3 h to CO was similar to that in 15 corresponding control animals from five litters for both male and female rats. Residential Maze. The circadian locomotor activity of l-month-old male animals in residential mazes is shown in Table 3. During the exploratory period, these CO-exposed animals were hyperactive relative to control animals. During the subsequent diurnal and nocturnal periods, however, there were no significant differences in locomotor activity between the two groups of animals. When the same rats were tested again at 4 and 6 months of age, the exploratory hyperactivity observed at the earlier age was not evident (Table 3). Locomotor activity throughout the circadian period was similar in control and CO-exposed animals. In female rats, differences were not evident at any of the ages tested. Swimming. No effect on motor coordination or function in CO-exposed animals was found in swimming tests conducted at 6 weeks of age. Both control and CO-exposed rats showed that coordinated leg movements and body position were adapted to effective swimming. In control male and
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Circadian
Locomotor 6-Month-Old
Exploratory Diurnal Nocturnal
336 + 31 81 + 10 137+ 6
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3
ActivitqP in Residential Mazes for I-, 4- and Control and CO-Exposed Rats
I-Month Control
PRENATAL
4-Month CO, 3 h 425 f 24: 75 f 14 146? 9
6-Month
Control
CO,
410 k 24 140 + 26 142 f 16
3 h
Control
421 f 39 132 f 27 130* 8
’ Values represent hourly photocell counts (? SE) for 10 control * Significantly different from controls, P < 0.05, t test.
CO,
336 + 33 108 + 17 106+ 6
3h
370 f 45 107 f 29 103 f 10
and 10 CO-exposed
animals.
female rats from five litters, the mean body angles (*SE) relative to the water surface while swimming were 19.1 -t 1.7 and 20.4 + 2.3 degrees, respectively. In CO-exposed male and female rats from six litters, the mean body angles (&SE) were 20.0 + 1.5 and 17.8 + 1.7 degrees, respectively. Exploratory Hole Board. Exploratory activity of 7- to %week-old rats in the hole board is shown in Table 4. After saline injection, control and COexposed male rats had a similar number of head-dips. Amphetamine challenge significantly elevated the total number of head-dips of both groups three- to fourfold. However, both control and CO-exposed animals responded in a similar manner to amphetamine. The same pattern was observed in female rats to follow saline and amphetamine injections. Residential Maze. Locomotor activity of 7-month-old control and COexposed rats (3 h) was recorded in residential mazes for 2 h foilowing saline or amphetamine injection. Baseline activity of 12 CO-exposed animals in groups of 2 after saline (1002 f 140 counts/h) was not significantly different from that of 12 control animals in groups of 2 (897 +- 82 counts/h). An TABLE Exploratory
4
Hole-Board
Behavior” Saline
Male Female
rats rats
Control CO, 3 h Control CO. 3 h
’ Values represent the total number and 12 CO-exposed animals. b 2.5 mg/kg, S.C.
17.8 19.8 22.7 31.1 of head-dips
(*SE)
zk + k +
Amphetamineb 2.1 2.7 2.6 4.6
in a IO-mm
84.1 74.9 67.3 66.5
f 12.4 f 11.5 f 9.7 k 12.5
test session for 12 control
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amphetamine challenge caused the expected increase in activity from baseline values for both groups of animals. Values for control and CO-exposed animals were 1750 + 256 and 2038 f 282 counts/h, respectively. There was large variability in the response and, although the mean response to amphetamine (amphetamine activity minus saline activity) of the CO-exposed group was somewhat greater than that of the control group, the difference was not significant. DISCUSSION It has been shown that acute exposure to CO on gestational day 15 results in severe hemorrhagic infarcts in the germinal matrix of the developing caudate nucleus (6). The present study documents the occurrence of caudate malformations in newborn and developing rats after exposure to CO for 2 or 3 h on gestational day 15. The probability of caudate damage increases markedly with the longer exposure time. The abnormalities in caudate structure are proposed to result from the hypoxic damage to the germinal matrix in utero. The disruption of the germinal neuroepithelium and the germination of caudate ectopias from hypoxia may be analogous to formation of cortical ectopias after death of the dividing cells of the germinal matrix of the cortex from antimitotic agents (9, 15, 24). Whether or not the ectopic caudate is capable of functioning normally is a matter of speculation. However the presence of typical bipolar spiny neurons and bundles of axons in the ectopic tissue suggests that the neurons might receive and transmit impulses. Although the structure of the remainder of the caudate nucleus was not grossly abnormal, dendritic branching of caudate Golgi type II neurons was reduced in l-day-old animals exposed to CO prenatally. The reduction in dendritic branching was not permanent and, by 1 week of age, branching in control and CO-exposed animals was similar. This recovery differs from persistent reduction in dendritic branching of caudate neurons in rats to 4 months of age reported