INTRODUCTION:

Flouxetine (FLX) is the first selective serotonin reuptake inhibitor (SSRI) and is as effective as other existing triclyclic antidepressants and at present FLX is the most widely prescribed antidepressant drug all over the World3. Women of child bearing age are at high risk of developing depression sometime during their pregnancy¹, and often Flox is the choice of drug for treatment of their depression. Overall, approximately 5% pregnant women use SSRIs during their pregnancy^2-4. Safe use of Flox during pregnancy and lactation has not been established⁵. It is well established that FLX crosses the placental barrier in rats and mice, producing a comparable level of fetal exposure^6,7. FLX and its metabolite norfluoxetine also cross the rodent fetal blood-brain barrier, leading to detectable FLX and norfluoxetine levels in the blood and brain of rodent pups (see www.karger.com/doi:10.1159/000355709).

Craniofacial malformation, extensive cell death in mesenchymal tissue (located 5-6 layers deep), other major malformation, spontaneous abortions and premature birth have been reported in humans after using Flox during pregnancy^{5, 8}. The studies have also reported deleterious effects of FLX on one or more measures, including decreased pregnancy duration⁹, litter size^7,10 and birth weight^7,9,11-14 in non-human animals. In addition, minor abnormalities and developmental delays have also been noted in FLX-exposed rats, such as transient impairment of the negative geotaxis reflex and delayed eruption of the incisor teeth¹², as well as an increase in the rate of ultrasonic vocalizations¹³.

Potential interference with developing neurotransmitters has been also found in some cases, but no dysmorphology and behavioural teratology have been reported so far in humans^15-17. Prenatal FLX exposure has been shown to cause emotional behavioural alterations in rodent models. Prenatal FLX exposure induced alterations in emotionality¹³, decrease in male impulsivity and increase in females depressive-like symptoms¹⁸ and increased anxious behaviours⁷, however, some contradictory reports are also on record ^18-20. In addition, prenatally FLX-exposed male rats showed increased memory in a shock-induced place aversion test⁷. Prenatal FLX exposure has been linked to decreased frontal lobe neurons^{21, 22}, downregulation of 5-HT1A receptors¹⁸ and altered 5HT1A signaling⁷.

The studies have indicated that SSRIs including FLX may be safe when taken during the first trimester of pregnancy although a large number of women need antidepressants sometime during their pregnancy^23-26 and there is a lack of information about the effects of antidepressants when clinically indicated during later periods of pregnancy²⁷. The long-term behavioural effects of prenatal FLX exposure are not well explored. As such, the present study was planned to assess the effects of prenatal FLX treatment during third trimester of gestation on anxiety parameters in rat offspring.

MATERIALS AND METHODS:

Female Charles Foster rats weighing 150 - 200 g were mated with adult Charles Foster male rats. The presence of sperm in the vaginal swab was taken as day one of conception. Flox (ProdepR; Sun Pharmaceuticals Industries, Gujrat, India) (5 and 10 mg/kg/day) with saline administered intraperitonially once daily to the pregnant rats at 0900 hrs from day 13 to day 20 of gestation as this is the critical period for brain development. Another group of pregnant rats were treated with the vehicle i.e., normal saline during the same period and route. Within 16 hrs of delivery, the litters were culled selectively to 8 male pups/dam and were foster nursed by well nourished lactating control mothers. The pups were weaned at 3 weeks of age and weighed weekly. Thereafter, at 8 weeks of age, one rat pup from each litter of a particular treatment was randomly selected to form groups of rats for each treatment to control the possible litter effects on behavioural measures²⁸. These rat offspring were subjected to a battery of behavioural tests of anxiety at 8 weeks of age. Animals were housed in colony cages at an ambient temperature of 25 ± 10C and 45-55% RH, with 12:12 hr L:D cycle. Animals were fed ad libitum with standard pellet chow (Brook Bond-Lipton, India) and given drinking water through drinking bottles. Experiments were conducted between 0900 and 1400 hrs. The experiments were performed following ‘principles of laboratory animal care’ (NIH publication No. 86-23, revised 1985).

Behavioural experiments:

1. Elevated plus-maze test:

The maze consisted of two opposite open arms, 50 x 10 cm, crossed with two enclosed arms of the same dimension but having 40 cm high walls. 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 room and elevated 50 cm above the floor. Naive rats were placed individually in centre of the maze, facing an enclosed arm. Thereafter, the 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 arm¹⁹.

2. Elevated zero-maze test:

The maze comprised of 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. The two opposite quadrants were enclosed by a black perspex wall (27 cm high) 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 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. The maze was cleaned with 5% ethanol/water solution and dried thoroughly between test sessions. 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 divide³⁰.

