Early phenobarbital-induced alterations in hippocampal acetylcholinesterase activity and behavior

Developmental Brain Research, 22 (1985) 113-123 Elsevier

113

BRD 50265

Early Phenobarbital-Induced Alterations in Hippocampal Acetylcholinesterase Activity and Behavior NURIT KLEINBERGER and JOSEPH YANAI

Department of Anatomy and Embryology, The Hebrew University Hadassah Medical School, 91010 Jerusalem (Israel) (Accepted March 12th, 1985)

Key words: acetylcholinesterase - - cerebellum - - cholinergic transmission - - hippocampus - - hippocampus-related behaviors - mouse - - phenobarbital - - prenatal exposure - - neonatal exposure

Early exposure to phenobarbital (PhB) causes marked destruction of large neurons which are then forming both in the hippocampus and in the cerebellum. Such exposure to PhB also reduces the achievements of mice in hippocampus-related behaviors such as radial 8-arm maze performance. Experimental evidence suggests that these behaviors are partially mediated by cholinergic transmission. We studied the performance of mice, exposed to PhB prenatally or neonatally, in radial 8-arm maze. Both treatments caused significant impairments in the animals' performance in the maze. Acetylcholinesterase (ACHE) and pseudocholinesterase (pChE) activities were studied in the hippocampus and cerebellum of mice who were exposed to PhB prenatally or neonatally. These enzymes are involved both in cholinergic transmission and in neuronal development. A significant decrease (13-16%, P < 0.01) in hippocampal AChE specific activity was found between days 15 and 22 in animals exposed to PhB neonatally. The total hippocampal activity of AChE was also greatly reduced (25-39%, P < 0.01) during that period as a result of both the reduction in specific activity and a reduction in hippocampal weight of the treated animals. These alterations were transient and were not detected in adulthood. No changes in hippocampal AChE or pChE activities were found in animals treated prenatally. Cerebellar AChE and pChE activities were not altered after prenatal nor after neonatal exposure to PhB. It is possible that the short-term effect of neonatal treatment on AChE specific activity might mediate the long-term impairments in hippocampus-related behaviors. INTRODUCTION

be one of the important neurotransmitters in the hippocampusl3, 34,36. Several studies suggest that cholin-

Previous findings in our laboratory suggested that early exposure to phenobarbital (PhB) causes numerous long term alterations in the CNS. Experiments concerning PhB effects in various brain struc-

ergic mechanisms might be involved in mediating hippocampus-related behaviors such as spontaneous alternation3.6 and radial 8-arm maze m,35,40. Acetyl-

tures including the hippocampus and cerebellum showed that prenatal treatment resulted in n e u r o n a l deficits of prenatally forming n e u r o n s 43, whereas

by many authors to take part in developmental processes in the CNS 5,7.11d8 in addition to their role in hydrolising the A C h molecule3L

neonatal treatment caused marked n e u r o n a l losses in neonatally as well as prenatally formed neurons 42. In addition, early exposure to PhB significantly impaired several behaviors including those which appear to be related to the hippocampus such as spontaneous and delayed spontaneous alternation 26 and radial 8-arm maze 27.

Previous experiments have indicated that when animals are treated with barbiturates, n u m e r o u s parameters of cholinergic transmission are being altered in various brain areas including the hippocampus23, 29.

C o m p o n e n t s of the cholinergic system might be involved in the mechanisms by which PhB exerts these long term effects. Acetylcholine (ACh) is believed to

choline and cholinesterases (ChEs) were suggested

Thus, we studied the possible effects of early PhB on the activity of ChEs, i.e., acetylcholinesterase (ACHE) and pseudocholinesterase (pChE) in the hippocampus and in the cerebellum of animals exposed to PhB prenatally or neonatally. Studying possible effects of early PhB exposure on ChEs activity

Correspondence: N. Kleinberger, Department of Anatomy and Embryology, The Hebrew University Hadassah Medical School, Box 1172, 91010 Jerusalem, Israel. 0165-3806/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

114 might contribute to our understanding of the mechanisms underlying the above-mentioned long term effects of early exposure to PhB. MATERIALSAND METHODS

Chemicals ACh-bromide and the pChE-inhibitor tetraisopropylpyrophosphoramide (iso-OMPA) were obtained from Sigma. [3H]ACh-iodide (67 mCi/mmol) was obtained from New England Nuclear. All salts and buffers were Analar grade.

Early exposure to PhB Adult HS/Ibg mice used as parents were housed in mating groups of one male and 4 females. Their offspring (the subjects of these experiments) received PhB prenatally via the placenta (PreB offspring) or neonatally via daily injections (NeoB). Prenatal administration. Females were checked daily and those that had mated, as evidenced by the existence of a vaginal plug, were housed with other pregnant females [day of plug is gestation day (GD) 1]. On GD 9 the females were placed in seperate cages. Treated females then received milled mouse food containing 3 g/kg PhB in acid form (their only food source) and water, both available ad libitum. Control females received milled food and water (PreC pups). Drug administration continued until GD 18 when the PhB and control diets were replaced with regular mouse pellets. In experiments involving fetal brain, transplacental administration of PhB was continued until GD 19. Neonatal administration. Female mice were housed with the males until pregnancy became apparent. At this time, they were moved to individual cages. After delivery (delivery day is day 1) the pups in each litter were divided into control and barbiturate-treated groups. Tail-tip cuttings were used for identification. Treated pups (NeoB) received a daily subcutaneous injection of 50 mg/kg sodium phenobarbital in sterilized water (10 ml vehicle/kg mouse) on days 2-21. Control pups (NeoC) received vehicle injections.

Behavioral experiments The test procedure in the radial 8-arm maze is described in detail elsewhere 25. Briefly, a week before

the experiment commenced, mice were put on a regimen of water deprivation that consisted of the administration of water for 30 min once a day. After a week, at age 50 days, the mice were introduced individually into the maze for 10 min of habituation without reinforcement of water. The first 16 entries were recorded. In the following 5 days of the test the mice received reinforcement with water drops of 50 ul. Unlike the habituation day (day 0), the animals were left in the maze until they had either entered all the 8 arms, or until they had made 16 entries, whichever occurred first. Animals were considered to have reached criterion if there were 8 correct entries out of the first 8 entries for two consecutive days. Thus, it became possible to determine: (1) the number of trials needed to enter all arms. The maximum allowed was 16 entries for an animal that did not enter all arms through 16 trials. (2) The number of days it took to reach criterion-. Animals that reached criterion after day 1 (8 correct responses out of 8 trials in habituation day + day 1) received the score 0. Animals that reached criterion after day 2 received 1, etc. Animals that did not reach criterion during the 5 days received the score 5.

Preparation of brain tissue Prenatally and neonatally treated mice and 'their controls were sacrificed on postnatal days 8, 15, 22 and 50 by decapitation. Brains were rapidly removed, weighed and put on an ice dish with the hemispheres facing upwards. The cerebellum was separated at the level of the peduncles and weighed. The two hemispheres of the cortex were separated by cutting the commissures. Each cortical hemisphere was peeled in a caudal-rostral direction and the hippocampus was revealed. It was then gently peeled away from the fornix-fimbria. The two hippocampi were weighed together. All tissues were stored at -70 °C until use. In experiments involving fetal brains, fetuses were removed from the mothers on GD 19 and whole brains were dissected out, weighed and frozen in -70 °C. Two adult male mice (used to establish adult levels of whole brain ChEs activity) were sacrificed and whole brains were dissected, weighed and frozen. Cerebelli and fetal whole brains were homogenized in 30 vols. of 1 M NaCI, 1% Triton X-100, 0.1 M Tris chloride, pH 7.0, for 7 s using MSE sonicator.

115 Hippocampi were homogenized in 40 vols. of the same buffer for 7 s in the same sonicator. The homogenates were centrifuged for 10 min in an Eppendorf 5414 centrifuge and the supernatant was used in the assay. Cholinesterase assay

