Neurobehavioural effects of prenatal sodium valproate exposure in rat offspring

 

Arun Kumar Jaiswal*

Neuropharmacology Laboratory, Department of Pharmacology, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221005, India and Department of Psychology, Mahatma Gandhi Kashi Vidyapith, Varanasi 221002, Uttar Pradesh, India

 

ABSTRACT:

Pregnant rats were administered sodium valproate (12.5, 25 and 50 mg/kg/day) orally once daily during the gestation days 13 to 20. Maternal sodium valproate treatment produced a dose dependent significant reduction in litter size at birth and reduced body growth during postnatal days. Sodium valproate treatment had no significant effect on maternal weight gain during pregnancy and pups mortality during postnatal period. The pups born were subjected to open-field exploratory behaviour and elevated plus maze tests of anxiety at 8 and 9 weeks of age. Prenatal sodium valproate treatment (12.5, 25 and 50 mg/kg/day) induced significant increase in open-field ambulation, rearings and self-grooming in rat offspring. Prenatal sodium valproate treated rat offspring displayed significantly increased anxiogenic behaviour on elevated plus maze (spent less time on open arms, more time on enclosed arms and made fewer number of entries on open arms)  comparison to control rat offspring. The results suggest that prenatal sodium valproate exposure induces neurobehavioural toxicity and indicate persistent behavioural alterations in the rat offspring.

 

 

 

INTRODUCTION:

Valproic acid (VPA) is a widely prescribed anticonvulsant first introduced in France in 1994, in Britain in 1973, and in the United States in 1978¹. The antiepileptic drug valproic acid (sodium valproate (SV), 2-propyl-pentanoic acid) has been implicated as a human teratogen by prospective and retrospective epidemiological studies suggesting that use of VPA in pregnancy may be associated with an increased incidence of spina bifida along with other malformations^2-7.

 

Neural defects (excencephaly) have been observed in vivo after maternal conventional treatments with VPA only in the mouse and hamster^8-10 but an increased incidence of spina bifida has been produced in the mouse only after three consecutive administrations of high doses of valproic acid on day 9 of gestation¹¹. Reports also suggest that treatment with SV during a critical time in neurogenesis in the CD-1 mouse embryo alters the neural architecture of the neuroepithelium, with a loss of integrity at both the basal and apical surfaces¹². Prenatal VPA exposure also caused malformations of the skeletal system in various animal species (mouse, rat, rabbit, and monkey)^13-15. Recently, prenatal exposure to SV has been reported to result in a very high frequency of malformed embryos (84%) including open brain folds (73%), somite defects (36%) and malformations (20%) in mice and relatively less marked effects in rats¹⁶.

 

Some experimental studies have examined the functional effects of developmental exposure to VPA in animals. Offspring exposed to VPA during gestation days 7-8 showed decreased locomotor activity, increased swimming maze errors and reduced tactile response¹⁷. Prenatal exposure to valproic acid has also been reported to cause increased auditory startle response in rat offspring¹⁸. VPA (200 and 300 mg/kg) exposure during prenatal days 7-18 in rats has been reported to decrease activity, increase errors in a multiple-T water maze, and reduce startle responding to both auditory and tactile stimuli in the rat offspring^19,20.  In a study, mouse offspring were exposed to SV (160-180 mg/kg) during the prenatal and postnatal days and these offspring exhibited increased social investigations when paired with other mice in adult age²¹.   Prenatal exposure to VPA induces long-term behavioural changes in rat pups which include accelerated maturation of negative geotaxis, convulsive behaviours, enhanced seizure like activity, increased hind limb movement, delayed startle response, abnormal body rotation and strub tail in comparison to age matched controls²².

 

VPA is an established CNS teratogen in humans and animal experiments have suggested that CNS teratogens are behavioural teratogens at lower, nonmalforming doses²³. However, behavioural teratological effects of SV on many other behaviour forms are not available. As such, the present investigation was planned to assess the effects of prenatal SV treatment in rats on open-field behaviour test and elevated plus maze test of anxiety in rat offspring.

