Prenatal haloperidol exposure: Effects on brain weights and caudate neurotransmitter levels in rats

036I -9230/92$5.00 + .OO

Brain ResearchBulletin.Vol. 29, pp. 449-458, 1992

Copyright 0 1992 Pergamoo Press Ltd.

Printed in the USA. All rights reserved.

Prenatal Haloperidol Exposure: Effects on Brain Weights and Caudate Neurotransmitter Levels in Rats R. WILLIAM&*-f+

S. F. ALI,*

F. M. SCALZO,§

K. SOLIMANt

AND

R. R. HOLSON*’

*Division of Reproductive and Developmental Toxicology, National Center for Toxicological Research, Jeflerson, AR 72079 tCollege of Pharmacy and Pharmaceutical Sciences, Florida A & A4 University, Tallahassee, FL 32307 $Agency for Toxic Substances and Disease Registry, Atlanta, GA 30329 JDepartment of Pediatrics, University qf Arkansas for Medical Sciences, Jeflerson, AR 72079 Received

20 December

199 1; Accepted

16 April

1992

R.. S. F. ALI, F. M. SCALZO, K. SOLIMAN AND R. R. HOLSON. Prenatui haloperidol exposure: Effects on brain weights and caudate neurotransmitler levels in rafs. BRAIN RES BULL 29(3/4) 449-458, 1992.-Monoamines may exert

WILLIAMS,

a trophic effect on early brain development. To assess the role of dopamine in prenatal neurological development of the rat, haloperidol (HAL) was given in daily 2.5 or 5 mg/kg SC doses to dams over gestational days 6 to 20. This treatment regime did not enhance fetal mortality, but did produce reliable, if modest, stunting of the body and brain weight of offspring. The 5 mg/kg HAL dose consistently reduced offspring brain weight to roughly 90% of controls. This effect was probably permanent, in that it was seen throughout maturation and in adults as late as 140 days of postnatal age. Appropriate controls showed that this effect was not due to drug-induced reductions in food intake, to the presence of HAL in maternal milk, or to behavioral abnormalities in HAL-exposed dams. These effects had, at best, modest regional specificity, in that most brain regions were affected, independently of degree of dopaminergic innervation. Closer investigation of HAL effects on the striatum suggested that this permanent weight reduction was not accompanied by alterations in striatal concentrations of monoamines. monoamine metabolites, amino acids, choline, acetylcholine. DNA, protein, or water. It is concluded that prenatal HAL does stunt growth, but that this effect may not involve a direct drug influence restricted to the fetal dopamine system in the brain. Brain

Haloperidol

Growth

Development

Dopamine

of neurotransmitters regulate brain growth and development. Such claims have been advanced for serotonin (33,64), dopamine (32,46), norepinephrine (6,18,6 I), acetylcholine (28), and fi-endorphins (57,65). These trophic neurotransmitter effects are as yet controversial, and their mechanisms are poorly understood. Given that an especially wide range of prescription drugs and substances of abuse affect dopaminergic neurotransmission, an improved understanding of the developmental role of this neurotransmitter is of particular importance. To date, the most compelling evidence for atrophic role for dopamine in CNS development has been provided by Patel, Lewis, and their colleagues (3,43-46). These researchers have repeatedly demonstrated that in vivo exposure to compounds which block dopaminergic neurotransmission on postnatal day 11 results in a substantial reduction in cell division over the next 36 hours (3,34,43-45). This effect is seen in both dopamine-enriched and dopamine-poor brain regions (3,43,44), and is evidently not a result of drug effects on

for reprints

should be addressed

Rat

Acetylcholine

nutrition or plasma corticosterone (43). However, these authors did not determine whether or to what degree exposure at this age results in long-term growth stunting. They also did not assess the effects of prenatal blockade of dopaminergic neurotransmission on brain growth. Other studies utilizing prenatal exposure to compounds which block dopaminergic neurotransmission have produced conflicting results. Prenatal reserpine, for example, has been reported to stunt body weight of offspring (10,20,27,62,63), but this effect is not seen by all investigators, sometimes even within the same laboratory (5-7,13,14,22,26,29,30). Similarly, prenatal chlorpromazine can reduce offspring body weight, especially after prolonged exposure or at very high doses (8,4 1,42,48,53-55,60), but this is by no means a universal finding ( l214,2 I ,23,25,26,29,39), with some labs even reporting weight increases in treated pregnant dams (27,62). Prenatal HAL exposure has produced similar confusion, with reports of reduced offspring body weight (9,15,17,56), no alterations in weight (24,37,49),

THERE is increasing evidence that a number

’ Requests

Striatum

to R. R. Holson.

