INTRODUCTION:

The benzodiazepines (BDZs) anxiolytics are widely prescribed psychoactive drugs and rank among the most commonly used class of therapeutic agents ^1-2. BDZ anxiolytics are also widely prescribed during pregnancy, with 30 to 40% of all pregnant women being given antianxiety drugs at some stage of pregnancy³. Prenatal exposure to BDZs induces a variety of functional disturbances in rodents. In general, motor activity, exploratory behaviour, learning, anxiety and operant responses may be altered in adult animals exposed in utero to BDZs like diazepam, chlordiazepoxide, lorazepam, and alprazolam, etc ^4-9.

Clobazam (CLB) is a benzodiazepine, whose structure differs from that of other BDZs in that the nitrogen atom in the heterocyclic ring is in the 1,5- instead of 1,4- position ¹⁰.

The drug was initially developed as an anxiolytic agent for the treatment of anxiety neurosis¹¹. In contrast to other BDZs, it has been shown in placebo-controlled trials to have antidepressant properties^12-14. Its antiepileptic property was first reported in mice¹⁰. In 1979, Gadtaut and Low¹⁵ reported the clinical effects of CLB use as adjunctive therapy for all types of refractory epilepsy. Since then, considerable animal and clinical anti epileptic properties have been reported in clinical and experimental studies that distinguish it from the 1, 4-benzodiazepines. CLB is indicated as an adjuvant in partial seizures, typical and atypical absences, myoclonic, and secondary generalized, atonic seizures and in Lennox-Gastaut syndrome. It is also used in catamenial epilepsy and cluster seizures and refractory epilepsy¹⁶. In animal models of chemical induced seizures, CLB is more potent antiepileptic drug than phenytoin phenobarbital, carbamazepine or valproate^17,18. In other animal models, the effects of CLB are more specific and seemingly superior to the anticonvulsant activity of the 1, 4-benzodiazepines¹⁸. Depression, irritability, and tiredness are the common side effects of CLB treatment in patients of epilepsy. Deterioration in behaviour and mood disturbances has also been reported particularly in patients with learning disabilities¹⁹.

So far, only one study has shown teratogenic effect of CLB in the developing central nervous system reported alteration in cytoarchitecture of the cerebral and the cerebellar cortex in rats²⁰, and behavioural teratological effects of CLB in humans and neonatal withdrawal symptoms are also on record²¹. As such, the present investigation was planned to assess the effects of prenatal CLB treatment in rats on behaviour of rat offspring in an open-field and anxiety parameters.

MATERIALS AND METHODS:

Subjects and general procedure

Primiparous Charles Foster (CF) strain albino rats, procured from Institute’s Central Animal House, were used in the 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. All the experiments were performed beginning at 09.00 hours. 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 housed singly and randomly assigned to four maternal drug treatments: clobazam 0.5, 1.0, 2.0, and 5.0 mg\kg and control groups. Clobazam (FRISIUM, Torrent Pharmaceuticals, Ltd, India) solution was prepared in 0.2% carboxy methyl cellulose (CMC) suspension. At 10.00 hours pregnant females were administered CLB 0.5, 1.0, 2.0 and 5.0 mg/kg once daily orally with the help of an orogastric cannula during gestation days 13 to 20 this being the critical period for neural development in rats²². 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 and the control treatment. 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 measure²³. These rat offspring were subjected to following behavioral tests of anxiety at 8 weeks of age.

Behavioral experiments:

Open- field exploratory behaviour test²⁴

An open open-field apparatus made of plywood and consisted of a square (61 x 61 cm) with high walls (61 x 61 cm) was used to study exploratory behaviour in rats. The entire apparatus was painted black except for 6 mm thick white lines, which divided the floor into 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 ambulation, number of rearings, self-groomings and faecal droppings were noted for every animal.

Elevated plus-maze test:²⁵

The plus maze, made of wood, 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 the shape of a plus sign. The maze was kept in a dimly lit room (light intensity – 10 lux) and elevated 50 cm above the floor. Naive rats were placed individually at the 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 observations.