to follow perinatal exposure to an antithyroid drug (2). As visualized by the Golgi technique, about 95% of caudate neurons have the same morphology, although functional differences have been reported. The reduction in branching in CO-exposed rats was uniform in the population of neurons examined, for the variation was no greater in COexposed than in control rats as reflected in the standard errors in Table 2. The results obtained with functional tests in the young rats are consistent with the rapid recovery in morphology. No consistent behavioral deficits in the CO-exposed offspring were found. The control values recorded for the tests in the present study agree well with means reported elsewhere (3, 23). The group of developmental tests in this study have been used with variable
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success in detecting functional damage to the developing nervous system after prenatal insults which result in morphologic defects. Both no behavioral effect (1) and behavioral changes (17, 18) have been reported from prenatal exposure of rats to antimitotic agents. However, in the absence of gross morphologic changes, profound delays in behavioral maturation are produced in animals made hypothyroid during the perinatal period (3). Thus morphologic changes have been reported in the absence of functional changes, as well as the converse situation. It seems unlikely that the failure to find behavioral changes in CO-exposed rats of this study can be attributed to the insensitivity of the tests used to detect behavioral changes, although this possibility cannot be completely dismissed. The tests used in the present study involved assessments of locomotor activity and motor function. Experimental studies in adult animals have established the involvement of the caudate-putamen in motor control. Hyperactivity is the most commonly reported effect of basal ganglia lesions in animals. Hyperactivity as recorded in running wheels, open field environments and residential mazes has been reported to follow experimental lesions of the substantia nigra, globus pallidus, and caudate-putamen (4, 8, 14, 25). Because the CO-exposed animals in the present study suffered caudate damage, it was reasoned that tests of sensory-motor coordination and motor function would be the type of behavioral evaluation most likely to detect changes in central nervous system function. However, in spite of the structural abnormalities, behavioral changes in these animals were minimal. Although striking behavioral changes were not detected in the CO-exposed rats of this study, functional deficits might be displayed under other circumstances of environmental challenge, such as occur during aging. REFERENCES 1. BRUNNER, R. L., M. MCLEAN, C. V. VORHEES, AND R. E. BUTCHER. 1978. A comparison of behavioral and anatomical measures of hydroxyurea induced abnormalities. Teratology 18: 379-384. 2. COMER, C. P., AND S. NORTON. 1981. Persisting effects of perinatal hypothyroidism on neuronal morphology and motor behavior in rats. Toxicologist 1: 57. 3. COMER, C. P., AND S. NORTON. 1982. Effects of perinatal methimazole exposure on a developmental test battery for neurobehavioral toxicity in rats. Toxicol. Appl. Pharmacol. 63: 133-141. 4. COSTALL, B., R. J. NAYLOR, AND J. E. OLLEY. 1972. On the involvement of the caudateputamen, globus pallidus and substantia nigra with neuroleptic and cholinergic modification of locomotor activity. Neuropharrnacology Ii: 3 17-330. 5. CULVER, B., AND S. NORTON. 1976. Juvenile hyperactivity in rats after acute exposure to carbon monoxide. Exp. Neurol. 50: 80-98. 6. DAUGHTREY, W. C., AND S. NORTON. 1982. Morphological damage to the premature fetal rat brain following acute carbon monoxide exposure. Exp. Neural. 78: 26-37.
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7. FILE, S. E., AND A. G. WARDHILL. 1975. Validity of head-dipping as a measure ofexploration in a modified hole board. Psychophnrmacologia 44: 53-59. 8. HANNON, R., AND A. BADER. 1974. A comparison of frontal pole, anterior-median and caudate nucleus lesions in the rat. Physiol. Behav. 13: 5 13-521. 9. HICKS, S. P., AND C. J. D’AMATO. 1966. Effects of ionizing radiations on mammalian development. In D. H. M. WOOLLAM, Ed., Advances in Teratology, Vol. 1. Logos Press, London. 10. KLING, A., AND T. J. TUCKER. 1968. Sparing of function following localized brain lesions in neonatal monkeys. In R. L. ISAACSON, Ed., The Neuropsychology of Development. Wiley, New York. Il. KOLB, B., AND A. J. NONNEMAN. 1978. Sparing of function in rats with early prefrontal cortex lesions. Brain Res. 151: 135-148. 12. KONIG, J. F. R., AND R. A. KLIPPEL. 1963. The Rat Brain. A Stereotaxic Atlas of the Forebrain and Lower Parts of the Brain Stem. Williams & Wilkins, Baltimore. 13. MAKANJUOLA, R. 0. A., G. HILL, I. MABEN, R. C. Dow, AND G. W. ASHCROFT. 1977. An automated method for studying exploratory and stereotyped behavior in rats. Psychopharmacology 52: 27 l-277. 14. NORTON, S. 1976. Hyperactive behavior of rats after lesions of the globus pallidus. Brain Res. Bull. 1: 3 17-33 1. 15. NORTON, S. 1979. Development of rat telencephalic neurons after prenatal X-irradiation. J. Environ.