449

450

WILLIAMS

TABLE EXPERIMENTAL Experiment I

No. of Treatments Vehicle

mg/kg HAL 5.0 mg/kg HAL Uninjected controls Vehicle controls Controls reared by dams exposed to 5 mg/kg HAL on GD 6-20 Pair-fed controls 5 mg/kg HAL-exposed progeny. reared by own mothers 5 mg/kg HAL-exposed progeny. reared by control mothers Vehicle 5.0 mg/kg HAL 2.5

z

3

4

Vehicle Pair-fed 5.0 mg/kg HAL

or even increased weight (52). an effect also reported in human progeny (47). Reports of the effects of prenatal exposure to antidopaminergic compounds on brain monoamine levels are similarly contradictory. Some laboratories have produced evidence for a reduction in various monoamines following such exposure (5.24.58.59). Other laboratories report no such effects (10.15.16,50). In view of the considerable confusion in this important area of research, we decided to carefully investigate the effects of prenatal HAL exposure on long-term neural and somatic growth of offspring. Care was taken to include controls for a variety of potential confounds (including drug effects on maternal food intake and behavior), and to assess growth of body, internal organs. and a variety of brain regions. All positive findings were fully replicated, to control for the intralaboratory variability often seen in such studies. Finally. a series of studies of HAL effects on striatal neurotransmitter content was conducted in an attempt to detect any dopamine-specific effects on development of this most dopamine-enriched brain region. METHOD

.-ltliWlUl.\ Rats were a Sprague-Dawley derived, caesarean-delivered albino strain maintained at the National Center for Toxicological Research. All rats were housed in (20.5 X 4 1 X 2 I cm) cages of transparent acrylic, with wood chips for bedding and stainless steel rod lids which held water bottles and rat chow (both of which were available ad lib). Vivariums were maintained on a I2 h light/12 h dark cycle, with temperature 23 * 1“C and humidity 50% + 10%. Females were placed overnight with experienced male breeders. If copulatory plugs were found the next morning, females were designated plug positive at gestational day (CD) 0 of pregnancy (in this colony roughly 80% of plug-positive rats deliver live litters). On the day of birth (postnatal day or PND 1). all offspring were counted, weighed, then randomly culled to a litter size of 8 pups (4 & I of each sex). Pups stayed with the dam

ET AL.

I DESIGN Litters

MeZlSUreS

14 12

Whole brain, body weights on PNDs 15, 30, 58, and 140 Caudate monoamine content on PNDs I. 15. 30. and 58

II 14

Whole regional brain and body weights, PND 30

14 13

PND 30 and PND 100 whole brain, body weights

13 18 13 14 18

12 II I2

Regional brain weights. PND 30 Regional brain content of DNA, protein on PND 30 Water content of whole brain Caudate content of ACh, Ch, and amino acids PND I organ weights PND 30 brain. body weights

until PND 21. at which time they were caged with littermates of the same sex.

The results of four experiments are reported here and outlined in Table I All four experiments had vehicle-exposed dams and dams exposed to 5.0 mg/kg HAL on GDs 6 through 20. The experiments differed in respect to the presence of other treatment groups (such as a 2.5 mg/kg HAL group in Experiment 1) and controls (e.g., pair-fed controls in Experiments 2 and 4). They also differed as outlined in Table I on the types of measures conducted on the offspring.

All HAL-exposed dams were injected subcutaneously daily between 0800 and 1000 h over GD 6 through GD 20 with a I ml/kg solution of haloperidol. Drugs were prepared weekly at a concentration of either 5.0 or 2.5 mg/ml. These solutions were made by dissolving haloperidol (Sigma Scientific, St. Louis, MO) in a few drops of lactic acid, then bringing to volume with distilled water, while adjusting pH to 4.0 with I N sodium hydroxide. Injection sites were rotated daily in the order left flank-neckright flank-left flank, etc. This prevented ulceration at the injection site in all but a few minor cases. Vehicle was prepared as above, but of course without the haloperidol.

Pair feeding was conducted on Experiments 2 and 4. This was done by mating potential pair-fed dams 24 h later than the HAL dams, then matching pairs on GD 0 body weight. Twentyfour-hour food intake was measured for the HAL group from GD 5 (the day prior to the first HAL dose) through GD 20. Pairfed controls then received the amount eaten each day by their matched HAL pair mates 24 h later. No attempt was made to match water intake. Not all HAL or pair-fed plug-positive females were actually pregnant but, of course, this was not known when dams were paired early after mating (GD 5). In consequence. a few pair-fed dams turned out to have been matched

PRENATAL

HALOPERIDAL

451

EXPOSURE

TABLE GESTATIONAL

HALOPERIDOL

EXPOSURE

2

(5 mg/kg, GD 6-20) HAD HIGHLY OFFSPRING

ExperimentI

REPLICABLE

Experiment2

EFFECTS

ON DAMS AND

Experiment3

Experiment4

Litter size (n of pups per litter)