Elevated zero-maze test:²⁶

The maze comprised a black perspex annular platform (105 cm in diam, 10 cm width) elevated to 65 cm above the ground level, divided equally into four quadrants. A black perspex wall (27 cm high) enclosed the two opposite quadrants on both the inner and outer edges of the platform, while the remaining two opposite quadrants were surrounded by perspex “lip” (1 cm high) which served as a tactile guide to animals on these open areas. The apparatus was illuminated by dim white light (10 lux) arranged in such a manner as to provide similar lux levels in open and enclosed quadrants. Rats were placed on one of the enclosed quadrants for a 5 min test period. During the 5 min test period, time spent on open arms, number of `head dips’ over the edges of platform, and number of `stretched attend postures’ from closed to open quadrants were recorded. Animals were scored as being in the open area when all four paws were in the open quadrants and in the enclosed area only when all four paws had passed the open-closed divide. The open-field and the mazes were cleaned with 5% ethanol / water solution and dried thoroughly between test sessions.

Statistical analyses:

The behavioral data were analyzed using the statistics software SPSS, version 16.0.The data are expressed as means ± SD for each treatment group. The data for body growth was analysed by repeated measure ANOVA and behavioural data were analysed by one-way MANOVA. Post hoc group comparisons were made by Newman-Keuls test for only those responses that yielded significant treatment effects in the analysis of variance²⁷.

RESULTS:

Body growth

Repeated measure ANOVA (with 5 treatment conditions and 17 ages as repeated measures) performed on body growth yielded significant main effects of ‘treatment’ [F (4, 33) = 35.15, P<.01], ‘age’ [F (16, 528) = 1220.00, P<.01] and interaction effect ‘treatment x age’ [F (64, 528) = 7.45, P<.01]. The results indicated that the body weight of rat offspring increased significantly with increase in age in all the 5 treatment groups. CLB treatment had adverse effects on the body growth of rat offspring. Significant interaction between ‘treatment x age’ also indicated that CLB treatments (0.5, 1, 2 and 5 mg) had differential effects on the body growth of rat offspring. At birth rat offspring exposed prenatally to CLB (1mg) weighed less than control rat offspring and this significant difference persisted only for a week.. By the age of 3 weeks offspring treated with CLB (2 and 5 mg) weighed significantly less than control offspring and by the age of 4 to 5 weeks rat offspring treated with CLB (0.5, 1, 2, 5 mg) weighed significantly less than control offspring. However, rat offspring of the four CLB treatment groups did not differ significantly from each other with respect to body weight up to the age of 7 weeks. Furthermore, by the age of 8 weeks rat offspring of the CLB (0.5, 1, 2, 5 mg) treatment groups not only weighed significantly less than the rat offspring of control group but also showed dose dependent significant reduction in body growth and this pattern of body growth persisted thereafter. From 8 weeks onwards rat offspring treated with different CLB doses also differed significantly from each other with respect to body weight.

Open-field exploratory behaviour test:

A one-way MANOVA performed on the scores of open-field test revealed a significant multivariate main effect for treatment, Wilks’ λ = 0.161, F (16, 92.289) = 4.727, p <0.01, partial eta squared = 0.367. Power to detect the effect was 0.999. Given the significance of the overall test, the univariate main effects were examined. Significant univariate main effects for treatment were obtained for ambulation [F(4,33) = 5.561, p <0.01] rearings [F(4,33) = 4.218, p <.01], grooming [F(4,33) = 15.715, p <0.01], feacal pellets [F(4,33) = 4.415, p <0.01] and central squares crossed [F(4,33) = 3.056, p <.05]. Rat offspring treated prenatally with CLB (0.5 and 1 mg/kg) displayed significantly increased open-field ambulation and rearings in comparison to control rat offspring. CLB treatment in the dose of 2 and 5 mg/kg as compared to control treatment had no significant effect on open-field ambulation and rearings. CLB treatments in all doses (0.5, 1, 2, and 5 mg) also caused dose dependent significant increase in self-grooming behaviour in comparison to control treatment. However, CLB treatment significantly increased the faecal droppings only in the doses of 0.5 mg and the higher doses (1.0, 2.0 and 5.0 mg) of CLB had no significant effect on faecal droppings (Table 1).

