Psychopharmacology 74, 349-352 Psychopharmacology �9 Spr inger-Ver lag 1981

Prenatal Maternal Phenobarbital Increases Reactivity and Retards Habituation of Mature Offspring to Environmental Stimuli

Lawrence D. Middaugh, Larry W. Simpson, Thomas N. Thomas, and John W. Zemp

Departments of Biochemistry and Psychiatry and Behavioral Sciences, Medical University of South Carolina, Charleston, South Carolina 29425, USA

Abstract. Adul t female offspring of C57BL/6J mice injected daily with phenobarbi ta l for the last third of pregnancy were more active than control offspring during a 3-rain test period in an open field arena, thus confirming previous reports of lasting effects of prenatal exposure to phenobarbi tal . These offspring habi tuated less rapidly than control offspring to the open field and were more reactive to sudden changes in environmental stimuli. The behavioral changes were not accompanied by body or brain weight deficits. The maternal drug injections did not alter brain concentrations of do- pamine or norepinephrine in the adult offspring or the degree of reduction in these transmitters produced by the synthesis inhibitors e-methyltyrosine. Al though activity was reduced by the catecholamine synthesis inhibitor, the effect was similar for offspring of both drug-treated and control dams.

Key words: Barbiturates - I n b r e d mice - Catecholamines - Prenatal drugs - Open-field activity

A number of studies completed in our labora tory (Middaugh et al. 1975a, b; Zemp and Middaugh 1975) using mice and others (Armitage 1952; Maur i 1966; Mart in et al. 1979) using rats indicate that prenatal maternal injections of phenobar- bital alter the behavior of developing and mature offspring. Other studies indicate that changes in biochemical measures frequently correlate with behavior for offspring of dams injected with the drug during pregnancy. Middaugh et al. (l 979) reported altered corticosterone concentrat ion in plas- ma of mice prenatal ly exposed to phenobarbi ta l , and Gupta et al. (1980) reported changes in gonadal hormones of female rats prenatal ly exposed to the drug. Some neurochemical measures also appear to be altered in offspring of mice injected with the drug during pregnancy. Brain levels of nucleic acids and protein are slightly reduced in young offspring of mice injected with phenobarbi ta l (Zemp and Middaugh 1975) and a similar finding has been reported for rats injected neonatal ly with the drug (Diaz et al. 1977). Recently, we have found that synaptosomal preparat ions from brain tissue of young mice prenatal ly exposed to phenobarbi ta l accumulate dopamine (DA), norepinephrine (NE), serotonin (5-HT), and 7-aminobutyric acid (GABA) more rapidly than do those from control offspring (Middaugh et al. unpublished data). Al though behavioral studies indicate that the effects of prenatal exposure to

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phenobarbi tal extend into adulthood, neurochemical evi- dence of long-lasting effects is lacking.

The experiments reported here further characterize the activity changes previously reported for mature offspring of dams exposed to phenobarbi tal , and demonstrate that mature female offspring of these dams are more reactive to change in environmental stimuli. The relationship of behavioral changes to possible neurotransmit ter changes was assessed by determining brain concentrations of D A and NE in animals that had been tested behaviorally and pretreated with the synthesis inhibitor c~-methyltyrosine (AMT) or its vehicle prior to the behavior test.

Materials and Methods

Subjects. C57BL/6J mice (Mus musculus) were obtained from our colony and maintained in a temperature-regulated room (23~ + 2~ under a 12-h light-dark cycle with access to food and water. Pregnancies were determined by sperm plug detection and were verified by weight gains (_> 4g) 12 days after plug detection. Animals in the drug groups were SC injected each morning with 20 mg or 40 mg sodium phenobar- bital per kg body weight (P 20 and P 40, respectively) for the last 6 or 7 days of pregnancy. Saline control (SC) animals were injected for the same time period with equivalent volumes of 0.9 % saline (0.01 ml/g body weight). Litters were culled to seven and offspring were reared by their biological mothers until 24 days of age, at which time they were separated according to sex and maintained six per cage until the experiments took place at 160-175 days of age.

Brain Concentration of Catecholamines. Catecholamines were determined by a modification of the procedure reported by Shellenberger and Gordon (1971). Animals were decapitated and brains were rapidly removed, weighed, and homogenized in 6 ml ice-cold 0.4 N HCIO 4 containing 0.1% sodium metabisulfite (w/v) and 0.05 % NA 2 EDTA (w/v) using a polytron tissue homogenizer. The homogenates were left on ice t0 min, and then centrifuged at 22,000g for 15 rain at 4~ The supernatant was removed and stored at -20~ until assay. All samples were assayed within 2 weeks. Concentrations were determined by comparing fluorescence from the tissue samples to that of standards prepared in the 0.4 N HC104-sodium metabisulfite-EDTA solution and run through the entire assay procedure.