Radiometric assays were performed according to the method of Johnson and Russell 12. Reaction mixtures in a 5-ml scintillation vial were composed of 30 pl of homogenate supernatant, 10/tl of 1.2 M NaC1, 0.5 M Tris HC1, pH 7.0, 10/4 of 100 nM radioactive ACh (made by diluting [3H]ACh into 100 nM AChbromide in order to yield 25,000-30,000 cpm total counts in each sample) and double distilled water to a final volume of 110/tl. Adding the radioactive substrate determined the time of reaction initiation. Triplicates were taken from each supernatant for measuring ChEs activity (ACHE + pChE) in the absence of inhibitors. Three other samples were taken for determining AChE activity only. These triplicates were incubated in room temperature for 30 min (in the case of cerebellar homogenates) and 40 min

(in the case of hippocampal homogenates) prixor to reaction initiation in the presence of iso-OMPA (a specific inhibitor of pChE) at final concentrations of 10 -4 M. Preincubation was used to ensure that covalent binding of the inhibitor to pChE was completed prior to reaction initiation. Assays were run at room temperature for 20, 30 and 40 min (in experiments with hippocampi, cerebelli and fetal whole brains respectively). The reaction was terminated by adding 100pl of stopping mixture (1 M chloroacetic acid, 0.5 M NaOH, 2 M NaCI). Scintillation fluid (4 ml of 90% toluene, 10% isoamylalcohol, 4 g 2,5 diphenyloxazole (PPO), 50 mg 1,4 bis[5-phenyl-2-oxazolyl]benzene-2,2'-p-phenylene-bis[5-phenyloxazole] (POPOP)), was then added. Samples were counted in Packard Tri Carb Liquid Scintillation Spectrometer. Protein was determined by the procedure of Lowry et a1.15. Results of enzymatic specific activity were expressed in nM/mg tissue/min or in nM/mg prot./min. In order to obtain whole tissue activity levels, the specific activity of each tissue as expressed in nM/mg tissue/min was multiplied by the tissue's weight in mg. Data was analyzed by analysis of variance

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116 RESULTS

TABLE l

Phenobarbital concentrations in the treated mother, her fetuses and treated neonates were described before 42,43. Briefly, blood PhB concentration in the

Number of days until learning criterion was reached (mean _+ S.E.M.) Numbers in parentheses represent sample sizes.

pregnant mice and in their fetuses averaged 113 _+ 12 ug/ml (mean + S.E.M.) during most of the PhB feeding period. PhB clearance rate in neonates was accelerated during the period of treatment. O n day 2, PhB brain levels (45 pg/g) remained almost unchanged

Control PhB

Prenatal exposure

Neonatal exposure

1.8 _+0.5 (14) 4.6 + 0.2*** (11)

1.8 -+ 0.5 (13) 3.3 -+ 0.5* (12)

* P < 0.05, *** P < 0.001 for the differences from control levels (ANOVA41).

throughout the 24 h postinjection period. O n day 10 no PhB remained in brain at 24 h postinjection. O n day 20 PhB brain concentration was a third of the peak concentration on days 2 and 10. No PhB was found in the brain at 4 h postinjection.

NeoB animals were 1.8 x slower than NeoC animals in reaching criterion (P < 0.05). Biochemical experiments

Behavioral experiments Fig. 1 shows the n u m b e r of trials needed by control and treated animals to enter all arms either after prenatal or neonatal exposure to PhB. Significant differences between PreB and PreC animals were found through the whole experiment and ranged between 24% and 48% (P < 0.01). Differences between NeoB and NeoC animals ranged between 28% and 48% (P < 0.05) and were significant on days 0, 3 and 4 of the experiment. N u m b e r of days until learning criterion was reached is presented in Table I. PreB animals were 2.6 × slower than PreC animals in reaching criterion (P < 0.001).

Effects o f prenatal admin&tration. Scores of body weight, brain weight, cerebellar and hippocampal weights of PreB and PreC animals are presented in Table II. On day 22 there was a 10% reduction (P < 0.05) in the weight of PreB animals as compared to PreC animals. At this age, brain weight of PreB animals was reduced by 8% (P < 0.001). Cerebellar weight of PreB animals was significantly reduced on day 50 (9%, P < 0.01). The data on specific activities of ChEs (ACHE + pChE) and A C h E only are presented in Fig. 2 (hippocampus) and in Fig. 3 (cerebellum).