Elevated plus maze behaviour test

A one-way MANOVA performed on the scores of elevated plus maze behaviour test revealed a significant multivariate main effect for treatment, Wilks’ λ = 0.060, F(16, 92.289) = 8.704, p < 0.01, partial eta squared = 0.505. Power to detect the effect was 1.00. Given the significance of the overall test, the univariate main effects were examined and significant univariate main effects for treatment were obtained for entries in open arms [F(4,33 ) = 44.471, p <0.01], entries in enclosed arms [F(4,33 ) = 8.964, p < 0.01] time in open arms [F (4,33 ) = 4.269, p < 0.01] and time in enclosed arms [F(4,33 ) = 47.1786, p < 0.01]. Rat offspring expopsed prenatally with CLB (0.5 and 1, 2.0, and 5.0 mg/kg) displayed significantly decreased entries and time in open arms and increased time spent in enclosed arms than control rat offspring. Rat offspring treated prenatally with 2.0 and 5.0 mg / kg CLB also exhibited significantly decreased entries in enclosed arms than control rat offspring (Table 2).

Elevated zero maze behaviour test

A one-way MANOVA performed on the scores of elevated zero maze test revealed a significant multivariate main effect for treatment, Wilks’ λ = 0.143, F (16, 92.289) = 5.142, p < 0.01, partial eta squared = 0.385. Power to detect the effect was 0.999. Given the significance of the overall test, the univariate main effects were examined and significant univariate main effects for treatment were obtained for entries in open arms [F(4,33) = 8.167, p <0.01], time in open arms [F(4,33) = 20.308, p < 0.01], head dips [F(4,33) = 16.789, p < 0.01] and stretched attend postures [F(4,33) = 10.388, p < 0.01]. Significant treatments differences were obtained in entries in open arms, time in open arms, head dips and stretched attend postures between rat offspring of control and CLB treatment groups. Rat offspring treated prenatally with CLB (1, 2 and 5 mg/kg) displayed dose dependent significantly decreased entries in open arms and stretched attend postures.

Table 1- Effect of prenatal clobazam treatment on open-field exploratory behaviour in rat offspring. (Values are mean ± SEM)

Groups	n	Ambulation	Rearings	Self-Groomings	Faecal pellets
Control	8	73.63±8.55	17.75±2.31	4.38±1.41	2.88±1.46
CLB (0.5 mg)	8	96.75±15.82^aa	25.38±4.90^aa	7.63±1.30^aa	5.25±1.83^aa
CLB (1 mg)	8	91.13±10.38^a	23.75±4.23^aa	8.38±1.41^aa	4.75±1.04a^a
CLB (2 mg)	7	85.29±7.57	21.71±4.99	8.86±1.22^aa	5.29±1.11^a
CLB (5 mg)	7	82.29±12.85	20.71±2.93	9.57±1.51^aa	4.57±0.98

Superscripts ^aand ^aa indicate statistical significance in comparison to control respectively at P<0.05 and 0.01 (Newman-Keuls test).