BehavioralActivity andReactivity. Activity and reactivity to environmen- tal stimulation was assessed in an open field arena. For both measures, a 40.6 x40.6 cm arena with 10.2 cm sides was used. The interior was painted flat-black and the floor was divided into 10.2 cm squares with thin red lines. The top was constructed of clear Plexiglas and contained a tone generator (Sonalert, BRS/LVE-TechServ, Beltsville, MD, USA) which produced a 2.8 kHz tone. A tensor lamp positioned 15 cm above the tone generator provided light for a tone-light compound stimulus. Both tone and light, when presented, were pulsed 10/s. Animals were individually tested at 10AM-12:30 PM by a person unaware of the

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treatment history. The animal was placed in one corner of the arena and activity was indexed by pressing a touch switch for each square entered. Switch closures were accumulated and printed at l-min intervals across the 3-min test. The procedure for assessing reactivity differed from the above only in that the compound stimulus was presented continuously during the final minute of the activity assessment.

ExperimentalDesign. The study consisted of two separate experiments. In the first experiment the effects of prenatal exposure to phenobarbital on locomotor activity and central catecholamine (CA) systems was exam- ined. Fifteen pregnant mice, five per treatment group, were injected with saline or phenobarbital (20 or 40 mg/kg) as already described. Female offspring were weighed and individually caged the day prior to the experiment with food and water continuously available. Seven of the 14 offspring in each prenatal treatment group were injected IP with the methylester hydrochloride of AMT (200 mg/kg) 3 h prior to the de- scribed activity test. An equal number of littermates of these offspring were injected with the water vehicle. Thus, the design consisted of four groups with seven animals per group. Activity was accumulated and printed at 1-min intervals to determine changes across time.

The second experiment was to determine the influence of prenatal exposure to phenobarbital on reactions to sudden changes in the stimulus environment, as described. The experiment utilized seven female off- spring per treatment group (SC, P 20, and P 40). Each offspring was obtained from a different litter. The reactivity test has been described. Animals were treated identically to those in the CA experiment, with the exception that injections were not given and the complex stimulus was presented during the final minute of the activity test.

Data Analysis. Data were subjected to analysis of variance (ANOVA) using litter values as the unit of analysis. In cases where more than one offspring was present in a particular treatment group, the average value was used as a data point. The number of litters associated with each treatment group and the type of ANOVA employed is specified in Results.

Resul t s

Catecholaminergic Systems and Locomotor Activity. Body and bra in weights, concent ra t ions o f D A and NE, l o c o m o t o r activity, and the effects o f A M T on the these three measures for female offspring o f SC, P20, and P40 dams are sum- mar ized in Table 1. Each t rea tment condi t ion represents five litters, and l i t termates were used to assess the effects o f A M T on offspring f rom the three prenata l t rea tment groups. Body and bra in weights did no t vary as a funct ion o f prenatal exposure to phenobarb i ta l [F(2, 14) = 0.400 and 0.350 for body and brain weight data, respectively]. L o c o m o t o r ac- tivity, D A , and N E data were analyzed using repeated measures A N O V A , since litters were evenly dis tr ibuted between the A M T and vehicle t reatments . Prenatal exposure to phenobarb i ta l did no t alter concent ra t ions of D A [F(2, 12) = 0.060] or N E [F(2, 12) = 0.275]. L o c o m o t o r activity of P20 and P40 offspring, however , was approx- imately 1.5-times higher than that of SC offspring [F(2, 12) = 4.943, P < 0.05]. Fu r the r analysis o f the activity data across t ime established a significant in teract ion of prenata l condi t ion with t ime of activity measuremen t [F2 ,24) = 3.946, P < 0.05]. These da ta are p lo t ted on the left g raph in Fig. 1 and will be compared with da ta f rom animals in the react ivi ty test discussed below. Fo r present considerat ion, it is no ted that the activity of SC, P20, and P40 offspring differs par t icular ly dur ing minute 3 of the test. Dur ing this period, activity o f SC offspring declined, whereas that o f P20 and P40 offspring was not reduced f rom that observed dur ing minute 1 o f testing.

Table 1. Effects of daily maternal phenobarbital or saline injections for the last trimester on mature female offspring locomotion, catechol- amines, and reaction to c~-methyltyrosine (AMT). Means _+ SE were calculated from data on five litters per group. Vehicle offspring were injected with water and AMT littermates with AMT 3 h prior to a 3-min activity test. Activity scores represent the number of squares entered in an open field arena. Statistical analysis of data is described in the text. The important finding was that prenatal exposure to phenobarbital elevated activity in the absence of altered body weight, brain weight, or brain concentrations of catecholamines

Saline Phenobarbital

20 mg/kg 40 mg/kg

Body weight (g) 26.8+_1.1 26.5_+1.3 27.2+-0.7

Brain weight (mg) 451 +- 5 444 _+ 9 449 +_ 2

Dopamine (ng/g brain)

Vehicle 1,105_+159 1,139+_83 1,093+- 59

AMT 568_+74 4 9 5 _ + 4 6 503+_34

Norepinephrine (ng/g brain)

Vehicle 534_+ 16 515 +_ 12 531 _+ 9

AMT 328 +- 6 328 + 3 340 _+ 7

Locomotor activity

Vehicle 97_+ 9 1 t9 + 10 124+_ 17

AMT 68+_6 116_+20 104_+13

0 o

03

- - 2O o

/

40 /

I I I

I 2 3

4 0

2 0

Minutes

~dk

I I I

I 2 3

Fig. 1. Open field activity scores for mature female offspring of dams injected daily for the last third of pregnancy with saline (0 ~,) or phenobarbital at 20 mg (k A) or 40 mg ( r e - ' t ) per kg. Data points on the left graph represent mean scores per minute based on five litters per treatment group. Data summarized on the right graph represent similar data from different animals (seven per treatment group), which were exposed to a pulsating tone-light stimulus during minute 3 of testing

A M T injections reduced bra in concent ra t ions of D A and N E to abou t the same extent for all p renata l t rea tment groups (approximate ly 55 % and 37 % for D A and NE, respectively). A M T injections also reduced activity for all but the P20 offspring. A N O V A on these data, however , established no significant interact ions of A M T with the prenata l drug condi t ion. Fu r the r analysis of activity data, consider ing the dis t r ibut ion of activity across t ime by A M T - and vehicle- injected animals, p rov ided no addi t ional useful informat ion .

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Reactivity to Environmental Stimuli. The activity of SC, P20, and P40 offspring in the absence and presence of the compound stimulus described above is summarized in Fig. 1. As noted on the left graph and already discussed, activity of P20 and P40 offspring was higher than that of SC offspring, particularly during the final minute of the test, when SC activity had declined. The data summarized in the right graph (Fig. 1) were obtained from seven offspring per group (one per litter) of SC, P20, and P40 dams. These animals were treated identically to those used in the CA experiment, with the exception that the AMT injections were omitted and the compound stimulus was presented during minute 3 of the activity measure, as described. Data summarized in Fig. 1 were initially analyzed with a 2 - x - 3 - x - 3 ANOVA using repeated measures on the time factor (program BMDP2V, Health Sciences, UCLA). As noted by considering both graphs in Fig. 1, the change in activity across time was dependent upon both prenatal treatment condition and stimulus condition, an observation statistically supported by a significant prenatal condition-• condition-x- time interaction IF(4, 60) = 3.18, P < 0.019]. Data sum- marized in the right graph indicate that activity was sub- stantially reduced during minute 3 when the tone-light stimulus was presented. Further analysis of data from minutes 2 and 3 statistically supported this reduction [F1,18) = 101, P < 0.01]. Although the graph suggests that activity may have been more extensively reduced for pheno- barbital offspring, the prenatal condition- x -time interaction was not significant. The differential effect of the stimulus on activity of SC versus P20 and P40 offspring is striking, however, when activity during minutes 2 and 3 on both graphs is considered. For SC offspring, activity was lower during the third compared to the second minute in both the absence (left graph) and presence (right graph) of the stimulus (latter reduction 21% greater). In contrast, activity of phenobarbital offspring was reduced during minute 3 only in the presence of the stimulus. Thus, the activity reduction during minute 3 in the presence of the stimulus was 50 % and 67 % greater than when the stimulus was absent for P20 and P40 offspring, respectively. Statistical support for the differential effect of the stimulus on activity of saline and phenobarbital offspring is provided by an ANOVA on data during minute 3, indicat- ing a significant prenatal condition-x-stimulus condition interaction [F(2, 30) = 9.74, P < 0.01].

Discussion

This study provides further evidence that injections of phenobarbital during the last third of pregnancy produce behavioral changes in offspring that extend well into adult- hood. The female offspring of drug-treated dams used in the present study were more active than saline controls in an open field arena and were more reactive to an abrupt change in environmental stimuli. Heightened open field activity has previously been reported for male (Middaugh et al. 1975a), but not female (Zemp and Middaugh 1975) offspring of mice injected with phenobarbital at doses and gestation periods identical to the present study. The only apparent differences between these studies is that offspring in the present study were approximately 2.5-times older. Whether or not the effect on females is indeed age-dependent or the difference reflects variability of outcome commonly noted in prenatal drug studies (Hutchings et al. 1979; Wilson 1973) remains to be determined. The heightened activity observed in the present

study appears to be at least partially related to slower habituation to the open field than was observed for SC animals.

Increased reactivity (i.e., a sharp reduction in activity) to abrupt changes in environmental stimuli observed in the present study has been recently noted in a similar experiment of 21-day old offspring of dams injected with phenobarbital (Middaugh et al. unpublished data). From the present study, it is apparent that the effect extends into adulthood. The greater disruption of ongoing activity for phenobarbital offspring upon presentation of the stimulus compared to controls in the present study is consistent with the greater disruption of an escape response to shock for rats prenatally exposed to the drug, as reported by Martin et al. (1979). It is possible that the greater disruption in behavior of rodents prenatally exposed to phenobarbital produced by changes in environmental stimuli reflects slower habituation to en- vironmental change, as suggested by the activity studies. Unfortunately, the study by Martin et al. (1979) and the present one do not allow us to factor out the contribution of altered habituation to the overall reduced scores. In contrast to results obtained in the present study, 18- or 19-day old rats injected with phenobarbital for 14 days prior to testing had lower activity levels than control rats, except during pre- sentation of a compound visual-auditory stimulus when their activity was higher than that of controls (Diaz and Schain 1978). The behavioral differences observed in the two studies could be due to several differences in methodology. Aside from differences in stage of development during which the drug was administered, the age of the animal at the time of testing, etc., a major difference was that, in contrast to the Diaz and Schain report (1978), there was no phenobarbital present in our animals at the time of behavioral testing. Thus, the effect noted in the present study is clearly due to alterations other than the immediate drug effect or with- drawal from the drug.

Neurochemical data obtained in the present experiment provide no evidence that neural systems utilizing DA or NE as a transmitter were altered in offspring of dams injected with phenobarbital. Brain concentrations of both transmitters were similar for the prenatal treatment groups and the extent of reduction in concentration following pharmacological inhibition of tyrosine, the rate-limiting enzyme, was inde- pendent of prenatal treatment. Reduction of transmitter concentration following synthesis inhibition under certain conditions serves as an index of transmitter turnover, which in turn reflects neural activity of tracts utilizing the particular transmitter (Costa and Neff 1969). AMT injections reduce brain concentrations of CA over a 6-h period (Corrodi and Hanson 1966). Thus, the concentration 3 h after injection of AMT in the present study allows testing for group differences in turnover of DA and NE. The similar reductions in DA or NE concentrations for the three groups following synthesis inhibition thus provides no evidence that catecholaminergic neural systems are functionally altered by prenatal exposure to phenobarbital. The same degree of AMT-induced activity reduction for the three groups again suggests that DA and NE neural systems are not extensively altered in offspring of mice injected with phenobarbital.

In summary, the present study indicates that maternal injections of phenobarbital increase activity and reactivity of mature female offspring to sudden changes in environmental stimuli. The behavioral changes were noted in the absence of readily observable physical changes and could not be related

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to a l te red ca t echo l amine rg i c sys tems wi th in the res t r ic t ions of the p resen t s tudy. Acknowledgements. This research was supported by NIDA grants DA 00041 and DA 01750. The authors thank Dr. C.L. Loadholt, Department of Biometry, for his assistance with statistical analysis of data.

R e f e r e n c e s

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Gupta C, Sonawane BR, Yaffe SJ (1980) Phenobarbital exposure in utero: Alterations in female reproductive function in rats. Science 208:508-510

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Middaugh LD, Boggan WO, Wilson-Burrows C, Zemp JW (1979) Prenatal maternal phenobarbital alters plasma concentrations o f corticosterone in developing offspring. Life Sci 24:999-1002

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Received July 8, 1980; Final version April 20, 1981