Statistical analysis:

The data are expressed as mean ± SD values for different-treatment groups. The data of elevated plus maze and elevated zero maze tests were analysed by one-way MANOVA. Post hoc mean comparisons were made for significant between groups effects by Tukey’s test³¹ using SPSS 7.5 version.

RESULTS:

A one-way MANOVA performed on the scores of elevated plus maze test revealed a significant multivariate main effect for ‘groups’, Wilks’ λ = 0.06, F4, 28 = 1168.18, p < 0.01, partial eta squared = 0.99, 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 ‘between groups’ were obtained for ‘time spent in enclosed arms’ (F2,33 = 11.64, p <.01), ‘time spent in open arms’ (F2,33 = 49.80, p < 0.01), ‘entries made in enclosed arms’ (F2,33 = 3.42, p < 0.05) and ‘entries made in open arms’ (F2,33 = 51.35, p < 0.01).

Post hoc mean comparisons for significant ‘groups’ effect revealed that FLX 5 and FLX10 treatments caused dose dependent increase in time spent and entries made in enclosed arms than control treatment, however, only FLX10 treatment significantly increased the time spent in enclosed arms in comparison to control treatment. Conversely, FLX5 and FLX10 treated rat offspring spent significantly less time and made less number of entries in open arms in comparison to control rats (Table-1), and these findings indicate presence of heightened anxiety in prenatally FLX treated rat offspring.

Table – 1: Effect of prenatal fluoxetine treatment on elevated plus maze behaviours in rat offspring

Groups	N	Time spent in		Entries made in
Groups	N	Open arms	Enclosed arms	Open arms	Enclosed arms
Control	12	30.63 ± 2.13	215.44 ± 5.15	9.92 ± 0.95	6.50 ± 0.72
FLX 5 mg	11	9.41 ± 2.00^aa	221.63 ± 8.88	1.65 ± 0.41^aa	7.27 ± 0.62
FLX 10 mg	11	6.18 ± 1.59^aa	257.69 ± 5.90^aa	1.55 ± 0.45^aa	8.91 ± 0.67^a

Superscripts ^a and ^aa indicate statistical significance in comparison to Control group respectively at p < 0.05 and p < 0.01

A one-way MANOVA performed on the scores of elevated plus zero test demonstrated a significant multivariate main effect for ‘groups’, Wilks’ λ = 0.01, F4, 28 = 564.25, p < 0.01, partial eta squared = 0.98, 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 ‘between groups’ were obtained for ‘time spent in open arms’ (F2,33 = 32.65, p <.01), ‘entries made in open arms’ (F2,33 = 1.06, p > 0.05), ‘number of stretched attend postures’ (F2,33 = 33.14, p < 0.01) and ‘number of head dips’ (F2,33 = 0.53, p > 0.05).

Post hoc mean comparisons for significant ‘groups’ effect indicated that prenatal FLX5 and FLX10 treatments caused dose dependent decrease in time spent in open arms on comparison to control treatment and both FLX5 and FLX10 treated rat offspring displayed significantly reduced amount of time spent in open arms and number of stretched attend postures in comparison to control rat offspring. However, prenatal FLX5 and FLX10 treatment had no significant effects on number of entries made in open arms and number of head dips behavioural measures of elevated plus maze test (Table -2).

Table – 2: Effect of prenatal fluoxetine treatment on elevated zero maze behaviours in rat offspring

Groups		Time Spent in Open arms	Entries in Open arms	Stretched Attend Postures	Head Dips
Control	12	114.66 ± 22.91	7.67 ± 3.26	4.75 ± 1.76	6.25 ± 1.60
FLX 5 mg	11	54.88 ± 27.12^aa	6.27 ± 3.13	1.18 ± 0.75^aa	5.82 ± 2.40
FLX 10 mg	11	46.79 ± 15.98^aa	5.09 ± 2.26	1.18 ± 0.87^aa	5.45 ± 1.63

Superscripts ^aa indicates statistical significance in comparison to

Control group at p < 0.01

DISCUSSION:

The obtained results demonstrated that prenatal FLX treatment during day 13 to 20 of gestation caused significant increase in anxiogenic behaviour patterns in rat offspring as indicated by reduced entries and time spent on open arms of elevated plus maze and significant decrease in stretched attend postures and time spent on open arms of elevated zero maze. Earlier also prenatal FLX exposure has to been documented to induce anxiety like behaviour⁷, impulsivity¹⁸ and altered emotionality¹³ in rodents and. The available evidence on open-field test and elevated plus maze indicates that FLX exposure either does not alter anxiety in adolescent or adult animals^{12,18,20,32-36} or causes an increase^6,7,37-39. Altered behavioural patterns as delay in eye opening, incisor eruption, negative geotaxis, cliff avoidance reflexes, increased emotionality in adults, reduction in brain DNA content, impairment in maze learning and increase in locomotion has been observed in prenatally and perinatally imipramine (a serotonin reuptake inhibitor) treated rat offspring⁴⁰.

Prenatal exposure to other pharmacological agents like phenobarbitone, diazepam, haloperidol, and chlordiazepoxide has also been reported to cause increased emotionality and impaired cognition in rodent offspring^{10, 41-43}.

FLX has been associated o long-term neurochemical and physiological changes in the brains of animals exposed in early developmental period and mostly the central serotonergic system have been investigated in these studies. The outcomes of FLX exposure differed based on when exposures occurred and when outcomes/ effects were assessed. The findings are contradictory and prenatal exposure reduced serotonin in the frontal cortex of adolescent rats⁴³ while postnatal exposure increased serotonin in adolescent and adult rats³⁶. Similarly, in adolescents postnatal FLX exposure increased serotonin in ventral and not in dorsal hippocampus³⁶ whereas prenatal FLX exposure had no effect on serotonin in hippocampus, hypothalamus, striatum or midbrain⁴³, however, by adulthood midbrain serotonin decreased and frontal cortex serotonin normalized⁴³. This decrease in midbrain serotonin could be accounted for changes in central serotonergic neurons themselves, as postnatal FLX exposure has been found to reduce the number and size of neurons in raphe nuclei⁴⁴ which could also be related to the observed reduction in serotonin transporter (SERT) in this region^6,33. Furthermore, in prenatally FLX exposed rats, SERT density decreased in the dorsomedial hypothalamus but increased in the lateral hypothalamus, hippocampus and amygdale⁴⁵. The research also indicate that there is little effect on serotonin receptors at this stage evidenced by no difference were observed in hypothalamic 5-HT_2A and 5-HT_2C density¹⁴ or prefrontal 5-HT_1A and 5-HT_2A mRNA expression³⁶. However, by adulthood changes in receptor density appear, at least in case of 5-HT2A and 5-HT2C receptors within the hypothalamus, which are down regulated¹⁴.

Since, reports are not available on the possible effects of prenatal exposure of FLX on behavioural development, brain neurochemistry and histology in the offspring; it is difficult to explain the behavioural alterations observed in the rat offspring induced by prenatal administration of FLX in the present study. However, prenatal exposures to other pharmacological agents like phenobarbitone, diazepam, haloperidol and chlordiazepoxide have also been reported to result in heightened emotionality and impaired cognition in rodent offspring^46-49. In humans, gestational use of these drugs has been reported to cause extrapyramidal dysfunction as tremors, hypertonus, poor suckling ability irritability, urinary retention, breathlessness, tachypnoea, cyanosis, increased emotionality, smaller brain and cortical size^50-53.

From both neurochemical and behavioural observations following, prenatal drug exposure the most sensitive period for inducing long term effects on behaviour of the offspring appeared to be the last week of gestation in rats. This period is most vulnerable to the actions of neuroactive drugs because this is the critical period for synaptogenesis formation of specific neural circuits^22,23 and functional maturation of central monoaminergic system in rats²⁴. Midbrain dopamine neurons undergo differentiation over the 11th to 15th day of gestation in rats²⁵, DA receptors appear in the embryonic rat brain^{9, 26} and have been demonstrated in the substantia nigra by GD 17 and in the caudate putamen by GD-14⁹ (22). The development of NE neurons has also been reported to take place during the 3rd trimester of prenatal development in the rodents¹². Serotonergic neurons begin their own differentiation relatively early (GD-13) both in terms of cessation of cell proliferation and neurotransmitter synthesis¹². In the developing hippocampus, 5-HT axons are first visible by GD 18¹². Prenatal exposure of PCPA (5-HT antagonist), 6-OHDA (catacholamine depletor) and haloperidol (DA antagonist) has been reported to impair development of 5-HT, NA and DA neurons in the rodent brain¹².

In the present study, prenatal FLX treatment given during the 3rd trimester of pregnancy, when 5-HT and catacholamine neurons undergo differentiation and cell proliferation. Therefore, it is likely that FLX treatment may alter the development of these neurons and thereby result in impaired behavioural development. These findings indicate that prenatal FLX treatment may lead to hyperemotionality in the progeny; however, further studies are needed to elucidate the role of specific neurotransmitter systems and functional elements of the developing brain underlying anxiogenic behaviour patterns in the offspring.

CONFLICT OF INTEREST:

Authors declare no conflict of interest.

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Received on 31.07.2017 Modified on 11.08.2017

Res. J. Pharmacology & Pharmacodynamics.2017; 9(3): 137-141.

DOI: 10.5958/2321-5836.2017.00023.4