Table 2 - Effect of prenatal clobazam treatment on elevated plus maze behaviour in rat offspring. (Values are mean ± SEM)

Groups	n	Time spent on (in sec)		Number of entries
Groups	n	Open arms	Enclosed arms	Open arms	Enclosed arms
Control	8	21.63±1.57	223.63±11.68	4.63±0.92	10.38±1.60
CLB (0.5 mg)	8	10.55±2.22^aa	259.08±8.96^aa	1.75±0.46^aa	8.88±2.80
CLB (1 mg)	8	8.30±1.69^aa	267.75±6.93^aa	1.50±0.53^aa	8.13±1.46
CLB (2 mg)	7	7.11±2.50^aa,bb	275.61±9.16^aa	1.43±0.53^aa	7.00±1.83^a
CLB (5 mg)	7	6.09±1.98^aa,bb	278.13±6.83^aa,b	1.14±0.38^aa	4.71±1.60^aa,bb,c

Superscripts a,b and c indicate statistical significance respectively in comparison to control, CLB (0.05mg) and CLB (1 mg). a, b, c and aa, bb denote p < 0.05 and p < 0.01 respectively (Newman-Keuls test).

Table 3 -Effect of prenatal clobazam treatment on elevated zero maze behaviour in rat offspring. (Values are mean ± SEM)

Groups	n	Time spent on open arms	No. of entries on open arms		No. of Head dips	No. of stretched attend postures
Control	8	23.59±2.35		3.63±1.41	4.63±1.06	7.88±1.46
CLB (0.5 mg)	8	13.28±2.68^aa		2.25±1.17	1.75±1.28^aa	7.63±1.68
CLB (1 mg)	8	11.89±2.58^aa		1.88±0.83^aa	1.50±0.76^aa	4.88±2.36^aa,b
CLB (2 mg)	7	8.97±3.21^aa		1.29±0.49^aa	1.57±0.79^aa	4.29±0.95^aa,bb
CLB (5 mg)	7	7.96±1.74^aa		1.14±0.38^aa	1.29±0.49^aa	4.14±0.69^aa,bb

Superscripts ^aand ^b indicate statistical significance respectively in comparison to control and CLB (0.5 mg). ^band ^{aa, bb} denote P<0.05 and 0.01 respectively (Newman-Keuls test).

Similarly, rat offspring treated prenatally with CLB (0.5, 1, 2 and 5 mg/kg) exhibited dose dependent significant decrease in time in open arms and number of head dips in comparison to control rat offspring. However, mo significant difference emerged among the rat offspring of different CLB treatment groups with respect to all the behavioural measures of elevated zero maze test (Table 3).

DISCUSSION:

The results of the present study indicate that prenatal CLB treatment led to increased anxiety, as assessed by the various paradigms used. Prenatal CLB treatment in the dose of 0.5 and 1 mg/kg/day caused increased ambulation and rearings and all the doses of CLB treatment caused increased self-grooming in the open field. Increased and decreased time spent in enclosed and open arms, respectively, fewer numbers of entries made in open arms on the elevated plus maze by rat offspring treated with different doses of CLB during the prenatal period and also the ratio of open/enclosed arms time and entries indicate presence of heightened anxiety in these rat offspring. The finding that the number of closed arm entries was unaffected by the prenatal CLB treatments diminishes the possibility that the drug has non-specific inhibitory effect on elevated plus maze behaviour. Reduced time spent, number of entries, head dips and stretched attend postures on open arms in the zero maze test also show increased anxiety in prenatal CLB treated rat offspring.

There is only one report on the psychoteratological effects of prenatal CLB treatment²¹, which supports our observations that prenatal CLB exposure induces neurobehavioural alterations in the offspring. Direct comparisons of the findings of the present study are not possible because of paucity of studies on behavioural effects of prenatal CLB exposure in both experimental and clinical settings. However, there are a number of studies available on the behavioural teratological effects of 1, 4-benzodiazepines. Prenatal exposure to diazepam, a BDZ anxiolytic, has been reported to increase open-field behaviour⁸and reduce locomotion in rodents²⁸. Prenatal exposure to lorazepam during days 13 to 20 of gestation increased open-field activity at 3 weeks of age in mice⁴. Prenatal alprazolam treatment during days 13-20 of gestation has been reported to increase open-field ambulation at 8 weeks of postnatal age in rats⁹. Similarly, prenatal phenobarbitone, alcohol²⁹ and prenatal haloperidol⁸ treatment has 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 represent more primitive responses and have been considered to be an index of increased emotionality³⁰. It has been suggested that this abnormal increase in open-field activity of treated animals may be due to slower habituation of the rat offspring to the novel environment³¹.

