Brain biochemical changes in rats treated with chlorpromazine and electroshocked during early postnatal development

BRAIN RESEARCH

403

BRAIN BIOCHEMICAL CHANGES IN RATS TREATED WITH CHLORPROMAZINE AND ELECTROSHOCKED DURING EARLY POSTNATAL DEVELOPMENT

ANTONIA VERNADAKIS Departments of Psychiatry and Pharmacology, University of Colorado School of Medicine, Denver, Colo. 80220 (U.S.A.)

(Accepted January 18th, 1972)

INTRODUCTION Numerous studies have shown that extrinsic stimulation during early periods of postnatal development affects adult behavior and physiology17. Furthermore, chlorpromazine, a CNS depressant, has been shown to attenuate sensory stimulus inputs 10. The present study further investigates this interaction of chlorpromazine and extrinsic stimulus input during early postnatal development of the central nervous system (CNS) of the rat. Electroshock was the stimulus used in this study. Previous studies have shown that electroshock stimulation during early periods of CNS maturation increases both DNA content and cholinesterase (ChE) activity in the cerebral cortex ~7. Thus, nucleic acid content and cholinesterase activity were two biochemical parameters also used in this study. MATERIALSAND METHODS Animals

Experiments were performed on Sprague--Dawley female rats. Sixteen litters, 8 females per litter, were used. Rats younger than 21 days were kept with their mothers. Drug treatment

Because of the generally long sojourn of chlorpromazine (CPZ) in the body 10, and the known low ability of immature hepatic tissue to metabolize drugs, single doses instead of chronic administration of this drug were used in this study. Chlorpromazine HC1 was prepared immediately prior to treatment and was kept in a bottle wrapped with aluminum foil to avoid deterioration as a result of exposure to light1°. The following treatments were used: (1) at 2 days after birth, 8 litters of Brain Research, 42 (1972) 403-411

404

A. V E R N A D A K I S

female rats, 6 rats per litter, received subcutaneously a single dose of CPZ, either 6 mg or 15 mg CPZ/kg body weight, or the vehicle (water). Each litter consisted of two control rats, two rats receiving 6 mg/kg CPZ and two rats receiving 15 mg CPZ; (2) at 13 days after birth 4 different litters received either 6 mg or 15 mg CPZ/kg body weight or the vehicle. No toxic effects were observed with these doses. These postnatal age periods of treatment were used because several studies have shown them to be critical periods of CNS development18,19. Electroshock procedure At 12 days after birth, the 8 litters of rats treated with CPZ or vehicle at 2 days after birth (CPZ-2 day) were further separated into two subgroups; one subgroup consisting of 4 litters was electroshocked beginning at 12 days after birth every 2 days up to 25 days of age, and every 5 days thereafter up to the end of the experimental period of 60 days of age; the remaining 4 litters were not electroshocked and served as controls. Similarly, the 4 litters of rats treated with CPZ or the vehicle at 13 days (CPZ-13 day) after birth were further separated into two subgroups; one subgroup consisting of two litters was electroshocked beginning at 18 days according to the same schedule as the earlier group. The remaining two litters were not electroshocked and served as controls. A non-shocked group treated with 6 mg/kg CPZ was not ineluded in the original experimental design. We have found subsequently (but not included here) that the values were not different from that treated with 15 mg/kg. Since no differences were found between non-shocked controls in the CPZ-2 day or CPZ-13 day groups the results were combined. Maximal electroshock seizures were produced by a stimulator delivering a current of 60 c/sec, for 0.2 see20. Stimulus intensity was 50 mA up to 21 days of age and 150 mA thereafter 1s,19. Brain samples The brain, excluding olfactory bulbs, was rapidly removed, stripped of grossly visible blood vessels, blotted free of moisture, and weighed, identical portions from both hemispheres of the cerebral cortex (grey matter), as well as the cerebellum were immediately removed, weighed and retained for nucleic acid, enzyme, and protein determinations. Nucleic acid extraction DNA was extracted according to the method of Schneiderla as modified by Gee1 and Timiras7. Tissue sp~mens were transferred to a l l - # s s tissue ~inders con' taining trichloroacetic acid (TCA, t 0 ~ w/v), h o m o g e ~ and left on ice f o r m min, Homogenates were centrifuged for 30 min in a refrigerated centrifuge. The TCAinsoluble fraction was subjected to su_,~__~ssivewashings of 5 ~ TCA and 95 ~ ethanol Brain Reaearch, 42 (1972) 403-411