TABLE II Effects of prenatal administration of PhB on body, brain, cerebellarand hippocampal weights Results are expressed in g, means ± S.E.M. GD 19 Body weight PreC PreB

Day 8

Day 15

1.14_+0.05 (10) 4.181±0.109(27) 6.483±0.339(13) 1.21 ±0.02 (9) 4.331_+0.126(29) 7.092_+0.289(11)

Day 22 9.248_+0.297 8.344±0.318'

Day 50 (24) 19.923 ± 0.826 (18) (17) 20.278 ± 0.457 (28)

Brain weight PreC PreB

0.069 ± 0.003 (10) 0.257± 0.005 (26) 0.359± 0.008 (13) 0.402± 0.006 (14) 0.073 +_0.002 (9) 0.262± 0.005 (31) 0.379± 0.007 (11) 0.370± 0.007*** (16)

0.429 ± 0.010 (15) 0.424 ± 0.006 (25)

Cerebellar weight PreC PreB

-

Hippocampal weight PreC PreB -

0.020 _+0.001 (26) 0.038± 0.001 (13) 0.043± 0.001 0.018 ± 0.001 (31) 0.042± 0.001 (11) 0.041 ± 0.001

(31) (27)

0.056 ± 0.001 (22) 0.051 ± 0.001"*(32)

0.014 ± 0.001 (21) 0.017_+0.001 (13) 0.012 ± 0.001 (27) 0.016± 0.001 (11)

(28) (24)

0.018± 0.001 (21) 0.019± 0.001 (28)

0.016 ± 0.001 0.016 ± 0.001

* P < 0.05. ** P < 0.01. *** P < 0.001. See Table I for the explanations of the symbols.

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118 TABLE III

TABLE IV

Enzymatic activity per whole hippocampus following prenatal treatment

Enzymatic activity per whole cerebellum following prenatal treatment

Results are expressed in nM/min, means ± S.E.M.

Results are expressed in nM/min, means ± S.E.M.

Whole cerebellar activity of ChEs (AChE and pChE)

Whole cerebellar activity of A ChE

Whole hippocampal activity of ChEs (A ChE and pChE)

Wholehtppocampal activity of A ChE

Day8 PreC PreB

16.506+1.386 (18) 13.330± 0.691" (23)

14.036±1.303(13) 12.586± 0.856 (13)

Day8 PreC PreB

21.940 ± 1.651 (22) 22.88I ± 1.877 (28)

20.131 ± 1.634 (7) 21.847 ± 0.289 (9)

Day 15 PreC PreB

42.499 ± 4.617 (10) 38.260 _+ 2.233 (10)

36.241 ± 3.777 (10) 31.863 ± 1.849 (10)

Day 15 PreC PreB

71.317 ± 3.626 (10) 77.257 ± 3.225 (11)

60.040 ± 3.144 (10) 65.549 ± 2.956 (11)

Day 22 PreC PreB

60.867 ± 4.941 (19) 56.017 ± 5.363 (19)

46.093 ± 3.486 (11) 41.688 ± 3.615 (12)

Day22 PreC PreB

110.446 ± 4.130 (19) 106.123 ± 3.153 (24)

90.674 ± 4.446 (9) 89.542 ± 2.616 (10)

Day50PreC PreB

76.354 ± 4.821 (14) 92.804 ± 5.096* (20)

65.411 ± 5.393 (10) 69.583 ± 5.951 (10)

Day50 PreC PreB

115.015 ± 4.493 (18) 103.047 ± 2.487* (23)

94.507 ± 5.052 (9) 81.958 ± 2.617" (10)

* P < 0,05. See Table I for the explanation of the symbols.

* P < 0,05. See Table I for the explanation of the symbols. U n l i k e the results of n e o n a t a l l y t r e a t e d animals,

specific activity levels d e c r e a s e d by 13% until the an-

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imal m a t u r e d .

animals at any of the ages e x a m i n e d e i t h e r in the hip-

about 85% of C h E s activity at all ages.

p o c a m p u s or in the c e r e b e l l u m .

T h e results of w h o l e - t i s s u e activity are p r e s e n t e d in T a b l e s III ( h i p p o c a m p u s ) and I V ( c e r e b e l l u m ) , A

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A C h E activity c o m p r i s e d a b o u t 85% o f C h E s activity

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b o t h the h i p p o c a m p u s and the c e r e b e l l u m .

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T h e r e are no changes in h i p p o c a m p a l A C h E activ-

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TABLE V

Effects of neonatal administration of PhB on body, brain, cerebellarand hippocampal weights Results are expressed in g, means ± S.E.M.

Day 8

Day 15

Day 22

Day 50

Body weight NeoC NeoB

4.215 ± 0.105 3.847 ± 0.138"

Brain weight NeoC NeoB

0.261 _+ 0.004 (12) 0.236 ± 0.005*** (11)

0.368 ± 0.005 (9) 0.333 + 0.009** (10)

0.391 ± 0.007 (9) 0.357 ± 0.006*** (13)

0.450 ± 0.019 (4) 0.380 ± 0.017" (4)

Cerebellar weight NeoC NeoB

0.018 ± 0.001 0.019 ± 0.002

(13) (11)

0.042 ± 0.002 0.038 ± 0.002

(8) (10)

0.046 ± 0.002 (14) 0.038 ± 0.001"** (14)

0.059 ± 0.002 (21) 0.051 ± 0.002**(24)

Hippocampal weight NeoC NeoB

0.013 ±0.001 0.011 ± 0.001

(11) (10)

0.019±0.002 0.016 ± 0.001

(9) (10)

0.014±0.001 (15) 0.010 ± 0.001"* (13)

0.017 ± 0.001 0.016 ± 0.001

(14) (12)

6.660 ± 0.373 5.598 ± 0.436

(8) (9)

9.646 ± 0.680 7.738 ± 0.398*

* P < 0.05, ** P < 0.01, *** P < 0.001. SeeTable I for the explanation of the symbols.

(8) (13)

21.792 ± 0.689 21.633 ± 2.028

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120 0.05, Table IV). This decrease results from the significant decrease in cerebellar weight (9%, P < 0.01), which is observed in PreB animals at this age (Table II). Effects o f neonatal administration. Table V presents scores of the weights of body, brain, cerebellum and hippocampus of neonatally treated animals. NeoB animals had reduced body weights on days 8 (9%, P < 0.05) and 22 (20%, P < 0.05). A constant decrease of 10-16% was found in brain weights of NeoB animals during all ages (P < 0.01). Cerebellar weight was decreased in NeoB animals on days 22 (17.4%, P < 0.001) and 50 (13.5%, P < 0.01). Hippocampal weight was decreased by 28.5% (P < 0.01) in 22-days-old NeoB animals. Figs. 4 and 5 show the specific activity of ChEs and of A C h E only in the hippocampus (Fig. 4) and in the cerebellum (Fig. 5). General patterns of enzymatic activity development resembled the patterns observed in prenatal experiments. Specific activity of A C h E in the hippocampus was significantly decreased in NeoB animals during the period of treatment. A reduction of 16.3% (P < 0.001) was found on day 15. On day 22 there was a reduction of 13% (P < 0.01). The same percentage of reduction was found in specific activity of ChEs. No differences were found in values of p C h E activity in all ages (results not shown). Thus, the differences in specific activity of ChEs reflect solely the differences in specific activity of ACHE. None of these changes persisted until day 50. In the cerebellum there were no differences in enzymatic activity of NeoB and NeoC offspring during all ages. Activity values per whole tissue after neonatal treatment are presented in Tables VI (hippocampus) and VII (cerebellum). Whole hippocampal enzymatic activity was significantly decreased during the time of treatment. On day 15 there was a 25% reduction (P < 0.05) in whole hippocampal A C h E (and thus ChEs) activity. On day 22 hippocampal A C h E and ChEs activity values were reduced by 39% (P < 0.01). The decrease on day 22 reflects the additive effects of the reduction in hippocampal weight (28.5% as indicated in Table V) and the reduction in specific activity (13% as indicated in Fig. 4) on values of whole hippocampal enzymatic activity. No differences between treatments were found in adult animals.

TABLE VI Enzymatic activity per whole hippocampus following neonatal treatment

Results are expressed in nM/min, means _+S.E.M. Whole hippocampal activity of ChEs (ACHE and pChE)

Whole hippocampal activity of A ChE

Day8 NeoC NeoB

16.176_+ 1.382 12.841_+ 1.218

(8) (9)

14.098_+1.195 (8) 11.165+_ 1.067 (9)

Day 15 NeoC NeoB

48.752_+4.250 (9) 36.530_+3.110" (9)

41.328+ 3.932 (9) 30.949+ 2.291" (9)

Day 22 NeoC NeoB

51.345+ 4.686 (9) 31.483+_3.055** (9)

43.182_+4.037 (9) 26.461_+2.700** (9)

Day 50NeoC NeoB

73.028_+ 3.917 (10) 61.118_+ 3.977 (10) 76.466+ 5.703 (10) 64.163 + 4.258 (10)

* P < 0.05, ** P < 0.01. See Table I for the explanation of the symbols.

TABLE VII Enzymatic activity per whole cerebellum following neonatal treatment

Results are expressed in nM/min, mean + S.E.M. Whole cerebellar activity of ChEs (,4 ChE and pChE)

Whole cerebellar activity of A ChK

Day 8 NeoC Neo B

22.728_+ 1.834 (9) 18.681_+ 1.404 (9) 26.759_+4.874 (10) 22.989_+3.927 (10)

Day 15 NeoC NeoB

74.025+ 2.537 (7) 74.335_+4.531 (8)

63.414+ 2.602 (6) 62.645_+4.066 (8)

Day22NeoC NeoB

107.697+ 4.391 (11) 93.251 + 4.828 (11) 92.055+ 4.056* (12) 81.363+ 3.855 (12)

Day50NeoC NeoB

115.783_+4.562 (12) 93.733_+3.842 (6) 102.159+ 3.212" (13) 86.102_+2.291" (13)

* P < 0.05. See Table I for the explanation of the symbols. Differences in the cerebellum between NeoB and NeoC animals were found on days 22 and 50. On day 22 there was a reduction of 14.5% (P < 0.05) in whole cerebellar activity of ChEs. Respective values of whole cerebellar A C h E activity did not reach significance. A reduction of 12% (P < 0.05) was found in whole cerebellar ChEs activity and A C h E activity of 50-days-old NeoB. The decrease in whole-tissue activity results from the reduction of cerebellar weight in NeoB animals (Table V).

121 DISCUSSION Prenatal as well as neonatal exposure to PhB resulted in significant impairment of the treated animals' performance in the radial eight-arm maze as compared to controls. Neonatal exposure to PhB significantly reduced the hippocampal A C h E specific activity of animals aged between 15 and 22 days. This transient reduction was not found on day 50. In contrast, no differences in A C h E or pChE specific activity were found in hippocampi of prenatally treated animals. The specific activity of cerebellar ChEs was not altered after prenatal nor after neonatal treatment at any of the ages examined. However, ChEs activity per whole cerebellum was significantly reduced on day 50 after both prenatal and neonatal treatments, due to the reduction in cerebellar weight after these treatments. ChEs activity per whole hippocampus was reduced between days 15 and 22 after neonatal exposure to PhB but remained normal after the prenatal treatment. The changes in hippocampal specific activity of AChE may possibly mediate the long-term behavioral effects observed in animals which were exposed to PhB. ChEs are involved in two different phenomena in the CNS: (a) cholinergic neurotransmission - - ChEs hydrolize ACh molecules in the synaptic cleft32; (b) possible involvement in neuromorphological development - - a close temporal correlation exists between the formation of neural tube and neural crest cells and the appearance of ChEs activity in these forming areasa.5,7,11A8,19. Whereas only transient ChEs activity is evident in developing cerebellar Purkinje cells 1, hippocampal ChEs activity is found throughout life beginning on the third postnatal day 16,20. Thus, the cerebellum might serve as a suitable example for a possible PhB influence on the suggested 'developmental functions' of ChEs, whereas PhB effects in the hippocampus might interfere with both functions of ChEs. Previous results from our laboratory show that, like the effects of early PhB exposure on A C h E activity, behavioral impairments after neonatal exposure to PhB are also greater than after prenatal treatment. Pick and Yanai26 tested prenatally and neonatally treated mice for spontaneous and delayed spontaneous alternation in a T-maze. Part of their results are cited in Table VIII.

TABLE VIII Alternations percent of prenatally 42-day-old mice

and neonataUy treated

Adopted from C. G. Pick and J. Yanai 1984 (ref. 26); mean ± S.E.M.

PreC PreB NeoC NeoB

Percent alternations on day I in spontaneous alternation

Percent alternations on day I in delayed spontaneous alternation

79 ± 4 (26) 69±4 (22) 75 ±7 (7) 48 ± 8** (14)

88 ± 4 (15) 61±4"** (15)

** P < 0.01, *** P < 0.001. See Table I for the explanation of the symbols. While NeoB animals had reduced levels of alternation compared to controls in both tests, it took the more sensitive test - - delayed spontaneous alternation - - to detect behavioral impairments in PreB animals. Various experimental procedures which include application of cholinergic agonists and/or antagonists i.p.3,6,10,35,4° or to the lateral ventricle 39, lesion experiments aimed at the cholinergic septohippocampal pathway 24 and transplantation experiments applying septal transplants to lesioned ratsS, 14 strongly suggest an important role of cholinergic transmission in mediating hippocampus-related behaviors. In view of this evidence it appears likely that the short-term effect of neonatal treatment - - namely the transient but significant reduction in AChE levels of NeoB animals during days 15 to 22 - - might mediate the long-term impairments in the above-mentioned hippocampus-related behaviors. Prenatal treatment also causes milder behavioral impairments. Although no differences in hippocampal AChE levels were found in PreB animals compared to controls at all ages, it should be remembered that enzymatic activities in earlier ages than GD 19 were not determined. However, a transient reduction in AChE level might have occurred earlier during prenatal hippocampal development (which starts on G D 102). Indeed autoradiographic studies have demonstrated a marked reduction in the number of hippocampal pyramidal labelled cells of PreB fetuses on GD 1344. It is possible that a concomitant reduction in hippocampal AChE activity occurred, but this possibility has not yet been studied. Possible effects of barbiturates on ChEs activity

122 were studied in animals who had acute or chronic exposure to these drugs in adulthood, and no changes in A C h E activity were found 1721,38. Thus, it seems that the transient reduction observed by us is unique in that it appears in periods of CNS development and

or neonates during the time of exposure. A concomitant increase in A C h E activity is observed in the neonates 28. This completely differs from what is known about barbiturate effects on parameters of cholinergic transmission.

maturation only. Yet it seems unlikely that transient reduction in A C h E activity mediates the neuronal losses as well as the behavioral impairment. Early ex-

Barbiturates were shown to inhibit K-stimulated release of endogenous A C h from slices of various brain areas including the hippocampus30, 3l. High-af-

posure to PhB destroys cerebellar and hippocampal neurons 42.43. Although n e u r o n a l loss in the cerebel-

finity choline uptake ( H A C U ) - - one of the factors determining the rate of A C h synthesis - - was inhibited in hippocampal synaptosomes in vitro after in

lum after both prenatal and neonatal exposure is much more severe than in the hippocampus, no changes in cerebellar AChE-specific activity are detected after either prenatal or neonatal treatments at any of the ages observed. Cholinergic systems are involved in the mechanisms of other sedative hypnotics, e.g., alcohol. Acute alcohol administration results in a decrease in ACh release both in vivo and in vitro, whereas it has no effects on the activities of A C h E or cholinacetyltransferase or on brain choline levels 37. Chronic alcohol administration, which results in ethanol tolerance, reduces the inhibitory effect of ethanol on electrically stimulated release of A C h 37. Brain ACh levels are decreased during and after chronic

vivo pretreatment of rats with p e n t o b a r b i t a P 3. A n increase in the n u m b e r of muscarinic binding sites was found in various brain areas during the period of abstinence after long-term barbital administration22,23. Those experiments were conducted in adult animals. Prenatal or neonatal exposure to barbiturates may cause different p h e n o m e n a . Still changes in H A C U , ACh release or receptor populations might take place during early exposure to PhB. Further studies should determine if such changes occur in the hippocampus and whether they are transient or persist to adulthood.

ethanol treatment, but no changes are observed in A C h E levels9-37. In contrast, prolonged ethanol con-

ACKNOWLEDGEMENTS

sumption by pregnant or lactating rats results in significantly reduced levels of ACh in brains of fetuses

We wish to acknowledge the assistance of Chaim Pick in the behavioral experiments.

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