 

MATERIALS AND METHODS:

Subjects and general procedure

Primiparous Charles Foster (CF) rats, procured from the Central Animal House of the Institute, were used in the present study.  Rats were acclimatized to the laboratory for a minimum of 20 days prior to drug administration and were maintained on a 12 h light/dark cycle with rodent chow (Brook-Bond, Lipton, India) and tap water available ad libitum.  They were housed in standard polypropylene cages in groups of 4-5, at 25°±1°C; 45-55% relative humidity. The experiments were performed following ‘Principles of laboratory animal care’ (NIH publication No. 86 – 23, revised 1985).

 

All the pregnant rats (presence of sperm in vaginal swab was taken as day one of conception) were randomly assigned to four maternal drug treatments: sodium valproate 12.5, 25 and 50 mg\kg and control groups. Sodium valproate [Epilex^® Abbot Laboratories (India) Pvt Ltd, Mumbai, India] solution was prepared in 0.2% carboxy methyl cellulose (CMC) suspension. Pregnant females were administered SV 12.5, 25 and 50 mg/kg/day orally with the help of an orogastric cannula once daily during gestation days 13 to 20 this being the critical period for neural development in rats²⁴.  Pregnant control rats were similarly treated with vehicle i.e., 0.2% CMC. Beginning with the morning of day 21, the gravid rats were checked twice daily for deliveries.  Animals were allowed to deliver normally and newborns were culled to 8 pups / dam and were foster nursed.  The pups were weighed weekly and weaned at three weeks of age.  Thus, several litters were obtained for every dose of the drug and control treatments. Thereafter, at eight weeks of age, one male rat pup from each litter of the several litters was randomly selected to form a treatment group to control the possible litter effects on behavioural measure²⁵. These rat offspring were subjected to open-field exploratory behavior test and elevated plus maze test of anxiety respectively at 8 and 9 weeks of age.

Behavioral experiments

 

Open- field exploratory behaviour test

An open open-field apparatus similar to that of Bronstein²⁶ was used to study the open field exploratory behaviour in rats. It was made of plywood and consists of a square (61 x 61 cm) with high walls (61 x 61 cm).  The entire apparatus was painted black except for 6 mm thick white lines, which divided the floor onto 16 squares.  Open-field was lighted by a 40W bulb focussing onto the field from a height of about 100 cm. The entire room, except the open-field, was kept dark during the experiment.  Each animal was centrally placed in the test apparatus for 5 min and the following behavioural aspects were noted:

 

(i) Ambulation: this was measured in terms of the number of squares crossed by the animal; (ii) Rearings: number of times the animal stood on its hind limbs; (iii) Self groomings: number of times the animal groomed its facial region, and licked / washed / scratched various parts of its body; and (iv) Faecal droppings: number of faecal droppings excreted during the period.

 

Elevated plus-maze test

The wooden plus maze consisted of two opposite arms, 50 x 10 cm, crossed with two enclosed arms of the same dimension with walls 40 cm high.  The arms were connected with a central square, 10 x 10 cm, giving the apparatus shape of a plus sign.  The maze was kept in a dimly lit (light intensity – 10 lux) room and elevated 50 cm above the floor.  Naive rats were placed individually in centre of the maze, facing an enclosed arm.  Thereafter, number of entries and time spent on the open and enclosed arms were recorded during the next 5 min.  An arm entry was defined when all four paws of the rat were in the arm. A neutral ‘blind’ observer made observations²⁷.

 

Statistical analyses

Days of conception, litter size, and birth-weight data were analysed by one-way ANOVA with the help of SigmaStat, while maternal weight gain and body-growth data of rat offspring were analysed by two-way ANOVA with one repeated measure using SPSS 7.5. Frequency data of offspring mortality was analysed using Fisher’s test for uncorrelated proportions (Two-Tailed). The behavioural data for the open-field and elevated plus maze tests were analysed by multivariate ANOVA with the help of SPSS 7.5. Post hoc comparisons were analysed by Tukey’s test for only those responses that yielded significant treatment effects in the ANOVA²⁸.

 

RESULTS:

Maternal and birth measures

The data obtained for maternal and birth measures are given in Table 1, 2 and 3. The control and various treatment groups did not differ significantly with respect to number of days of conception (F_{3, 44} = 0.56, P > 0.05) and maternal weight gain during pregnancy. Neither the “treatments” (F_{3, 44} =1.24, P > 0.05) nor the interaction “treatment × gestational age” (F_{9, 132} =1.11, P > 0.05) have been found to be statistically significant. The main effect of “gestational age” (F_{3, 132} = 1298.91, P < 0.05) was noticed to be significant which can be attributed to normal weight gain due to pregnancy. However, the mean values among the treatment groups differed significantly for litter size (F_3,44 = 7.60 < 0.01) and the post hoc comparisons further indicate that SV in the dose of 25 and 50 mg/kg caused significant reduction in the litter size in comparison to control and SV (12.5 mg/kg) treatments.

 

Table 1-  Effect of prenatal sodium valproate on gestational weight gain during pregnancy (in g) (Values are mean ± SEM)
Groups	n	Days of conception	Gestational age
			1st day of conception	7th day	14th day	21st day
Control	12	22.00 ± 0.12	187.58 ± 2.10	210.08 ± 2.27	231.25 ± 1.87	284.58 ± 2.69
SV(12.5 mg)	12	22.25 ± 0.13	189.63 ± 2.88	210.18 ± 2.90	233.54 ± 2.64	282.09 ± 2.54
SV (25 mg)	12	22.08 ± 0.15	185.58 ± 2.46	205.83 ± 2.25	230.00 ± 2.20	279.67 ± 1.91
SV (50 mg)	12	22.17 ± 0.17	190.00 ± 2.78	208.75 ± 2.50	229.58 ± 2.32	279.42 ± 2.25

 

 

 

Birth-weight and Growth rate of treated and control pups

Prenatal SV treatment produced a doses dependent significant reduction in birth weight of the rat pups (F_3,32 = 8.28, P < 0.01) and the rat pups treated with SV 25 and 50 mg/kg during  the prenatal period weigh significantly less than rat pups of control and SV (12.5) groups. The obtained results for growth rate of treated and control pups are presented in the Fig -1. Analysis of variance with one repeated measure performed on the body weight for 10 weeks yielded significant effects of “treatment” (F_3,32 = 125.58, P < 0.01), “age” (F_10,320 = 12933.82, P < 0.01) and “treatment × age” interaction (F_30,320 = 43.84, P < 0.01) on body-growth of rat offspring. A dose dependent significant growth retardation was observed from the very first week of age in rat offspring treated with 12.5, 25, and 50 mg/kg dose of SV in comparison to control rat offspring and this significant difference persisted afterwards also. The “age” effect reflected growth and the “treatment × age” reflected the differential growth among treatment groups. No significant difference was observed between groups with respect to number of dead foetuses and also the number of deaths of pups during the weaning period.

 

 

Fig. 1- Effect of prenatal sodium valporate exposure on body growth of male rat offspring.

 

 

 

 

Open-field exploratory behaviour test

Multivariate ANOVA performed on the four response measures of OFT indicate that there exist statistically significant treatment effect (F_{12, 124}= 7.05, P < 0.01) and the univariate tests were also found to be statistically significant for ambulation [F_3,32 = 23.31, P < 0.001], rearings [F_3,32 = 15.04, P < 0.001], self-groomings [F_3,32 = 8.66, P < 0.001]  and faecal pellets [F_3,32 = 8.95, P < 0.001]  responses. Post-hoc multiple comparisons among treatment mean values indicated that rat offspring treated in utero with SV (12.5, 25 and 50 mg/kg) displayed significantly increased open-field ambulation and rearings in comparison to control rat offspring. SV (25 and 50 mg) treatments also caused significant increase in self-grooming behaviour in comparison to control treatment. However, SV (12.5) had no significant effect on open-field self-groomings in rats. SV treatment significantly increased the faecal droppings only in the doses of 25 and 50 mg but the lower dose of SV (12.5) had no significant effect on faecal droppings (Table 1).

 

 

 

 

 

 

 

 

 

Elevated plus maze behaviour test

Multivariate ANOVA performed on the four response measures of EPM test indicate that there exist statistically significant treatment effect (F_{12, 124} = 8.32, P < 0.01) and the univariate tests also produced statistically significant treatment effects for time spent in enclosed arms [F_3,32 = 42.12, P < 0.001],  time spent in open arms [F_3,32 = 10.08, P < 0.005], number of entries in enclosed arms [F_3,32 = 10.32, P < 0.001], and number of entries in open arms [F_3,32 = 14.48, P < 0.001]. Further inter group comparisons indicate that rat offspring exposed to SV (12.5, 25 and 50 mg/kg/day) during the prenatal period spent significantly less time on open arms and more time on enclosed arms, made significantly fewer number of entries on open arms and made more number of entries on enclosed arms in comparison to control rat offspring (Table 2). Ratio of open/enclosed arms for time spent and entries made also yielded similar results.

 

 

DISCUSSION:

The results of the present experiment demonstrate that prenatal exposure to SV has long-term effects on litter size, offspring birth weight, growth and behaviour. These effects were reduced litter size, low birth weight, stunted body growth, increased open-field exploratory behaviour and anxiety. No differences were found among groups of pregnant dams with respect to number of days of conception and also the maternal weight gain through the whole gestation period. Similar findings have been reported in a study, however,  in this study a much higher dose range of valproic acid  (200, 300, 400, 600 mg/kg) was used and the drug was given during 7th to 18th day of gestation¹³ . This study reported significant reduction in maternal weight gain and live fetuses per dam by 400 mg/kg dose of valproic acid and significantly low birth weight of pups by 300 and 400 mg/kg of valproic acid. This study also reported a dose dependent reduction in body weight of valproic acid treated pups at different postnatal ages. These observations also support the results of the present investigation. In the present study, a much lower dose of SV was administered through the oral route and the time of the drug treatment was also different i.e., day 13-20 of gestation. These differences in the observations of the present study and earlier reports may be attributed to the difference in drug administration protocol and also to the strain of the rats used, and at present it is difficult to make any generalization regarding the effects of these variables on maternal toxicity. Valproic acid has been shown to produce embryotoxicity and the pattern of drug administration is a major determinant of embryotoxicity⁸. Higher doses of valproic acid have been documented earlier to produce maternal toxicity in rats¹³.

 

There is only one report on the psychoteratological effects of prenatal SV treatment¹⁹ (Vorhees, 1987)¹⁹, which supports our observations that prenatal SV exposure induces neurobehavioural alterations like decreased activity, reduced spontaneous alternation, increased swimming time, increased maze errors and reduced startle responding in the rat offspring treated during gestation days 7-18 with 150 and 200 mg/kg doses of valproic acid¹⁹. In contrast, present findings that prenatal SV exposure, in much lower doses (12.5 to 50 mg/kg) caused significantly increased open field ambulation and rearings do not support the earlier observations. Direct comparisons of the findings of the present study are not possible because of paucity of studies on behavioural effects of prenatal SV exposure on anxiety parameters in both experimental and clinical settings. However, there are a number of studies available on the behavioural teratological effects of other centrally acing drugs. Prenatal exposure to diazepam, a BDZ anxiolytic, has been reported to increase open-field behaviour in rat offspring²⁹. Prenatal exposure to lorazepam during days 13 to 20 of gestation increased open-field activity at 3 weeks of age in mice³⁰. Prenatal alprazolam treatment during days 13-20 of gestation has been reported to increase open-field ambulation at 8 weeks of postnatal age in rats³¹. Similarly, prenatal phenobarbitone, alcohol^32, and haloperidol²⁹ treatments have also been found to increase open-field activity in rodent offspring. Earlier reports also indicate abnormally increased activity in the open-field such as ambulation, rearing, defecation and urination which represent more primitive responses and have been considered to be an index of increased emotionality³³. It has been suggested that this abnormal increase in open-field activity of treated animals may be due to slower habituation to the novel environment³⁴.

 

Contradictory reports are also available on the effects of prenatal benzodiazepine exposure on anxiety patterns in rodent offspring i.e., it reduced and increased social interaction in rat offspring respectively in familiar and unfamiliar environments in comparison to vehicle treatment³⁵. However, reports of increased anxiety state in rat offspring on elevated plus maze, elevated zero mazes and social interaction tests by prenatal exposure to diazepam (10 mg/kg/day)²⁹ and alprazolam (0.1 and 0.2 mg/kg/day) during gestation days 13 to 20 are also on record. Phenytoin, phenobarbital and trimethadione have been reported to produce behavioural teratogenecity wherein phenytoin and trimethadione produced hyperacivity in rodents^36,37.  The findings of the present study are in accord with the aforementioned observations suggest that prenatal SV exposure is behaviourally teratogenic at doses much below those causing embryotoxicity, maternal toxicity or malformations. These findings support the clinical observations that valproic acid may cause a fetal valproate syndrome in which CNS dysfunction is the most serious debilitating effect³⁸.

 

From both neurochemical and behavioural observations following BDZs exposure, the most sensitive period for inducing long-term effects of prenatal drug exposure appears to be the 3^rd week of gestation in rats²⁴. This period appears to be the most vulnerable to the action of neuroactive drugs because this is the critical period for synaptogenesis and formation of neural circuits^39,40.

 

Autoradiographic studies demonstrate that BDZ receptors appear earliest in the brain stem and hypothalamus in the rat, but by birth BDZ binding is present in all areas of the CNS⁴¹. Development of GABA and catecholamine receptors also takes place during the 3rd week of gestation in rats⁴². Acetylcholine, GABA and catecholamine neurotransmitters also appear during this period⁵. GABA is an inhibitory transmitter and acts on a receptor channel complex permeable mainly to chloride anions that act to reduce neuronal excitability. GABA is the principle excitatory neurotransmitter in the developing brain and acts as an epigenetic factor to control developmental processes⁴³, including cortical and hippocampal neuroblast migration^44,45. Therefore, GABAergic signalling plays a major role in brain physiology, and GABAergic signalling dysfunction may result in pathological conditions. Vigabatrin (VGB) and valproate (VPA) act on GABA signaling^{46, 47,} and prenatal-exposure to these antiepileptic drugs during gestation days 14 to 19 have been demonstrated to cause hippocampal and cortical dysplasias in rats, and embryonic day 14 to 19 correspond to the period of neurogenesis and migration of hippocampal (CA1) and neocortical (superficial) neurons⁴⁸. In the present study, SV was administered during embryonic days 13 to 20 and the actions of anticonvulsant drugs are mediated through GABAergic signaling, a similar cortical malformations may be hypothesized in rat offspring exposed in utero to SV, however, empirical studies are needed before making any conclusions.

 

The present investigation indicates that prenatal exposure to SV during critical developmental period of brain in rat can adversely affect the behaviour of the progeny and hence SV can be said to induce behavioural teratological effects.

 

ACKNOWLEDGEMENTS:

This work was supported by the grant-in-aid [F.6-1/93(SA)] from University Grants Commission, New Delhi, India to the author.

 

CONFLICT OF INTEREST:

Author declares no conflict of interest.

 

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Received on 05.08.2016       Modified on 17.08.2016

Accepted on 23.08.2016      ©A&V Publications All right reserved