Vehicle

12.6 f 0.5

13.7 f

I.0

12.0 f 0.8

13.9 f 0.8

HAL

[I41 13.2 + 0.7 1111

[I21 12.2 + 0.8

[I41 II.9 + I.0 [I81

1121 I I.8 ?I 0.7 [I21

399.4 * 5.9 [I41 345.6 f 5.3’ 1111

409.3 f 13.3

409.9 + 9.4 [I41 359.6 k 1.5* iI81

404.3 + 6.5 1121 363.0 k 8.7* [I21

86.4 f 2.5 [I41 16.3 f l.9* [I 11

17.1 + 3.2 [I21 74.6 f 1.4 [I21

109.9 * 4.4 [I41 93.6 f 2.9* 1181

106.0 + 4.8 [I21 93.1 zk 2.6* [I21

[I21

Maternal CD 20 wt. (g)

Vehicle HAL PND 30 male body wt. (g) Vehicle HAL

1121 353.3

f

6.0*

1121

PND 30 male brain wt. (g)

1.56 rt 0.01 [I41 I .39 ? 0.02 * [lOI

1.53 + 0.03 [I21 1.45 f 0.01* 1121

1.63 f 0.02 [I41 I .44 + 0.02* 1181

I.61 f 0.02 1121 I .44 t 0.02 * [I21

Vehicle

1.83 + 0.05 [I41

HAL

I.81 * 0.04

2.00 + 0.07 iI21 1.95 + 0.05 iI21

I.51 k 0.05 1141 I .56 + 0.04 [I81

1.54 f 0.06 1121 1.55 + 0.03 [I21

Vehicle HAL PND 30 male brain body wt. ratio (‘%)

1101

* Significantly different from vehicle controls. All values * SEM. Number of litters in brackets

to nonpregnant HAL females. Since pregnant rats typically eat more than nonpregnant, this meant that those few dams were fed conservatively and, thus, that pair-fed food intake was slightly lower than HAL intake. The conservative nature of this matching, it was felt, more than justified the inclusion of these pairfed litters in the study. Crossfostering was utilized in Experiment 2, by placing HAL and control litters on control and HAL dams (respectively), so that HAL-exposed pups were reared by control dams and vice versa.

two cerebral peduncles where they join with the forebrain. The hippocampi were then removed from the remaining posterior section (mostly dorsal and ventral cortex), which was added to the antero-ventral segment to make up the remainder. Each of these eight regions was weighed in the order removed, prior to freezing on dry ice. The entire dissection procedure took not more than 5 min/brain. Samples were then stored at -70°C until analysis.

HPLC Techniques Regional Bruin Weights Offspring were killed by decapitation. Brains were removed and dissected into eight regions by the method of Glowinski and lversen (19). These regions were pituitary, cerebellum, brain stem, hippocampus, diencephalon, head of the caudate nucleus, frontal cortex, and remainder. Briefly, the technique involved making a coronal razor-blade section at the posterior boundary of the optic chiasm. This cut sectioned the hypothalamus, with the anterior 20% or so in the frontal section, together with the head but not tail of the caudate nucleus. The head of the caudate was then removed from this anterior section, after which cortex dorsal to the rhinal sulcus was removed for the frontal cortex. Ventral cortex, including olfactory tubercles and septum, was pooled with the remainder. Cerebellum was prepared by first removing the paraflocculi, then severing the peduncles which attach this organ to the brain stem. The diencephalon was next removed using the edge of the infundibulum as a posterior boundary and the third ventricle as a dorsal boundary. Next, the brain stem was severed from the forebrain by sectioning the

Monoamine assay. Animals were sacrificed by decapitation, their brains were removed and quickly dissected on ice into different regions following the methods of Glowinski and Iversen (19) frozen over dry ice and stored at -70°C until analysis. DA, DOPAC, HVA, 5HT, and 5-HIAA were resolved and quantified by HPLC/EC as described by Ali et. al. (2). Briefly, each caudate was weighed and diluted with a measured volume (10 v/w) of 0.2 N perchloric acid containing 250 ng/ml of the internal standard 3,4_dihydroxybenzylamine (DHBA). Brain tissue was then disrupted by ultrasonication, centrifuged (1,000 X g, 5 minutes) and 150 ~1 of the supernatant removed and filtered through a 0.2 pm microfilter [MF- 1 microcentrifuge filter, Bioanalytic System (BAS), W. Lafayette, IN]. Aliquots of 25 ~1, representing 10 mg of brain tissue, were injected directly onto the HPLC system for separation of monoamines and metabolites. The analytical system included a Waters Associates M-6000A pump (Milford, MA), a Rheodyne@ 7125 injector (Rainin Instrument, Woburn, MA), a Biophasem ODS, 5 pm (250 X 4.6 mm) analytical column (BAS), a LC4A amperometric detector

452

WILLIAMS

35

400

A. Maternal Food Intake

B. Maternal Body Weight

1

1

ET AL.

T

30 t 25. 5 %

20 -

E -

15

z IL"

10 -

5.

01 0

2

4

6

J

I

6

10

I

12 14

!

16 16

/

2601

20

0

I 2

I 4

6

I 8

10

12

I 14

16

16 20

t

I

24h Aner First Dose

Gestational

Day

I 24h Pitter Firs, close

FIG. I. Gestational HAL exposure reduces maternal food mtakc and weight gain. A single daily SC injection of haloperidol(5 mg/kg) over gestational days h-20 reduced food intake and body weight gain of pregnant rats. Pair feeding produced identical effects on maternal weight. (A) Daily maternal food intake (g) + SEM. (B) Daily maternal body weight (g) f SEM. Standard errors are shown mostly on alternate days. to reduce visual clutter.

and LC- 17 oxidative flow cell consisting of a glassy carbon electrode (TL-5) versus Ag-AgCI reference electrode maintained at a potential of 0.65 V (BAS). The mobile phase consisted of 0.15 M monochloroacetate. pH 3.0, 4.5% acetonitrile and an ion pairing reagent of 0.5 rn,M octyl sodium sulfate. Chromatograms were recorded and quantitated on a 3380A integrator (HewlettPackard, Avondale, PA). The endogenous biogenic amine concentrations were calculated using a standard curve for each amine. The standard curves were generated by determining in triplicate the ratio between three different known amounts of each amine and a constant amount of DHBA. .dmino acid. Quantification of brain amino acids required that they first be extracted and derivatized (35). Each brain region was weighed and sonicated in I ml of 0.05 M HCI in 47.5% methanol after adding 20 ~1 of DL-homoserine (50 mM/ml) as an internal standard. The mixture was centrifuged at 3 1,000 X .r: at 0” for 10 min. A 400 ~1 aliquot of the supernatant was neutralized with 200 ~1 of 0.5 M KHC03 and centrifuged at 15,000 X g at 0” for 10 min. The supernatant was then filtered through a 0.45 pm pore-size microfilter by centrifuging at 4000 X
rahydrofuran (THF) and 20’;, acetonitrile. Solution B differed from A only in containing IO’? THF. The flow rate was I .5 ml per min at ambient temperature. The switch from A to B was made 5 min after beginning flow of A. After a chromatogram was completed, solution A was pumped through the column for at least 7 min before the next sample was injected. Chromatograms were integrated and recorded on a Perkin-Elmer LCI100 Integrator (Perkin-Elmer Corp.. Norwalk, CT). A 20 ~1 aliquot of tissue extract or standard amino acid was mixed in a I .5 ml plastic centrifuge tube with 100 ~1 of the OPA reagent, allowed to react for 2 min, then 10 ~1 was injected onto the column. The amino acid concentrations were calculated using a standard curve for each amino acid. Standard amino acid solutions were prepared in a water/methanol mixture (1: 1) containing I m&f Na2-EDTA. The standard curves were generated by determining in. at least triplicate. the peak area ratio between three different amounts of each amino acid and a constant amount of the internal standard. Concentrations of aspartate. glutamate. glutamine. glycine, taurine. and GABA were determined. HPLC‘ LI.S.CUJ~ ~!~‘~~(,(,/~‘/(.Ilolinr. Animals were sacriticed by microwave irradiation. Brains were removed quickly. caudate was dissected out and stored at ~70°C until analysis. The levels of acetylcholine and choline in the caudate nucleus were determined using a HPLC method with electrochemical detection. Briefly, caudate nuclei were homogenized in 5 volumes of 0.05 M perchloric acid and centrifuged at 14,000 rpm for 2 min. Twenty ~1 of the supernatant from each sample was injected onto the HPLC columns. The mobile phase consisted of 50 mM sodium phosphate dibasic, and the pH was adjusted to 8.5 with 1 N phosphoric acid. The mobile phase was filtered through millipore filters (0.45 PCM)(Millipore Corporation. Bedford, MA) and degassed under vacuum for 5 min. The mobile phase was pumped through a special acetylcholine analytical column (BAS Acetylcholine IO pcM, 100 mA4 X 4.6 mM) (Bioanalytical Systems Lafayette. IN) and then through an enzyme column connected to the analytical column at a flow rate of 0.8 ml/min. The glassy carbon working electrode was maintained at a po-

PRENATAL

HALOPERIDAL

453

EXPOSURE

TABLE

3

STUNTING OF WEIGHT IN ADULTHOOD IS NOT DUE TO GESTATIONAL UNDERNUTRITION

PND 30: (Experiment 2) Vehicle Pair-Fed HAL PND 30: (Experiment 4) Vehicle Pair-Fed HAL

BodyWeight (g)

Brain Weight (g)

Brain/Body Weight Ratio x 100

109.93 f 4.38* [I41 103.01 + 2.21* 1141 93.58 ? 2.87*’ 1181

I .63 + 0.024A 1141 1.54 f o.o19*a 1141 1.44 + 0.017*c [I81

1.51 + 0.046 1141 I .50 f 0.033 [I41 I .56 f 0.038 [I81

106.0 + 4.8A 1121 109.7 f 2.6A

1.61 f 0.02A [I21 I .59 + 0.02A [Ill I .44 i- 0.02* B [I21

I .54 f 0.006 [I21 1.45 * 0.033 1111 1.55 f 0.038 1121

2.03 + 0.025A 181 2.02 + 0.072A [lOI I .83 f 0.022* B 1101

0.42 + 0.008 [81 0.44 f 0.020 1101 0.43 f 0.010 [lOI

[Ill 93.1 + 2.6*’

iI21 PND 100: (Experiment 2) Vehicle Pair-Fed HAL

486.4 + 10.31A 181 456.7 f 9.8*’ [lOI 429.1 f 13.5*’ [IO1

*A.BMeans f SEM with different letters are statistically significantly different at p I 0.05. Numbers of litters in brackets.

tential of 0.5 V relative to the reference electrode. The concentrations of acetylcholine and choline in each sample were determined by using 3-4 point standard curve. Protein Drterminution Aliquots of membrane preparations were used for the determination of protein content by the method of Lowry et al. (36) using bovine serum albumin (Sigma Chemical Co.) as the standard. DN,4 Determimtion The DNA content was measured in different brain regions in the offspring using an established assay procedure by Labarca and Paigen (31). Various brain regions were weighed and homogenized in distilled water (1:6), then diluted with sodium phosphate buffer ( I : IO) and sonicated. Sodium phosphate buffer (900 ~1) was pipetted into all sample tubes. The DNA standard was diluted with sodium phosphate buffer (I: IO) and a standard curve was constructed. Next, the Heoscht dye was diluted (1: 10) with sodium phosphate buffer. The 50 ~1 of the prepared tissue sample and 50 ~1 of Heoscht dye was added to the sample tubes which contained 900 ~1 of sodium phosphate buffer to make a final volume of I ml. Each tube was vortexed immediately after adding Heoscht dye to the sample tube. Finally, all tubes were read on a fluorometer with the following settings: excitation 356 nM and emission 458 nM.

Statistical Analysis In all cases the litter was the unit of analysis. Typically one male and one female were sacrificed per litter and age. These data were analyzed treating sex as a correlated variable within litter. This approach essentially pools values for the male and female into a single mean per litter. Since litter means are difficult to interpret for variables which show large sex differences (e.g., adult body weight), data which did not show a treatment X sex interaction were also analyzed by one-way ANOVA for dose effects, separately by sex. This technique always preserved litter as the unit of analysis. For statistically significant one-way ANOVAs by sex, Duncans’s multiple range test was then conducted for post hoc comparisons between individual treatment groups. RESULTS Drug effects on offspring were highly similar for males and females; in no case did we obtain a significant treatment X sex interaction. Results, consequently, are generally given for males; this is because mean weights across sex are difficult to interpret, especially for adults. When comparisons are restricted to the vehicle and 5.0 mg/ kg HAL groups, all four experiments produced the same basic results: prenatal HAL did not reduce litter size, but did decrease maternal weight on CD 20, PND 30 male body weight and PND 30 male whole brain weight (Table 2). PND 30 brain/body weight ratios were unaltered in all four experiments (Table 2).

454

WILLIAMS

The maternal weight loss is examined more closely in Fig. 1. which contains pair-feeding data for Experiment 2. Haloperidol clearly reduced maternal food intake, and the fact that the pairfed weight profile over dosing was identical to that of the HAL dams shows that this weight loss was completely a function of drug-induced anorexia (Fig. I). The anorexia induced in HAL dams did not fully account for weight loss in the offspring. As Table 3 shows, PND 30 brain weights in HAL male offspring were significantly lower in both pair-feeding experiments than were pair-fed or vehicle control brain weights. These differences persisted into adulthood. At PND 100. brain weights of HAL males were still significantly lower than vehicle or pair-fed controls (Table 3): Fig. 2 tracks these differences in Experiment 1 still further. At all ages measured, including PND 140, there was a dose-response decline in weight of body and brain of HAL-exposed males. Haloperidol effects on body and brain were also not due to drug effects on maternal behavior. HAL-exposed pups reared by control dams had the same degree of stunting as HAL pups reared by HAL dams. while normal pups reared by dams exposed to HAL during gestation did not display these weight deficits (Fig. 3). The haloperidol effect on brain and body weight was not seen on all organs. Table 4 gives weights of body, brain. and major internal organs on the day of birth (Experiment 4). Again. body and brain weights of HAL-exposed male neonates were significantly lower than for vehicle or pair-fed controls. but weights of lungs, liver, kidneys, spleen. heart. thymus. and adrenals were not depressed. The effects of prenatal HAL on brain weight were distributed remarkably evenly within brain regions (Fig. 4). Only diencephalon did not weigh significantly less on PND 30 in HAL progeny compared to vehicle controls. Hippocampus, frontal cortex, cerebellum, pituitary, caudate nucleus, and remainder all were stunted at PND 30 relative both to vehicle and to pair-fed controls. Moreover, HAL-exposed cerebellum, remainder, pituitary. and caudate nucleus, while all slightly more stunted than whole brain, were within a few percentage points of each other relative

2.2 r

A. Brain Weight

0

to vehicle controls (Fig. 4). This does not suggest that dopamineenriched brain regions were especially vulnerable to HAL. The reduction in brain weight also did not appear to be at all selective within the striatum. Over GD I to 58 there was no reduction in concentration of dopamine, 5HT or their major metabolites in male striatum, with the possible exception of a transitory reduction in striatal HVA concentration on the day of birth but not thereafter (Table 5). Likewise, male striatal ACh and Ch concentrations were not altered by prenatal HAL. nor were concentrations of amino acids DNA, or protein (Table 6). Water content of whole brains was also not reduced in HALexposed offspring on PND 28 (data not shown). DNA and protein concentrations likewise did not show a HAL effect in cerebellum. frontal cortex. or hippocampus (data not shown).

Prenatal haloperidol exposure has a highly replicable doseresponse effect on body and brain weight of rat offspring. Across four independent experiments daily SC injections of 5 mg/kg HAL over the course of postimplantation pregnancy reduced body and brain weight of pups to roughly 90% that of controls: a 2.5 mg/kg dose produced about half this reduction in brain weight. This effect was of comparable magnitude in both sexes. It was also permanent. in that it was still seen as late as 140 days postnatally. The high reliability of this effect certainly suggests that HAL-induced stunting is a real phenomenon. The fact that not all laboratories report this effect (24.37,49.52) is probably attributable to the relatively high prolonged HAL exposure required to produce substantial stunting. Appropriate controls allow exclusion ofa variety of maternal haloperidol effects as a causal mechanism in this stunting. Haloperidol, at the doses used here, produces maternal catalepsy and consequent reductions in food intake. Dams almost stop eating and lose weight over the first few days of exposure. Some tolerance to haloperidol anorexia then develops. but food intake and body weight over the course of pregnancy never reach control levels. Pair-feeding experiments reported here show that drug-

B. Body Weight (% of Vehicle) ,oa ~ ~ _ _ _ _ _ _ _ __ _ _ _. _.

T

I

101

I

15

30

,

45

60

75

90

105

120

ET AL.

I601 135 140 0

15

/

,

30

45

I

60

75

90

105

120

135 140

Age (PND) FIG. 2. Gestational HAL exposure permanently reduces body and brain weight ofotfspring. (A) Shown are brain weights (g + SEM) of male littermate offspring, killed on postnatal day 15.30. 60, or 140. *Significantly different from vehicle controls. (B) Body weight of low (2.5 mg/kg) or high (5.0 mg/kg) HAL male littermate offspring as percent of vehicle control weights on PNDs 15. 30, 60. or 140. *Significantly different from vehicle controls.

PRENATAL

HALOPERIDAL

455

EXPOSURE 1.7

‘*O A. Body Weight

B. Brain Weight -I-

L I

ll

13 0

m

5 t? $ f

FIG. 3. HAL stunting in PND 30 male offspring is not due to a variety of direct drug efforts on the dam. HAL fostered = 5 mg/kg HAL on CD 6-20. Pups at birth are reared by vehicle control dams. Hal = 5 mg/kg HAL on CDs 6-20. Pups are reared by their genetic mother. Pair fed = dams are matched by weight to HAL dams, and given the same daily amount of food eaten by their HAL pair mates. Control = nontreated controls. Vehicle fostered = dams are given daily vehicle injections over GDs 6-20. At birth pups are reared by HAL fostered dams. Vehicle = dams are given daily vehicle injections, and rear their own pups. Shown are weights in g * SEM. *Denotes significant difference from all non-HAL-exposed offspring.

offspring were virtually identical at PND I (Table 4) and PND 100 (Table 3). We conclude that haloperidol stunting is not attributable to the substantial reduction in food intake of exposed dams. Drug-induced stunting was also not a consequence of hngering trace amounts of haloperidol in maternal milk, or of direct drug effects on maternal behavior. Exclusion of such long-term effects was suggested by the finding of maximal stunting in offspring on the day of birth. This indication was confirmed by crossfostering experiments. Control pups reared by haloperidolexposed dams displayed no abnormalities in growth. Similarly, infants exposed to haloperidol prenatally but reared by control

induced reductions in maternal weight are due solely to reduced food intake. These experiments also show that while a part of the PND 30 differences in offspring brain weight may be due to reduced maternal food intake (in Experiment 2 but not 4, pairfed brain weights were significantly lower than controls), a substantial part of the drug effect at this age is not due to undernutrition. Moreover, as has been repeatedly demonstrated (4) prenatally undernourished rats undergo prolonged postnatal catch-up growth, usually resulting in eventual normalcy. Such was the case for the pair-fed pups in these studies. In contrast, HAL-exposed progeny showed no signs of a compensatory rebound. The relative deficits in brain and body weight of treated TABLE

4

PRENATAL HAL EXPOSURE BUT NOT PAIR FEEDING SELECTIVELYREDUCES PND 1 WEIGHTS OF WHOLE BODY AND BRAIN WEIGHT BUT NOT WEIGHT OF MOST INTERNAL ORGANS Organ

Body (id Brain (mg) Lungs (mg) Liver (mg) Kidneys (mg) Adrenals (mg) Thymus (mg) Spleen (mg) Heart (mg) Testes (mg)

Vehicle (n = 12) 6.53 237.8 143.7 360.2 69.1 2.64 14.0 17.0 46.2 5.4

f 0.16” f 3.3A f 5.8 ?z 18.2 f 2.8 + 6.24 f I.17 f 0.9 * l.4A + 0.24

Pair Fed (n= II) 6.17 229.5 139.2 328.4 61.4 2.43 12.6 16.9 40.4 5.08

f 0.16A.B f 2.6A f 4.7 f 14.2 f 2.2 f 0.21 + 0.5 + 0.79 ?z 1.3*a f 0.23

HAL (n = 12) 5.72 212.17 127.4 349.7 64.9 2.87 14.4 17.3 43.4 4.9

f 0.14*’ ?z 4.0*’ f 5.5 z!z13.4 f 1.5 f 0.10 + 2.8 f 1.7 f 1.4A’B + 0.23

A,B,*Means + SEM with different letters are significantly different at p 5 0.05.

456

WILLIAMS

TABLE Diencephalon

ET AL.

6

PRENATAL HAL EXPOSURE DOES NOT ALTER THE CONCENTRATION OF AMINO ACIDS, DNA. PROTEIN. ACh. OR CHOLINE IN CAUDATE OF 30.DAY-OLD MALE OFFSPRING

Stem Hippocampus

Vehicle

HAL

Frontal Cortex Whole Brain Cerebellum Remainder

Aspartate

7.10 -t

0.14

2.23+

0.1s

GABA

1.36 +

0.08

1.39 k

0.06

Glutamate

X.67 ?

0.41

9.09 t

0.4X

Glutamine

4.67 i-

0.71

4.92 f

0.37

Glycine

0.62 +

0.07

0.66 k

0.07

Taurine

7.25 +

0.47

X.37 t

0.49

121.2

ACh Choline

Pituitary

X6.7 696.6

DNA (ng/mg) Protein (g/mg)

Caudate Nucleus 80

60

100

89.0

+ 74.9 i-

I I.9

+ 30.5 i

127.0

rt 10.0

109.6

f ‘4.5

671.6

5.7

t 31.4

79.7

i

3.7

120

Regional Brain Weight, % of Control heavily against it. Thus. pre-/neonatal treatments which stunt growth usually reduce body weight relatively more than brain weight, and also reduce weight ofinternal organs including liver. This is true for undernutrition (4) and for glucocorticoid-induced stunting (38). These well-documented findings demonstrate that brain weight in individual animals is not all that tightly linked to body weight and, hence, suggest that a precise 10% reduction in brain weight in response to a 10%’ reduction in body weight is biologically unlikelq. Further. none of the internal organs weighed at birth was affected by HAL (Table 4). This finding. too. argues against a totally nonspecific HAL effect on growth. Finally. HAL triggers substantial maternal glucocorticoid secretion (I). Glucocorticoids typically stunt growth and, hence. the HAL effect could hypothetically be mediated by the drug’s influence on the maternal adrenal. Thymus and liver are especially stunted by glucocorticoids. however, while HAL neonates displayed no such stunting. This finding militates against attribution of HAL effects on growth to the drug’s substantial effect on maternal corticosteroid secretion. In conclusion, prenatal haloperidol exposure produces a rather general stunting ofbrain and body. On balance. it is likely that the drug effect on brain is direct, and not a response to reductions in body weight. This effect is permanent and, consequently. more closely resembles effects of X-irradiation (5 I) or antimitotics (I I) than of treatments such as undernutrition or corticosterone exposure, where substantial postnatal catchup growth is seen. Hence. this prenatal drug effect may be the

FIG. 4. PND 30 regional brain weights are reduced more in HAL-exposed males than in pair-fed controls. Regional brain weights (tSEM) are shown as a percent of vehicle controls. *I, < 0.05, compared to vehicle controls. tl’ i 0.05. compared to pair-fed controls.

dams were as stunted as treated progeny reared by their biological mothers. While the above findings allow us to rule ouit some maternally mediated drug effects. the causal mechanism(s) of haloperidol stunting remain unclear. even puzzling. The drug’s effect on growth appears to be rather general, occurring in body as well as brain weight, and in dopamine poor as well as dopamine enriched areas of the brain. These findings. together with the lack of haloperidol effect on a range of caudate concentrations of neurotransmitters. metabolites, DNA. and protein. seem to rule out a direct effect of the drug on classical dopamine receptor sites in the fetus. Indeed, the generality of haloperidol stunting calls into question the specificity of reductions in brain weight. At no time in postnatal life did prenatal HAL alter brain to body weight ratios. Thus. it could be argued that the drug had no direct effect on brain growth. with the observed decrease simply passively reflecting somatic stunting. While nothing in these studies can totally rule out such a possibility, other consideration weigh

TABLE TIME

COURSE OF PRENATAL

Dopamine Postnatal Age (Days) I

I5 30 S8

Vehicle 0.57 2.51 3.75 5.47

i 0. I3 io.19 2 0.40 + 0.26

DOpaC HAI.

0 47 i

0.05 2.70 f 0.30 4.33 + 0.29 5.28 + 0.27

Vehicle 0.10 0.30 0.50 0.54

+ f k i-

5

HAL EXPOSURE EFFECTS ON STRIATAL AND THEIR MAJOR METABOLITES

O.OI 0.03 0.04 0.06

Homovanilic HAL

0.0x 0.30 0.52 0.50

Values represent mean, i SEM in rig/g wet tissue weight )I * Sigmficantly diferent from vehicle controls (p < 0.05). Dopac = Dihydroxyphen4lacetic acid. 5-HIAA 5-Hydrox)indoleacetic acid.

i- 0.0 I k 0.03 t 0.04 -i- 0.05

I2

Vehicle

0.08i 0.02 0.37 t 0.05 0.37 t 0.03 0.33 + 0.04

LEVELS OF DOP.4MINE.

Serotonin

,Acid HAL

0.04 f 0.0 I * 0.37 t 0.04 0.39 ? 0.03 (1.32 -1.0.02

SEROTONIN’.

Vehxlc

5.HIAA IIAL

0.07 i 0.0 I 0.08 k 0.02 0. I2 i O.O? 0.I t i 0.02 0.30 i 0.06 0.32 i 0.06 0.35 - 0.07 0.34 + 0.0x

Vehrle 0. IX 0.09 0.25 0.23

i i 2 2

0.09 0.02 0.05 0.05

HAL 0.07 k 0.02 0.05 k 0.02 0.26 + 0.05 0. IX + 0.05

PRENATAL

HALOPERIDAL

451

EXPOSURE

same as the antimitotic effect of this drug in PND 11 animals so thoroughly documented by Pate1 and Lewis (46) and also seen in cultured adult lymphocytes (40). If so, this would imply that haloperidol has a broad antimitotic effect, one almost certainly not attributable to blockade of classical dopamine receptors. The nature of such an effect very much remains to be elucidated. While reports of very similar effects prenatally and postnatally do not suggest a maternal mechanism for HAL stunting, further work is necessary in this area. We are currently evaluating the effect of haloperidol on embryo cultures. If the drug reduces embryonic growth in vitro at physiological concentrations, maternal mediation of the drug effect will become very unlikely. More important, perhaps, is the question of the specificity of the drug effect on growth. If haloperidol is stunting

growth through a direct effect on a dopamine-like receptor site, or even more specifically through blockade of dopaminergic neurotransmission in the fetus, then a variety of other compounds, from reserpine and a-MPT to more specific neuroleptics, should have very similar effects on growth. Pharmacological dissection of this haloperidol effect is also currently under way in our laboratory. ACKNOWLEDGEMENTS

The authors wish to express their thanks to Ms. Sullivan-Jones and Mr. Bobby Cough for their technical support throughout the project. Dr. Jo Ann Sweeney performed the analyses for caudate choline and acetylcholine content, and Mr. George Lipe conducted the amino acid analyses. Mrs. Edwina Martin and Ms. Rose Huber are also thanked for their expert secretarial help in producing this manuscript. Robert Williams was supported by the Florida Endowment Fund.

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