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 treatment³². However, increased anxiety state in rat offspring on elevated plus maze, elevated zero mazes and social interaction tests due to prenatal exposure to diazepam (10 mg/kg/day)⁸ and alprazolam (0.1 and 0.2 mg/kg/day)⁹ during gestation days 13 to 20 is also on record. The findings of the present study are in accord with the aforementioned observations and further add to the knowledge that prenatal CLB treatment induced anxiogenic behaviours on elevated plus and elevated zero mazes.

There is little information on prenatal exposure to BDZs and changes in neurotransmitter activity. However, it is now clear that early developmental exposure to BDZs can induce long-lasting effects on CNS and on behavioural functions in animal models. 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 3rd week of gestation in rats²². 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^{22, 33-34}.Developmentally, BDZ receptors have been identified in the rat³⁵. Autoradiographic studies indicate 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 CNS³⁶. Similarly, development of GABA and catecholamine receptors also takes place during the 3rd week of gestation in rats⁵. Acetylcholine, g-aminobutyric acid and catecholamine neurotransmitters also appear during this period⁵. g-Aminobutyric acid (GABA) is an inhibitory transmitter, acting on a receptor channel complex permeable mainly to chloride anions that act to reduce neuronal excitability. Conversely, GABA is the principle excitatory neurotransmitter in the developing brain and acts as an epigenetic factor to control developmental processes⁴², including cortical and hippocampal neuroblast migration^{43, 44}. As such, 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^{45, 46} and prenatal-exposure to these antiepileptic drugs during gestation days 14 to 19 have been reported to induce 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) neurons⁴⁷. Since, CLB was administered during embryonic days 13 to 20 in the present study and the actions of BDZs are mediated through GABAergic signaling, a similar cortical malformations may be expected in rat offspring exposed in utero to CLB, however, empirical investigations are required before making any conclusions.

Prenatal BDZ exposure during 3rd week of gestation appears to induce altered binding of BDZ in the offspring and a supersensitivity phenomenon at the level of BDZ-GABA complex has been consistently reported²². Gestational exposure to diazepam has been reported to reduce cerebellar and cortical norepinephrine levels³³. The effects of early diazepam exposure on the NE projection to the hypothalamus were not apparent until after 5 weeks of age and functional activity within the hypothalamic NE system is normally delayed^33,
37. Prenatal BDZ exposure has been reported to induce various functional deficits in rodent offspring like alterations in stress responses, EEG synchronization, startle response and complex maze learning tasks^{22, 38-39}. Studies have also documented reduced number of glial cells in the cortex of the offspring following prenatal diazepam exposure, indicating neural degeneration⁴⁰. These observations suggested that prenatal BDZ exposure interferes with the normal organization or development of specific neural systems or mechanisms responsible for mediation of various behavioural functions.

The results of the present study indicate that the effects of prenatal CLB treatment during gestation or early in life may be different from those of its anxiolytic/ antiepileptic effects. The clinical studies have also shown that prenatal drug exposure results in behavioural characteristics of a hyperkinetic child, a syndrome that includes excessive motor activity, excitement, irritability, tearfulness and aggression⁴¹. The present investigation indicates that prenatal exposure to CLB during critical period of brain developmental in rat can adversely affect the behaviour of the progeny and hence CLB can be said to induce behavioural teratological effects.

ACKNOWLEDGEMENTS:

This work was conducted in Neuropharmacology Laboratory, Department of Pharmacology, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221005, India when A K Jaiswal was working there as U.G. C. Research Scientist.

CONFLICT OF INTEREST:

Author declares no conflict of interest.

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Received on 04.04.2016 Modified on 10.04.2016

Research J. Pharmacology & Pharmacodynamics.2016; 8(2): 58-64

DOI: 10.5958/2321-5836.2016.00011.2: