Trichloroethylene affects learning and decreases myelin in the rat hippocampus

Neurotoxicologyand Teratology,Vol. 12, pp. 375-381. e Pergamon Press plc, 1990. Printed in the U.S.A.

0892-0362/90 $3,00 + .00

Trichloroethylene Affects Learning and Decreases Myelin in the Rat Hippocampus L. G. I S A A C S O N , S. A. S P O H L E R A N D D. H. T A Y L O R 1

Department of Zoology, Miami University, Oxford, OH 45056 R e c e i v e d 31 July 1989

ISAACSON, L. G., S. A. SPOHLER AND D. H. TAYLOR. Trichloroethylene affects learning and decreases myelin in the rat hippocampus. NEUROTOXICOL TERATOL l:t(4) 375-381, 1990.--The goal of the present study was to relate previously observed behavioral effects with changes in myelin in the hippocampus following exposure to the industrial solvent 1,1,2-trichloroethylene (TCE). Young adult rats exposed to TCE via their drinking water underwent tests to evaluate their ability to perform spatial navigational tasks, and their brains were examined for changes in myelin in the hippocampus. Exposure to an average daily load of 5.5 mg TCE first for 4 weeks and then to 8.5 mg for an additional 2 weeks (separated by a 2-week interval) resulted in an increased level of performance in spatial navigational tasks. Examination of the brains from these animals revealed a significant decrease in the amount of myelin in one layer of the hippocampus, the stratum lacunosum-moleculare. No increase in performance was observed in rats exposed to an average daily load of 5.5 mg TCE for 4 weeks only. A reduction in myelin was observed, however, in the stratum lacunosum-moleculare of these animals. This decrease was not as severe as that seen in the twice-exposed animals. The results of this study suggest that exposure to TCE results in a reduction of hippocampal myelin, and that this reduction may be related to the increased level of performance observed following a second exposure to TCE. Trichloroethylene

Spatial navigation

Myelin

Hippocampus

TRICHLOROETHYLENE (TCE), a chlorinated hydrocarbon (C2HC13), is used as an industrial degreaser, a solvent in the dry cleaning industry, and has been used previously as an anesthetic. The USEPA has estimated that, in 1984, the United States produced approximately 234,000 tons of TCE (30). Due to improper disposal techniques and subsequent release into groundwater from waste-disposal sites, TCE has become an increasingly common contaminant of human drinking water supplies (30). Until recently, TCE was known primarily as an occupational hazard. Following inhalation, TCE has been shown to cause central nervous system disturbances such as dizziness, lethargy, lightheadedness, headache, inability to concentrate, impairment of visual-motor response (32), trigeminal nerve impairment (2), pneumatosis cystoides intestinalis (24) and, in severe cases, coma and death (6). Among the reported behavioral effects resulting from inhalation exposure to TCE are a decrease in social activity (27) and swimming performance (9), an increase in exploratory behavior (4), and psychomotor slowing in a two-choice visual discrimination task (17). Inhalation exposure to TCE had no effect on seed-seeking ability of Mongolian gerbils in a symmetrical labyrinth, but a second TCE exposure to the same animals increased their ability to make correct choices (14,15). Following inhalation exposure, the brain is one tissue in which TCE uptake has been demonstrated (30). Biochemical changes which occur in the brain as result of this uptake include a decrease in brain RNA content (26), a decrease in soluble proteins (10) and

an increase in dopamine and norepinephrine synthesis (22). Baker (1) found that acute and chronic inhalation exposure to TCE affected Purkinje layer of the cerebellum in dogs. Similar central nervous system damage was observed in rabbits following IM administration of TCE (3). Other studies examining the effects of TCE on the central nervous system report a loss of myelin in the brainstem (5) and affected myelin sheaths in the temporal and occipital cortices in the anterior and lateral columns of the spinal cord (1). The hippocampus appears to be particularly vulnerable to inhalation exposure to TCE (1,10). Exposure via the drinking water also affects the hippocampus as evidenced by previous biochemical, histological, and behavioral studies in our laboratory. For example, prenatal and postnatal exposure to TCE via the dam's drinking water result in a decrease in glucose uptake in the rat pup hippocampus and cerebellum (21), an increase in exploratory behavior (29), and a decrease in myelin in the stratum lacunosum-moleculare of the dorsal hippocampus (13) and cerebellum (16). Also, using this same exposure paradigm, tests of the ability to perform spatial navigational tasks, a type of learning in which the hippocampus appears to be involved (7,19), revealed a facilitation or increased ability to learn in rats exposed to TCE during development (8). While previous studies examined the effects of pre- and postnatal exposure to TCE, the current study investigates the effects of TCE exposure via the drinking water on the hippocampus of the young adult rat. In the present study, our experiments

tRequests for reprints should be addressed to Douglas H. Taylor, Ph.D., Department of Zoology, Miami University, Oxford, OH 45056.

375

376

ISAACSON, SPOHLER AND TAYLOR

were designed to 1) determine the effects of exposure to TCE on spatial learning on young adult rats, 2) relate any changes in learning ability resulting from TCE exposure to structural changes in the hippocampus, and 3) compare the effects of TCE exposure on animals exposed once with the effects on those receiving a second exposure. METHOD

Subjects and Handling Procedures Male Sprague-Dawley rats from Charles Rivers Laboratory were received at 21 days of age. TCE dosing began on that day. Teklad Mouse/Rat diet (Winfield, IA) and distilled water (or water + TCE) were available ad lib. The holding room was maintained at 22°C on a 12:12 light:dark cycle.

Exposure Procedures In Experiment 1, 18 rats (21 days of age) were divided into 3 groups of 6 each and were randomly assigned to treatment groups. One group (nonexposed) received only distilled water for 8 weeks (21 days to 78 days) prior to behavioral testing. A second group (TCE1) received TCE for 4 weeks (21 days to 49 days) followed by distilled water for 4 weeks (50 days to 78 days). The third group (TCE2) received TCE for 4 weeks (21 days to 48 days), distilled water for 2 weeks (49 days to 62 days), and TCE for 2 weeks (63 days to 78 days). Experiment 2 duplicated this protocol, so that in total 36 rats were tested. This design yielded a total of 12 rats in each of the three treatment groups (six each from Experiment 1 and Experiment 2).

Trichloroethylene

1,1,2-Trichloroethylene (Mallinckrodt) was mixed initially in concentrations of 312 mg/1 in distilled water. The mixture was stirred for approximately 4 hours in a stoppered flask using a Teflon-coated stir bar and magnetic mixer. The TCE/water mixture was delivered to 250-ml dark brown bottles via a siphon in order to minimize volatilization. Distilled water for the nonexposed rats also was dispensed into 250-ml dark bottles. All bottles were changed approximately every 48 hr. Water consumption by the TCE1 rats averaged approximately 30 ml/day for 28 days. Consumption by TCE2 rats averaged approximately 30 ml/day for 28 days and 46 ml/day for the additional 14-day exposure period. The average TCE concentration in the bottles during any 48-hr period was determined by gas chromatography to be 185 mg/l. Total TCE loads were estimated using equation 1, and average daily loads were estimated using equation 2: CONS = DR (MCONC) NDAYS (1) AVDAY = DR (MCONC) (2) where CONS =estimation of total consumption (mg), D R = drinking rate (1/day), MCONC = average TCE concentration per 48 hours (mg/1), A V D A Y = average day load, and NDAYS = number of days in exposure period. The TCE1 rats received an average daily load of 5.5 mg TCE for 28 days, resulting in a total load of 154 mg TCE. TCE2 rats received an average daily load of 5.5 mg TCE for 28 days and 8.5 mg for 14 days, resulting in respective total loads of 154 mg and 119 mg TCE during the two exposure periods. While water consumption data were used from water containing TCE, it has been determined previously that no statistical difference in water consumption occurs between rats drinking TCE water at this concentration and distilled water (20).

Behavioral Assay All exposed rats (n = 36) were subjected to behavioral testing.

Daily testing began at the end of the exposure period in each experiment and took place for 14 consecutive days for a total of 28 days. So that each rat could be tested at approximately the same time each day, the 18 rats from each experiment were randomly divided into 3 subgroups of 6 each, consisting of 2 rats from each treatment group (i.e., 2 nonexposed, 2 TCE1, and 2 TCE2). These animals were tested daily within the same time period (1200-1600 hours). The Morris Swim Test (19), as modified by Whishaw (33), was used as the assay for evaluating the rats' ability to perform spatial navigational tasks. Briefly, a circular swim tank (118 cm in diameter and 59.5 cm high) was filled with water to a depth of 33 cm. A Plexiglas platform (15 c m x 15 cm) was submerged 2.5 cm below the water's surface at one of four locations. Location A was approximately 8 cm from the wall between the northeast and southeast quadrants. Location B was in the exact center of the tank while locations C and D were centered in the northwest and southwest quadrants, respectively. Approximately l0 ml of red food coloring was added to the water to obscure the platform. Four symbols (a vertical line, a horizontal line, a circle, and an " X " ) were placed at equidistant points on the pool wall. Their locations designated the 4 start positions. Eight experimental activity sets were conducted per subgroup per day, consisting of 2 trials each at each of the 4 start positions. In trial 1, a rat was given 120 sec to find the platform. If the rat succeeded, it was left on the platform for 5 sec; if it failed, the rat was placed on the platform by the experimenter for 5 sec. Using the same start position, trial 2 was initiated after a 5-sec rest period. Following trial 2, the rat was returned to its cage until all other rats in its subgroup had been tested at a particular start position. The rats then underwent additional sets of trials using the same protocol until all 4 start positions had been used. The entire sequence then was repeated, for a total of 16 trials/rat/day. Swim tests were videotaped using a JVC Saticon video-camera mounted over the swim tank and a Panasonic VHS Omivision videocassete recorder. The time required to find the platform (latency) was scored from videotape. Group differences were determined by analysis of variance (ANOVA), and mean separations were performed by the Bonferroni Multiple Comparison procedure (24).

Histology Within two days of the completion of behavioral testing, four animals from each experimental group were anesthetized with sodium pentobarbitol (35 mg/kg) and killed by aortic perfusion using a 10% formol-saline solution. Brains were removed, blocked in the coronal plane and stored in 10% formol-saline. In preparation for sectioning, brains were infiltrated with a mixture of 30% sucrose in 10% formol-saline. Frozen coronal sections (30 Ixm) were cut using a freezing microtome and were stored in 10% formol-saline until staining. Frozen sections containing the dorsal hippocampus were stained for myelin using a modification of the Heidenbaim procedure developed specifically for frozen sections (11). Sections were hydrated through a graded series of alcohols, rinsed, and placed in a mordant solution (2.5% ferric ammonium sulfate) for 30 minutes. Following a brief distilled water rinse, they were transferred to a hematoxylin staining solution for 1 hour. Sections were rinsed, mounted on subbed slides, dehydrated, cleared in xylene, and coverslipped. Myelinated fibers appeared blue to black against a white background and were easily visualized with a light microscope. Photographic montages of myelinated fibers located in the stratum lacunosum-moleculare of the dorsal hippocampus were

TCE AFFECTS LEARNING AND DECREASES MYELIN

377

30

14

~g A

~

control

0 0

12

20 A "O

0

10

0 0 @

41 10

O

@ 4.1 III

0

2

4

6

8

10

12

14

Days

t~ III

x

FIG. 1. Daily latency values for each treatment group.

made by photographing the fibers in a single plane of section at 100 x magnification in a series of overlapping photos and printing these photos on 8 × 10-in. photographic paper. Upon visual inspection, it was obvious that the number of myelinated fibers was reduced in TCE-exposed animals, yet systematic counts of the fibers were made to determine the extent of reduction. From each montage, four to nine quadrates were systematically marked off moving from the medial to lateral direction. An acetate grid (79 squares/cm) was placed over each quadrate, white squares (i.e., squares not containing myelinated fibers) were tallied, and the percentage of myelin coverage was calculated. Two sections per animal from each of four animals per treatment group were used in the quantification procedure. Counts of fibers, therefore, were obtained from a total of 24 photomontages (8 per treatment group). Data were subjected to analysis using ANOVA and the Duncan's Multiple Range Test (24). RESULTS

Spatial Navigational Tasks The time required to find the hidden platform (latency) each day during the 14-day testing period is shown in Fig. 1. These numbers represent an average of all latencies for the designated day. The TCE2 group outperformed both the TCE1 and nonexposed groups on Day 1 with a latency of 20 sec. While this latency decreased in subsequent days in all 3 groups, the TCE2 group continued to require less time to find the platform compared to TCE1 and the nonexposed group. This day effect was found to be significant using ANOVA, F(13,395)= 14.68, p = 0.0001. When the latency among the 3 groups was compared using ANOVA and the Bonferroni Multiple Comparison test (Fig. 2; Table 1), it was determined that animals receiving a second exposure of TCE found the hidden platform in significantly less time compared to the other two groups, F(2,395)= 14.19, p = 0 . 0 0 0 1 . This increased, or facilitated, performance in spatial navigational tasks was observed only in animals receiving a second TCE exposure.

Myelin in Hippocampus Stained coronal sections through the dorsal hippocampus of the nonexposed animals revealed a staining pattern similar to that observed previously in the adult rat (Fig. 3A). Heavily myelinated

Control

TCEI

TCE2

Treatment

FIG. 2. Mean latency ( ---S.E.) to fred the platform during the Morris swim test for nonexposed, TCE1 and TCE2 rats for 14 days. *Significance at p<0.05.

areas such as the internal capsule, cerebral peduncles, and optic tract stained dark blue. Other areas such as the corpus callosum and fornix also stained intensely for the presence of myelin. In the hippocampal formation, stained fibers were observed in the stratum lacunosum-moleculare, and to a lesser extent, in the hilus (Fig. 3A). Fibers were observed either in small groups or as individual strands which traversed several directions throughout the stratum lacunosum-moleculare (Fig. 4A). Stained fibers could be easily resolved for quantitation (Fig. 4A, B). Upon visual inspection of stained sections through the dorsal hippocampal formation from rats in both the TCE1 and TCE2 groups, myelinated areas such as the internal capsule, optic tract, and fornix appeared similar to nonexposed animals in staining intensity, numbers, and appearance (Fig. 3B). In the hippocampus, it was obvious that, in the TCE-exposed groups, a decrease or diminished staining was present in the stratum lacunosum-moleculare, particularly in the regio superior of the hippocampus proper

TABLE 1 p-VALUESRESULTINGFROM THE BONFERRONIMULTIPLE COMPARISONTEST COMPARINGTHE EFFECTSOF TCE ON LATENCY AMONGTREATMENTGROUPS(n = 12, EACH)

Nonexposed TCEI TCE2

Nonexposed

TCE 1

TCE2

-0.1111 0.0001 *

O. 1111 -0.0003*

0.0001' 0.0003* --

• Significance at p<0.05.

© < Z <

2

r~

© < <

O3

TCE AFFECTS LEARNING AND DECREASES MYELIN

379

FACING PAGE FIG. 3. (A) Photomicrograph of a frozen coronal section through the dorsal hippocampus which has been stained for the presence of myelin. Note the heavily myelinated regions such as the corpus callosum and fornix. In the hippocampus, stained myelinated fibers can be seen in the stratum lacunosum-moleculare and the hilus, with little staining elsewhere. CC, corpus callosum. F, fomix. G, granule cell layer. H, hilus. LM, str. lacunosum-moleculare. P, stratum pyramidale. Scale b a r = 0 . 2 mm. (B) Photomicrograph of a frozen coronal section depicting the appearance of myelinated fibers in the TCE2 rats. Myelinated tracts such as the fornix and corpus appear similar to that observed in the nonexposed animals. However, a reduction in the number of stained myelinated fibers is apparent in the stratum lacunosum-moleculare. For abbreviations, see above. Scale bar = 0.2 mm.

FIG. 4. (A) This high power photomicrograph, representing the rectangle in 3A, depicts myelinated fibers in the stratum lacunosum-moleculare and exemplifies a sample area which was used for quantification. Note the appearance of clumped as well as individual myelinated fibers in this region. Scale bar = 50 Ixm. (B) High power photomicrograph, representing the triangle in 3B, depicting the appearance of myelinated fibers in the stratum lacunosum-moleculare of a TCE2 rat. Note little clumping and reduced numbers of fibers when compared to the fibers observed in the nonexposed rat depicted in (A). Scale bar = 50 i~m.

380

ISAACSON, SPOHLER AND TAYLOR

TABLE 2 RESULTS OF DUNCAN'SMULTIPLERANGETEST COMPARINGTHE MEAN PERCENTCOVERAGEBY MYELINATEDFIBERSIN THE STRATUMLACUNOSUM-MOLECULAREOF THE DORSALHIPPOCAMPUS OF NONEXPOSED,TCE1, AND TCE2 RATS

Group

N

Number of Quadrates

Mean % (S.D.)*

Nonexposed TCE1 TCE2

8 8 8

49 34 43

97.06 (0.81 )A 93.52 (3.40) B 89.51 (4.13) c

*Means with the same letter are not significantly different at p<0.05.

(Figs. 3B, 4B). Fibers in the hilus, though not quantitated, appeared to be present in numbers similar to that observed in nonexposed animals. Though damaged fibers would be difficult to visualize at the light microscopic level, the myelinated fibers which were present appeared intact with no evidence of swelling or sheath breakdown. Results from statistical analyses of the number of fibers present in the stratum lacunosum-moleculare revealed a significant treatment effect, F(2,21)= 10.18, p = 0 . 0 0 0 8 . The Duncan's Multiple Range Test indicated that exposure to TCE significantly reduced the amount of myelin present compared to nonexposed rats. These results indicated that the number of stained myelinated fibers present in animals receiving the second exposure of TCE (TCE2) also was significantly less than in those receiving a single exposure (Table 2). DISCUSSION The present study was an attempt to relate, in the same animals, behavioral changes resulting from exposure to TCE with structural alterations in the hippocampus. Following tests designed to evaluate their performance in spatial navigational tasks, young adult rats that received a second exposure of TCE (TCE2) exhibited an increased level of performance, an effect also observed following TCE exposure during development (8). In addition, a diminished or reduced amount of stained myelin in one layer of the hippocampus, the stratum lacunosum-moleculare, was observed in brains examined from these same animals. These myelinated fibers in the hippocampus of young adult rats exposed to TCE via their drinking water are affected in a manner similar to that observed in rats exposed during development (13,28). Also, when compared to a single exposure, a second exposure to TCE apparently was sufficient to affect spatial learning and resulted in a more pronounced effect on myelin in the hippocampus. In their studies of seed-seeking behavior of gerbils following inhalation of TCE, Kjellstrand and his colleagues (15) found that a second exposure of a similar organic (1,1,1-trichloroethane) resulted in an increased ability to make choices. Their findings are similar to those reported here where observable changes in learning took place only following a second TCE exposure. It is possible that the decrease in myelin observed in TCEexposed animals may result from affected oligodendrocytes, the myelin-synthesizing cells of the central nervous system. Oligoden-

drocytes containing cytoplasmic vacuoles have been observed upon electron microscopic examination of the stratum lacunosummoleculare in the CA1 region of animals maternally exposed to TCE (Isaacson, unpublished). Vacuolation such as this indicates cell stress and may signify preliminary stages of cell death (23). Exposure to TCE may interfere with the metabolic processes of oligodendrocytes, rendering them incapable of synthesizing myelin in quantities necessary to carry out normal activities. While it is possible that these cells may be damaged in other areas of the brain, damage to oligodendrocytes in the stratum lacunosummoleculare of the hippocampus could affect the transmission of incoming impulses from the entorhinal cortex (34) and the synaptology of these cortical axons making contact with CA1 dendrites. Though it is difficult to ascertain what changes, if any, might result from an alteration in the input to the CA1 region, it is possible that a facilitation in learning might result from this structural alteration. In another study investigating the effect of lesions of hippocampal afferents on behavior in cats, a facilitated or increased performance was observed (12). The authors postulated that other, undamaged hippocampal afferents, which were suppressed under normal conditions, compensated for the loss, possibly by undergoing a type of synaptic rearrangement. Pharmacological studies have shown that synthetic benzodiazepines, agents which cause a reduction in arousal states, impair learning and memory (31). Conversely, benzodiazepine antagonists, which enhance arousal and anxiety, have been shown to enhance or facilitate learning (18). Because exposure to TCE during development also has been shown to result in a long-lasting increase in exploratory behavior (29), the facilitated performance observed in the present study may be accounted for by an increase in anxiety or arousal state. The results of the present study provide evidence that an alteration in myelinated fibers in the hippocampus could provide the anatomical basis, at least in part, for the enhanced performance observed following exposure to TCE. This alteration may aid in explaining the difference in the effects on behavior which result from single and double exposures to TCE. It may be that the alteration in myelinated fibers observed in TCE1 rats was not severe enough to affect spatial learning, resulting in no significant difference in latency, while the second exposure to TCE affected myelin severely enough to produce an observable behavioral effect. It is unknown at present why the enhanced learning occurred following the second TCE exposure. It may have been a consequence of the larger dose which the animals received. An alternate explanation for the effects observed in TCE2 animals is that a longer exposure period is required to produce observable changes in spatial learning. Long-term exposure studies and electron microscopic examination of the stratum lacunosum-moleculare will be necessary to determine the anatomical bases for the behavioral differences observed following TCE exposure. ACKNOWLEDGEMENTS We thank Drs. Craig Steele and James Otis, Department of Zoology, Miami University, for their helpful comments on the manuscript. This work was supported in part by a grant by the Ohio Board of Regents. This research also was supported in part by U.S. EPA Cooperative Agreement CR-8809618. The manuscript has not been subjected to the Agency's peer and administrative review and, therefore, does not necessarily reflect the views of the Agency and no official endorsement should be inferred.

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TCE AFFECTS LEARNING AND DECREASES MYELIN

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