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24. WATTERSON, R. L. 1965. Structure and mitotic behavior of the early neural tube. In R. L. DE HAAN AND H. URSPRUNG, Eds., Organogenesis. Hoh, Reinhart and Winston, New York. 25. WHITIER, J. R., AND A. ORR. 1962. Hyperkinesia and other physiologic effects of caudate deficit in the adult albino rat. Neurology 12: 526-539.
Caudate
NEUROLOGY
80,265-278
(1983)
Morphology and Behavior of Rats Exposed to Carbon Monoxide in Utero WAYNE
C. DAUGHTREY
AND STATA
NORTON’
Department of Pharmacology and Ralph L. Smith Mental Retardation Research Center, University of Kansas College of Health Sciences, Kansas City, Kansas 66103 Received June 24, 1982; revision received November 18, 1982 Postnatal morphologic damage was found in the caudate nucleus of rats exposed 2 or 3 h to carbon monoxide on gestational day 15. There were gross abnormalities in the form of ecotopic swellings of caudate tissue into the lateral ventricles. The incidence of caudate ectopias was about 20% in rats exposed 2 h as fetuses and 70% from 3 h of exposure. In addition, in the body of the caudate the number of dendritic branches was reduced in Golgi type II neurons. Postnatal behavior of the exposed rats was not significantly altered in a series of behavioral tests of motor function. Growth rate was not retarded. The failure to detect behavioral changes may be due to insensitivity ofthese tests detecting functional damage or to compensation by the developing brain, resulting in normal function. INTRODUCTION
The vulnerability of the central nervous system to hypoxic damage during development is well documented. In a study of hypoxic damage in human fetuses, Towbin ( 19) described two basic patterns of cerebral damage depending on fetal age. In the early fetus and premature newborn, the damage involved predominantly periventricular tissues, basal ganglia, and other deep neuronal aggregations. In the mature fetus and newborn the cerebral cortex became the primary target of injury. The functional consequences of hypoxic brain damage reflect the severity of the hypoxic event and the site of the lesion in the brain. Interest has generally focused on the easily recognizable, large destructive lesions encountered in cerebral palsy and mental retardation. Less severe hypoxic le’ Supported in part by U.S. Public Health Service grants MH 17279, ES 07079, and HD 02528. Please address correspondence to Stata Norton, Ph.D., Department of Pharmacology, University of Kansas Medical Center, 39th St. at Rainbow Blvd., Kansas City, KS 66103. 265 0014-4886/83 $3.00 Copyright All
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sions during the fetal period may contribute to more subtle forms of central nervous system disturbance, such as minimal brain dysfunction (20). However, the relationship between structural damage and function is complicated by the ability of the brain to compensate for damage through structural redundancy and plasticity. Therefore, functional deficits are not always observed to follow perinatal brain damage (5, 10, 11). It has been shown that exposure to carbon monoxide (CO) at a concentration causing severe hypoxia on gestational day 15 causes marked damage to the germinal matrix of the caudate nucleus of the fetal rat brain (6). The intent of the present investigation was to examine the consequences of COinduced caudate damage in parallel morphological and behavioral studies of the offspring of control and CO-exposed female rats, from birth through 7 months of age. The behavioral studies in preweaning offspring consisted of a series of developmental tests similar to those proposed for use in the evaluation of neurobehavioral status after prenatal exposure to toxic substances (3, 23). Behavioral testing of juvenile and adult animals was carried out to evaluate motor function and locomotor activity. The rationale behind the selection of these functional tests was the established involvement of the caudate nucleus in the regulation of motor behavior. In addition, behavioral testing with drug challenge was used in an attempt to unmask latent central nervous system damage. METHODS Breeding and Carbon Monoxide Exposure. Female, Sprague-Dawley-derived rats weighing approximately 150 grams were obtained from Charles River (Cambridge, Mass.). After 2 weeks of acclimation, female rats were placed with males of the same strain. Vaginal washings were examined daily until sperm positive (day 1 of gestation), then the female rat was removed from the breeding cage and housed individually for the duration of the experiment. On day 15 of gestation, the pregnant female rats were exposed to CO for either 2 or 3 h. Exposure to CO was carried out individually in a dynamic flow chamber (Bethlehem Model No. 6 15HP). The concentration of CO in the chamber was approximately 1000 ppm in filtered air; flow was 4 liters/ min. The exposure protocol produced maternal carboxyhemoglobin concentrations of about 50% at equilibrium, maintained for several hours (6). Control animals were exposed to air flow only. Exposure to CO at this concentration resulted in loss of righting reflex and coma after 2 h with recovery in less than 1 h. No deaths occurred from exposure with this protocol. At day 1 postpartum, the litters were culled to eight pups with equal distribution of gender. In instances where a litter contained fewer than eight
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pups of the same age were cross-fostered from sim-
Morphology Toluidine Blue. Offspring of control rats and rats exposed 3 h to CO were randomly selected for morphological evaluation at 1 day, 7 days, 14 days, 2 months, or 7 months of age. After decapitation, the brains were rapidly removed and fixed in neutral, buffered Formalin. Paraffin sections were cut at 8 pm and stained with toluidine blue and Luxol fast blue. For evaluating architectural integrity, every 10th transverse section through the forebrain showing the caudate nucleus, lateral ventricles, septum, and cerebral cortex was examined from 6.9 to 8.9 mm anterior to the external auditory meatus as described by Konig and Klippel (12). Golgi Stain. Brains of control and CO-exposed offspring were taken for Golgi analysis at 1 day, 7 days, 14 days, or 7 months of age. Pieces of forebrain were fixed in an osmic acid-dichromate solution followed by silver nitrate as described by Valverde (22). Sections were cut at 100 pm. Golgi type II neurons in the caudate nucleus were chosen for analysis of dendritic branching patterns, excluding neurons in ectopic caudate. Under low magnification (100X) neurons appearing to have intact dendritic fields, not cut in a plane of section, were selected for study. The neurons were selected randomly throughout the caudate nucleus. When a neuron was selected under low power, the most branched dendrite of that neuron was counted, again to avoid truncation of branches by a plane of section. At 100 pm the sections were thick enough to encompass the entire dendritic field of the early postnatal neurons. For older neurons the method was increasingly important in order to study intact dendritic fields. Branching patterns of dendrites were drawn schematically from Golgi-stained neurons using oilimmersion optics (1250X) and were assigned orders using the centrifugal method of Uylings and co-workers (2 1). All slides were coded and the analysis was conducted in a blind fashion. Litter Size. At 24 h postpartum, the number of live offspring in each litter was recorded. Behavioral
Protocol
Weight Gain. All animals were weighed daily from day 1 after birth until 2 1 days of age, and at 28 days of age. Surface Righting. In tests of surface righting ability, a pup was placed on its back on a smooth surface. The time required for the pup to turn and place all 4 feet in contact with the surface was recorded. A maximum of 60 s per trial was allowed. The criterion for the mature righting response was
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righting in 1 s on each of 3 consecutive trials on a given day. Pups were tested daily, from postnatal day 1 until the criterion response was achieved. Auditory Startle. The criterion used for the appearance of auditory startle was a characteristic jump (startle) in response to a loud, brief noise. In testing for this response, an animal was placed in a glass beaker and a brief noise was produced by a metal rod being tripped to fall 3 cm onto an aluminum plate suspended above the beaker. The response was recorded as either present or absent. Animals were tested once daily from postnatal day 10 until the auditory startle response appeared. Eye Opening. The onset of eye opening was recorded as the first day on which both eyelids were well separated. Reflex Suspension. As an evaluation of grip strength and motor development, a test of reflex suspension was carried out daily between postnatal days 7 and 14. The forefeet of an animal were brought into contact with a horizontal metal rod 2 mm in diameter. The length of time that an animal remained suspended was recorded. Each animal was given three consecutive trials with a maximum of 60 s per trial. The time of the longest of the three suspension periods was recorded. Open Field. Spontaneous locomotor activity was measured in 21-day-old animals placed in an open area surrounded by a 30-cm high wall. The 60cm2 floor was marked off into 5-cm squares. The total number of squares entered with both front feet during a IO-min period was recorded. Residential Maze. Locomotor activity of control and CO-exposed rats was assessedin residential mazes at 1,4, and 6 months of age. The maze consisted of two continuous corridors shaped like a figure 8 with a blind corridor. An electronic counter provided cumulative counts for all eight photocells in the maze (16). The rats were housed in the mazes individually for 23-h starting at 1000 h on a 0600 to 1800 diurnal light cycle. Motor activity recorded during the initial 2-h period after a rat was placed in the maze was designated exploratory activity. Activity was recorded throughout the dark cycle beginning at 1800. The rats were removed from the mazes at 0900 the following morning. Swimming. At 6 weeks of age all rats were evaluated for swimming ability. The rats were placed individually at one end of a clear plastic tank (90 cm long X 18 cm wide X 30 cm deep) filled with water at 22 to 25°C and allowed several practice trials in swimming to the opposite end from which they could exit. Following this brief learning period, each animal swam the length of the tank. At the midway point, the rat was photographed. From the photograph the body angle was measured relative to the surface of the water. The body angle was the acute angle between the line from the base of the head to the base of the tail and the surface of the water. Exploratory Hole Board. The use of this apparatus has been described
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elsewhere as a measure of exploration (7, 13). At 7 to 8 weeks of age, the rats were tested in the hole board apparatus, which consisted of an open field (40 cm2) with 16 evenly spaced holes into which the animal was able to dip its head. Each animal was tested on 2 consecutive days following a S.C. injection of either saline or d-amphetamine sulfate (2.5 mg/kg). Thirty minutes elapsed between the time of injection and the start of testing. The total number of head-dips was recorded for 10 min. Half of the animals received saline and half received amphetamine on the first day of testing. On the next day, treatments were reversed. Response to amphetamine was determined for each rat by subtracting the number of head-dips after saline from the number of head-dips after amphetamine. Residential Maze with Drug Challenge. At 7 months of age, the locomotor response of rats to an amphetamine challenge was tested in a residential maze. The animals were placed in pairs in the mazes for several days prior to commencing drug challenges to assure that they were acclimated to the environment. Activity was recorded for 2 h following injection of either saline or d-amphetamine (1 mg/kg, s.c.) on separate days. Half received saline and half received amphetamine first. Response to amphetamine was determined in each pair of rats by subtracting the activity recorded after saline from the activity recorded after amphetamine. Statistics Comparison of the incidence of ectopic caudate in control and CO-exposed groups was made using a chi-square test of independence. In the Golgi study, comparison of neurons was made using a Mann Whitney U test on the mean number of branches per dendrite in control and CO-exposed animals. For each dendrite examined, the total number of branches present was tabulated. A mean value for the number of branches per dendrite was then calculated for each animal based on the evaluation of either 10 or 20 neurons as indicated for the rats of various ages. These mean values, each representative of a single animal, were the basis for comparison of control and exposed groups. Litter size in control and CO-exposed female rats were compared using a Mann Whitney U test. Student’s t test was used to compare the behavioral measures of control and CO-exposed animals. RESULTS Morphology. Toluidine blue-stained sections from forebrains of CO-exposed animals were examined for malformations in the structure of the caudate nucleus. In control animals (Fig. la), the caudate nucleus outlined the smooth external border of the lateral ventricle, seen as a triangular area in cross section at a level about 7 mm anterior to the external auditory meatus
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TABLE Incidence
of Ectopic
Caudate
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in Control
and CO-Exposed
Offspring
Incidence Control
Age 1 day 7 days 14 days 2 months I months
No. of animals” 017 015 O/5 014
O/6
Litter? 4 5 5 4 5
CO, 3 h
Percentage 0 0 0 0 0
No. of animals” 9/12* 6/8* 317 5/8+ 9/13*
Litters 6 8 I 8 6
Percentage 75 75 43 62 69
’ Values represent the number of brains showing the abnormality over the number of brains examined. * Values represent the number of different litters from which the animals of that group were obtained. * Significantly different from control, P < 0.0 I, chi-square test of independence.
(12). In the CO-exposed rats (Fig. lb), the margin of the caudate nucleus bordering the lateral ventricle was disrupted. An abnormal mass of tissue, or ectopic caudate, projected from the main body of the caudate into the ventricular lumen. Ectopic caudate was present in CO-exposed offspring at all ages examined (Table 1). Ectopic caudate was never observed in control animals. In animals exposed 2 h to CO the incidence was about 20%. In animals exposed 3 h to CO, two-thirds of the animals had ectopic caudate. Although the locus and appearance of the malformation was consistent, the severity of the damage varied. In some CO-exposed animals, ectopic caudate was present bilaterally whereas in other animals it was present only on one side. In most cases, the abnormal tissue at the maximum size projected into the ventricular lumen a considerable distance, but some ventricular space was retained. In a few instances, however, the ectopic caudate at the maximum point nearly obliterated the ventricular lumen. Bundles of fibers were seen occasionally within the masses of ectopic caudate. In most instances the ectopia was hemispherical and covered a distance of 1 to 2 mm in the anterior-posterior dimension. In Golgi-stained preparations, the ectopic tissue contained Golgi type II bipolar spiny neurons typical of the caudate nucleus. The ectopic tissue FIG. I. Photomicrographs of forebrain sections from 2-month-old control and CO-exposed rats. A-control. B-CO-exposed showing caudate ectopia. Corpus callosum at top, septum at right and caudate nucleus at left side of photomicrograph. Magnification X29.
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TABLE 2 Dendritic Branching of Caudate Golgi Type II Neurons”
Age 1 day 7 days 14 days 7 months
Control
CO, 2 h
Control
CO, 3 h
12.8 f 0.8 14.4 f 0.7 11.9 + 0.6 -
10.5 k 0.78 13.7 f 0.9 12.2 + 0.8 -
14.6 f 1.0 13.9 + 0.7
10.3 f 0.5* 12.7 f 0.6
LIValues represent the mean number of branches per dendrite. For l-day-old, 7-day-old, and 7-month-old animals, counts were averaged from 10 neurons per brain. For 14-day-old animals, counts were averaged from 20 neurons per brain. Each value shown in the table is a mean + SE determined from at least four different brains. * Significantly different from control value, P i 0.05. Mann Whitney .!I test.
represented a small portion of the total caudate nucleus projecting from the dorsolateral surface and no differences in neuronal orientation within the body of the caudate was observed in those caudates with ectopias. However, the number of branches per dendrite on the bipolar spiny neurons in the body of the caudate was decreasedin the rats exposed either 2 or 3 h to CO (Table 2). This reduced differentiation in neurons of CO-exposed rats was found in neonatal rats only. In the rats of later postnatal ages,the branching in control and CO-exposed neurons was not significantly different. Litter Size. Exposure of pregnant female rats to CO for 2 or 3 h did not significantly reduce litter size. In 10 control litters, the median number of pups was 12 (range, 6 to 15). In 7 litters exposed 2 h to CO, the median number of pups was also 12 (range, 5 to 16). In 11 litters exposed 3 h to CO, the median number of pups was 10 (range, 3 to 15). The smallest litter with only 3 pups suggeststhat CO exposure wascausing death of somefetuses. Growth Rate. The rate of weight gain during the preweaning period was not significantly affected by prenatal CO exposure. The growth rate of control offspring and offspring exposed 3 h to CO is shown in Fig. 2. The mean weight of CO-exposed animals was approximately 5% lessthan that of controls at most ages.However, statistical comparisons using the litter mean as the unit of analysis did not indicate that these differences were significant. Physical and Rejlex Development. In tests of physical and reflex development there were no significant differences between control and CO-exposed animals (Fig. 3). The surface righting response matured about the eighth postnatal day in control animals and in animals exposed either 2 or 3 h to CO. The auditory startle responsedeveloped in all groups of animals around the 12th postnatal day. The average age at which the eyes opened in all three groups was approximately 15 days.
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FIG. 2. Growth of control rats (solid line) and CO-exposed for 3 h (dashed line). Points are mean weights f SE of pups from 5 control and 6 CO-exposed litters.
Reflex Suspension. In agreement with previous studies (5) there was a general improvement in the performance of this test in all groups of animals as they matured. On postnatal day 7, the average time for four control litters was 6.4 * 1.2 s and that for five litters of offspring exposed 2 h to CO was 7.7 + 1.0 s. Animals exposed 2 h to CO lagged behind controls on days 8, 9, and 10 postnatally. By postnatal day 11, they were performing somewhat better than control animals. The difference in performance was not significant. When these tests were repeated in animals exposed 3 h to CO, a similar trend was not observed. Six litters of CO-exposed animals did not lag behind animals in five control litters during the early stages of testing and performed significantly better than controls on day 13. On days 7, 8, 10, and 11, COexposed animals lagged behind controls and on days 9, 12, and 14 performed slightly better. The lack of a consistent pattern or dose response suggests that any of the differences observed between control and CO-exposed animals were not treatment-related effects. Open Field. Spontaneous locomotor activity recorded in an open field at 2 1 days of age was not significantly different in 8 control male rats from four
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16-
SURFKE RIRHTlR6
AIR
CO 3HR
AIR
CO 31
FIG. 3. Physical and reflex development in control rats (open bars) and CO-exposed for 3 h (dashed bars). Bars represent the age of the pups at the time the criterion was met (mean age f SE for 5 control and 6 CO-exposed litters).
litters and 9 CO-exposed male rats from five litters. Average activity in animals exposed 2 h to CO (329 + 26 squares crossed) was somewhat higher than that in corresponding controls (248 + 48 squares), although the difference was not statistically significant. Comparable data were obtained with female rats. Locomotor activity in 18 rats from six litters exposed 3 h to CO was similar to that in 15 corresponding control animals from five litters for both male and female rats. Residential Maze. The circadian locomotor activity of l-month-old male animals in residential mazes is shown in Table 3. During the exploratory period, these CO-exposed animals were hyperactive relative to control animals. During the subsequent diurnal and nocturnal periods, however, there were no significant differences in locomotor activity between the two groups of animals. When the same rats were tested again at 4 and 6 months of age, the exploratory hyperactivity observed at the earlier age was not evident (Table 3). Locomotor activity throughout the circadian period was similar in control and CO-exposed animals. In female rats, differences were not evident at any of the ages tested. Swimming. No effect on motor coordination or function in CO-exposed animals was found in swimming tests conducted at 6 weeks of age. Both control and CO-exposed rats showed that coordinated leg movements and body position were adapted to effective swimming. In control male and
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Locomotor 6-Month-Old
Exploratory Diurnal Nocturnal
336 + 31 81 + 10 137+ 6
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ActivitqP in Residential Mazes for I-, 4- and Control and CO-Exposed Rats
I-Month Control
PRENATAL
4-Month CO, 3 h 425 f 24: 75 f 14 146? 9
6-Month
Control
CO,
410 k 24 140 + 26 142 f 16
3 h
Control
421 f 39 132 f 27 130* 8
’ Values represent hourly photocell counts (? SE) for 10 control * Significantly different from controls, P < 0.05, t test.
CO,
336 + 33 108 + 17 106+ 6
3h
370 f 45 107 f 29 103 f 10
and 10 CO-exposed
animals.
female rats from five litters, the mean body angles (*SE) relative to the water surface while swimming were 19.1 -t 1.7 and 20.4 + 2.3 degrees, respectively. In CO-exposed male and female rats from six litters, the mean body angles (&SE) were 20.0 + 1.5 and 17.8 + 1.7 degrees, respectively. Exploratory Hole Board. Exploratory activity of 7- to %week-old rats in the hole board is shown in Table 4. After saline injection, control and COexposed male rats had a similar number of head-dips. Amphetamine challenge significantly elevated the total number of head-dips of both groups three- to fourfold. However, both control and CO-exposed animals responded in a similar manner to amphetamine. The same pattern was observed in female rats to follow saline and amphetamine injections. Residential Maze. Locomotor activity of 7-month-old control and COexposed rats (3 h) was recorded in residential mazes for 2 h foilowing saline or amphetamine injection. Baseline activity of 12 CO-exposed animals in groups of 2 after saline (1002 f 140 counts/h) was not significantly different from that of 12 control animals in groups of 2 (897 +- 82 counts/h). An TABLE Exploratory
4
Hole-Board
Behavior” Saline
Male Female
rats rats
Control CO, 3 h Control CO. 3 h
’ Values represent the total number and 12 CO-exposed animals. b 2.5 mg/kg, S.C.
17.8 19.8 22.7 31.1 of head-dips
(*SE)
zk + k +
Amphetamineb 2.1 2.7 2.6 4.6
in a IO-mm
84.1 74.9 67.3 66.5
f 12.4 f 11.5 f 9.7 k 12.5
test session for 12 control
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amphetamine challenge caused the expected increase in activity from baseline values for both groups of animals. Values for control and CO-exposed animals were 1750 + 256 and 2038 f 282 counts/h, respectively. There was large variability in the response and, although the mean response to amphetamine (amphetamine activity minus saline activity) of the CO-exposed group was somewhat greater than that of the control group, the difference was not significant. DISCUSSION It has been shown that acute exposure to CO on gestational day 15 results in severe hemorrhagic infarcts in the germinal matrix of the developing caudate nucleus (6). The present study documents the occurrence of caudate malformations in newborn and developing rats after exposure to CO for 2 or 3 h on gestational day 15. The probability of caudate damage increases markedly with the longer exposure time. The abnormalities in caudate structure are proposed to result from the hypoxic damage to the germinal matrix in utero. The disruption of the germinal neuroepithelium and the germination of caudate ectopias from hypoxia may be analogous to formation of cortical ectopias after death of the dividing cells of the germinal matrix of the cortex from antimitotic agents (9, 15, 24). Whether or not the ectopic caudate is capable of functioning normally is a matter of speculation. However the presence of typical bipolar spiny neurons and bundles of axons in the ectopic tissue suggests that the neurons might receive and transmit impulses. Although the structure of the remainder of the caudate nucleus was not grossly abnormal, dendritic branching of caudate Golgi type II neurons was reduced in l-day-old animals exposed to CO prenatally. The reduction in dendritic branching was not permanent and, by 1 week of age, branching in control and CO-exposed animals was similar. This recovery differs from persistent reduction in dendritic branching of caudate neurons in rats to 4 months of age reported to follow perinatal exposure to an antithyroid drug (2). As visualized by the Golgi technique, about 95% of caudate neurons have the same morphology, although functional differences have been reported. The reduction in branching in CO-exposed rats was uniform in the population of neurons examined, for the variation was no greater in COexposed than in control rats as reflected in the standard errors in Table 2. The results obtained with functional tests in the young rats are consistent with the rapid recovery in morphology. No consistent behavioral deficits in the CO-exposed offspring were found. The control values recorded for the tests in the present study agree well with means reported elsewhere (3, 23). The group of developmental tests in this study have been used with variable
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success in detecting functional damage to the developing nervous system after prenatal insults which result in morphologic defects. Both no behavioral effect (1) and behavioral changes (17, 18) have been reported from prenatal exposure of rats to antimitotic agents. However, in the absence of gross morphologic changes, profound delays in behavioral maturation are produced in animals made hypothyroid during the perinatal period (3). Thus morphologic changes have been reported in the absence of functional changes, as well as the converse situation. It seems unlikely that the failure to find behavioral changes in CO-exposed rats of this study can be attributed to the insensitivity of the tests used to detect behavioral changes, although this possibility cannot be completely dismissed. The tests used in the present study involved assessments of locomotor activity and motor function. Experimental studies in adult animals have established the involvement of the caudate-putamen in motor control. Hyperactivity is the most commonly reported effect of basal ganglia lesions in animals. Hyperactivity as recorded in running wheels, open field environments and residential mazes has been reported to follow experimental lesions of the substantia nigra, globus pallidus, and caudate-putamen (4, 8, 14, 25). Because the CO-exposed animals in the present study suffered caudate damage, it was reasoned that tests of sensory-motor coordination and motor function would be the type of behavioral evaluation most likely to detect changes in central nervous system function. However, in spite of the structural abnormalities, behavioral changes in these animals were minimal. Although striking behavioral changes were not detected in the CO-exposed rats of this study, functional deficits might be displayed under other circumstances of environmental challenge, such as occur during aging. REFERENCES 1. BRUNNER, R. L., M. MCLEAN, C. V. VORHEES, AND R. E. BUTCHER. 1978. A comparison of behavioral and anatomical measures of hydroxyurea induced abnormalities. Teratology 18: 379-384. 2. COMER, C. P., AND S. NORTON. 1981. Persisting effects of perinatal hypothyroidism on neuronal morphology and motor behavior in rats. Toxicologist 1: 57. 3. COMER, C. P., AND S. NORTON. 1982. Effects of perinatal methimazole exposure on a developmental test battery for neurobehavioral toxicity in rats. Toxicol. Appl. Pharmacol. 63: 133-141. 4. COSTALL, B., R. J. NAYLOR, AND J. E. OLLEY. 1972. On the involvement of the caudateputamen, globus pallidus and substantia nigra with neuroleptic and cholinergic modification of locomotor activity. Neuropharrnacology Ii: 3 17-330. 5. CULVER, B., AND S. NORTON. 1976. Juvenile hyperactivity in rats after acute exposure to carbon monoxide. Exp. Neurol. 50: 80-98. 6. DAUGHTREY, W. C., AND S. NORTON. 1982. Morphological damage to the premature fetal rat brain following acute carbon monoxide exposure. Exp. Neural. 78: 26-37.
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24. WATTERSON, R. L. 1965. Structure and mitotic behavior of the early neural tube. In R. L. DE HAAN AND H. URSPRUNG, Eds., Organogenesis. Hoh, Reinhart and Winston, New York. 25. WHITIER, J. R., AND A. ORR. 1962. Hyperkinesia and other physiologic effects of caudate deficit in the adult albino rat. Neurology 12: 526-539.