CPZ AND SHOCKON BRAINBIOCHEMISTRY

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(2 times), then incubated and extracted with 5 % TCA at 90 °C for 15 rain. A final extraction was made with 5 % TCA. The supernatant fluids of the final two washings were combined and retained for nucleic acid analysis. D NA analysis DNA was analyzed by the diphenylamine procedure described by Burton 4 as modified by Geel and Timiras 7. The reagent, prepared fresh daily, consisted of 1.5 ml concentrated H2SO4/100 ml glacial acetic acid (redistilled), and 0.5 ml acetaldehyde (10 ml/500 ml HzO). Determinations were made on 1 ml portions of the nucleic acid extract to which was added 0.1 ml of 60 % HC104 (final concentration of HCIO4 = 0.54 M) and then 2 ml of the reagent. Color was developed by incubating for 16-20 h at 35 °C along with a suitable blank and standards of calf thymus DNA. The color was read at 600 nm in a Beckman DU spectrophotometer. Total DNA content was expressed as #g/mg wet tissue. Enzyme analysis Acetylcholinesterase (ACHE) and butyrylcholinesterase (BuChE) activities were determined colorimetrically by means of a Beckman DU spectrophotometer, using the rate of hydrolysis of the substrates, acetylthiocholine (AcTCh), and butyrylthiocholine (BuTCh), respectively, according to the method of Ellman et aLL The determination of enzyme activities was carried out at 37 °C. Homogenates consisted of 10 mg tissue/ml of 0.07 M phosphate buffer, pH 8.0, prepared with 0.07 M Na2HPO4 and 0.07 M KH2PO4. The final reaction mixture for determining AChE activity consisted of 2.6 ml pH 8.0 buffer, 0.4 ml homogenate, 100/zl (0.01 M) dithiobisnitrobenzoic acid (DTNB), and 20 #1 acetylthiocholine iodide (0.075 M). For determination of BuChE the selective AChE inhibitor 1,5-bis-(4-trimethylammoniumphenyl) pentan-3-1-diiodide (62c47) was used (made available through the generosity of Wellcome Laboratories). At a final concentration of 2 × 10-5 M, the AChE inhibitor has been shown to inhibit AChE activity 100%, but BuChE activity only slightly 2. The final reaction mixture for determining BuChE activity consisted of 1.2 ml pH 8.0 buffer, 0.8 ml homogenate, 0.5 ml 62c47 (6 × 10-5 M), 100 ~1 DTNB (0.01 M) and 0.5 ml butyrylthiocholine iodide (0.033 M). Enzyme activity was expressed as #moles of substrate hydrolyzed/min/g wet tissue. Protein analysis An aliquot from the same sample used for enzyme analysis was utilized for protein analysis by the Lowry method 12 with bovine serum albumin used in the standard. Calculations of total protein based on this determination were expressed as mg/g wet tissue. Brain Research, 42 (1972) 403-411

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A. VERNADAKIS

Statistical methods To determine whether the means of the parameters measured in controls and CPZ-treated rats differed significantly, the t test for nonpaired data was applied 6. RESULTS Cerebral cortex: enzyme activity (Table I) Both AChE and BuChE activities were significantly higher in the control rats which were shocked beginning at 12 days, but only BuChE activity was higher in the control rats shocked from day 18 as compared to non-shocked controls. No changes were observed in AChE activity between non-shocked control and non-shocked plus CPZ-treated groups, whereas BuChE activity was significantly lower in the CPZ-treated groups. The activity of AChE was lower i n t h e shocked plus CPZ-treated rats at 2 days but not at 13 days as compared to the appropriate shocked controls, whereas no differences were found in BuChE activity. Cerebellum: enzyme activity (Table I) As in the cerebral cortex, a higher cerebellar BuChE activity was observed in shocked controls as compared to non-shocked controls. Except for a decrease in BuChE activity in shocked rats which had been treated with 6 mg CPZ/kg at 13 days, no differences were found among control and CPZ-treated groups. Cerebral cortex: protein content (Table I) Protein content was significantly higher in shocked control groups as compared to non-shocked controls and this is in agreement with previous observations 17. Protein content was significantly lower in non-shocked CPZ-treated rats at 2 days but not at 13 days as compared to non-shocked controls. Also, protein content was significantly lower in shocked CPZ-treated rats with 6 mg/kg at 13 days as compared to appropriate shocked controls. Cerebellum: protein content (Table I) Protein content was higher in shocked control rats beginning at 18 days but not at 12 days. Protein content was not affected by CPZ treatment at 2 days of age. When CPZ was given to rats at 13 days protein was lower in the non-shocked as compared to non-shocked controls and in shocked treated with 6 mg/kg CPZ as compared to their appropriate shocked controls. Cerebral cortex: D N A content (Table II) No changes were observed in D N A content between any of the groups studied. Brain Research, 42 (1972) 403--411

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TABLE I CHANGES IN CHOLINESTERASE ACTIVITY IN THE CEREBELLUM AND CEREBRAL CORTEX OF 6 0 - D A Y - O L D RATS AFTER EARLY POSTNATAL CHLORPROMAZINE TREATMENT AND ELECTROSHOCK

Treatment*

#moles AcTCh hydrolysed/ minis wet tissue

I~molesBuTCh hydrolysed/ minis wet tissue

Protein (mg/g wet tissue)

Cerebral cortex

Cerebellum

Cerebral cortex

Cerebral cortex

3.94 4- 0.06 3.59 4- 0.11

0.46 4- 0.025 0.68 4- 0.02 0.33 4- 0.05 0.56 4- 0.03 (< 0.05)** 0.34 4- 0.04 0.73 4- 0.03 ( < 0.05)**

98 4- 3 115 4- 1 90 4- 2 114 4- 2 ( < 0.05)** 82 4- 2 119 4. 2 ( < 0.001)**

5.17 4- 0.21 4.30 4- 0.18

0.14 4- 0.03 0.72 4- 0.04 (< 0.001)**

99 4- 6

105 4- 0.5 (<0.001)**

7.864-0.61 4.154-0.13 (< 0.001)**

0.824-0.06 0.794-0.02 (< 0.001)** (< 0.001)**

1094-1 ( < 0.01)**

1184-2

6.35 4- 0.26 4.46 4- 0.20 (0.05-0.1)*** 5.91 4- 0.16 4.30 4- 0.20 (< 0.02)***

0.75 4- 0.02

0.79 4- 0.02

110 4- 2

118 4- 8

0.79 4- 0.003 0.76 4- 0.03

109 4- 1

118 4- 2

Shocked Control

5.80 4- 0.61 4.03 4- 0.23

0.92 4- 0.09 0.77 + 0.03 (< 0.001)** (< 0.001)**

119 4- 2 121 4- 2 (< 0.001)** (<0.05)**

CPZ-13 day 6 mg

6.28 4- 0.72

4.29 4- 0.18

0.80 4- 0.05

15 mg

5.55 4- 0.56

4.36 4- 0.16

108 4- 5 113 4- 1 ( < 0.01)*** (<0.05)*** 117 4- 2 120 4- 3

Non-shocked Control CPZ-2 day 5.09 -4- 0.22 6 mg 4.66 4- 0.19 15 mg CPZ-13 day 15 mg

Shocked Control CPZ-2 day 6 mg 15 mg

4.38 4- 0.25

3.92 4- 0.01

Cerebellum

0.50 4- 0.04 ( < 0.01)*** 0.86 4- 0.05 0.85 4- 0.05

Cerebellum

* Chlorpromazine HCI (6 mg or 15 mg/kg body weight) was administered subcutaneously to female rats either at 2 days or 13 days of age (CPZ-2, CPZ-13). Each group was subdivided into non-shocked and shocked groups. ** Numbers in parentheses represent P values for comparison to non-shocked controls. *** Numbels in parentheses represent P values for comparison to appropriate shocked controls.

Cerebellum: D N A content ( T a b l e II) As with A C h E a n d B u C h E activities, D N A c o n t e n t was significantly higher in all shocked c o n t r o l g r o u p s as c o m p a r e d t o the n o n - s h o c k e d c o n t r o l g r o u p . N o differences were observed in D N A c o n t e n t b e t w e e n n o n - s h o c k e d c o n t r o l an d n o n - s h o c k e d C P Z - t r e a t e d animals. H o w e v e r , D N A c o n t e n t was significantly l o w er in all shocked C P Z - t r e a t e d g r o u p s as c o m p a r e d to a p p r o p r i a t e shocked controls, a l t h o u g h n o t statistically significant in 15 m g C P Z - 2 day.

Brain Research, 42 (1972) 403-411

408 TABLE

A. VERNADAKIS II

CHANGES IN

DNA

CONCENTRATION IN THE CEREBRAL CORTEX AND CEREBELLUM OF 6 0 - D A Y - O L D RA'[S

AFTER EARLY POSTNATAL CHLORPROMAZINE TREATMENT AND ELECTROSHOCK

Treatment *

DNA (mg/g wet tissue) Cerebral cortex

Cerebellum

1.15 ± 0.04

6.27 4- 0.12

1.17 4- 0.07 1.13 4- 0.04

6.26 ± 0.12 6.61 4- 0.14

1.17 4- 0.04

5.95 4- 0.15

Control

1.21 4- 0.05

7.19 4- 0.10 ( < 0.001)**

CPZ-2 day 6 mg

1.19 i 0.08

15 mg

1.14 4- 0.03

6.02 4- 0.24 ( < 0.01)*** 7.06 4- 0.30

Non-shocked

Control CPZ-2 day 6 mg 15 mg CPZ-13 day 15 mg Shocked

Shocked

Control

1.06 ± 0.005

6.72 4- 0.17 ( < 0.05)**

CPZ-13 day 6 mg

1.20 i 0.04

15 mg

1.19 4- 0.06

5.55 + 0.11 ( < 0.001)*** 5.90 + 0.01 ( < 0.01)***

* Chlorpromazine HC1 (6 nag or 15 mg/kg body weight) was administored subcutaneously to female rats either at 2 days or 13 days of age (CPZ-2, CPZ-13). Each group was subdivided inI0 non-shocked and shocked groups. * * Numbers in parentheses represent P values for comparison to non-~ocked controls. * * * Numbers in parentheses represent P values for comparison to appropriate shocked controls.

DISCUSSION T h e influence o f e l e c t r o s h o c k o n the activities o f A C h E a n d B u C h E a n d on D N A a n d p r o t e i n c o n t e n t s has been previously r e p o r t e d 17. It has been f o u n d , f o r example, t h a t A C h E activity is h i g h e r in the h y p o t h a l a m u s , the B u C h E activity a n d D N A a n d p r o t e i n c o n t e n t s a r e higher in t h e c e r e b r a l c o r t e x in rats electros h o c k e d b e g i n n i n g at 2 d a y s o f age a n d sacrificed at 90 d a y s o f age 17. O t h e r studies have also s h o w n increased cholinesterase activity in the c e r e b r a l c o r t e x o f r a t s h a n d l e d a t early infancy 14 a n d rats e x p o s e d to c o m p l e x e n v i r o n m e n t a n d t r a i n i n g a. I n this s t u d y the activity o f b o t h e e r e b d l a r a n d e o r e b r o c o r t i c a l B u C h E was higher in s h o c k e d rats as c o m p a r e d to n o n - s h o c k e d animals. C o n c o m i t a n t l y , w i t h this higher activity o f B u C h E , eerebellar D N A c o n t e n t was h i g h e r i n the s h o c k e d rats as c o m p a r e d to n o n - s h o c k e d rats. T h e h i g h e r D N A c o n t e n t m a y be i n t e r p r e t e d Brain Research, 42 (1972) 403-411

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AND SHOCK ON BRAIN BIOCHEMISTRY

409

to reflect a higher density (number of cells per unit volume) of neurons or glial cells, as the result of either an increase in the total number of cells (neurons and glial) or a decrease in intercellular space. While neurons are capable of limited postnatal proliferation in the rat and other animals 1, the evidence for glial proliferation is more substantiveg, 11. Since non-specific cholinesterase has been reported to be predominantly localized in glial cells s, the concomitantly higher BuChE activity is supportive evidence that the number of glial cells may be higher in electroshocked animals. Chlorpromazine treatment either at 2 or 13 days resulted in a decrease of cerebrocortical BuChE activity. The sensitivity of BuChE to chlorpromazine is in agreement with previous studies from this laboratory where it was found that BuChE is lower in the cerebral hemispheres of newborn rats treated with chlorpromazine during gestationlL This decrease, however, was not evident after electroshock (Table I, column 4). Thus, it appears that electroshock may compensate for the CPZ-induced decrease in BuChE activity in the cerebral cortex. Unlike that of the cerebral cortex, CPZ-treatment did not affect BuChE activity of the cerebellum (except for a transient decrease in the shocked CPZ-13 day with 6 mg/kg). The difference in the response of these two CNS structures to CPZ may reflect differences in their stage of maturation. The cerebellum in the rat matures postnatally predominantly; that is, the cerebellum does not reach complete maturation until after 30 days of agelL The cerebellar neural elements (i.e. glial cells) therefore, which might be sensitive to the drug, may not be present in rats until after two weeks after birth. However, CPZ treatment prevented the rise of DNA in the cerebellum induced by electroshock (Table II, column 3). Assuming that the rise in DNA in the cerebellum after electroshock may represent proliferation of the number of glial cells, CPZ appears to counteract this effect. These findings further support the view that CNS structures during maturation respond differentially both to afferent sensory and chemical inputs. That early neonatal CPZ treatment may attenuate the effects of afferent input is of importance and requires further investigation. SUMMARY

The activities of cerebral and cerebellar acetylcholinesterase (ACHE), butyrylcholinesterase (BuChE), DNA, RNA and protein contents were studied in female rats treated with chlorpromazine (6 mg/kg body weight or 15 mg/kg) either at 2 (CPZ-2 day) or 13 days (CPZ-13 day) after birth and electroshocked beginning at 12 days for the CPZ-2 day groups or 18 days for the CPZ-13 day groups. Both cerebrocortical and cerebellar BuChE were higher in all control shocked groups whereas only cerebellar DNA was higher as compared to non-shocked controls. Chlorpromazine treatment lowered only the cerebral BuChE activity but this effect was not apparent after electroshock. Although chlorpromazine alone did not affect DNA content in either the cerebral cortex or cerebellum, it prevented the rise of DNA induced by electroshock in the cerebellum. Brain Research, 42 (1972) 403-411

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A. VERNADAKIS

ACKNOWLEDGEMENTS This investigation was s u p p o r t e d by Research G r a n t M H

15931 from the

N a t i o n a l Institute o f M e n t a l Health, N a t i o n a l Institutes o f Health a n d a Research Scientist D e v e l o p m e n t A w a r d K O 2 M H 42473 from the N a t i o n a l Institute o f M e n t a l Health, N a t i o n a l Institutes o f Health. The technical assistance o f Judith A s t r i n is greatly appreciated.

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