ISSN 10623590, Biology Bulletin, 2014, Vol. 41, No. 4, pp. 372–377. © Pleiades Publishing, Inc., 2014. Original Russian Text © M.A. Volodina, S.A. Merchieva, E.A. Sebentsova, N.Yu. Glazova, D.M. Manchenko, L.A. Andreeva, N.G. Levitskaya, A.A. Kamensky, N.F. Myasoedov, 2014, published in Izvestiya Akademii Nauk, Seriya Biologicheskaya, 2014, No. 4, pp. 391–397.
HUMAN AND ANIMAL PHYSIOLOGY
Effects of Neonatal Fluvoxamine Administration to White Rats and Their Correction by Semax Treatment M. A. Volodinaa, S. A. Merchievaa, E. A. Sebentsovab, N. Yu. Glazovab, D. M. Manchenkoa, L. A. Andreevab, N. G. Levitskayab, A. A. Kamenskya, and N. F. Myasoedovb a Biological
b
Faculty, Moscow State University, Moscow, 119991 Russia Institute of Molecular Genetics, Russian Academy of Sciences, pl. akademika Kurchatova 2, Moscow, 123182 Russia email:
[email protected] Received August 9, 2013
Abstract—The aim of this work was to study the delayed effects of chronic neonatal administration of the selective serotonin reuptake inhibitor fluvoxamine (FA) to white rat pups and to estimate the possibility to correct these effects by treatment with semax. Fluvoxamine was injected intraperitoneally at a dose of 10 mg/kg from postnatal days 1 to 14, and semax was injected intranasally at a dose of 0.05 mg/kg from post natal days 15 to 28. It was shown that neonatal FA administration produced a significant delay in animal somatic growth. A loss in body weight was detected both during FA administration and 4–6 weeks after the last injection. Furthermore, FA administration increased the anxiety level and disturbed the learning ability of animals. The negative consequences of neonatal FA administration were largely compensated by Semax. DOI: 10.1134/S106235901403011X
INTRODUCTION The early neonatal period of life plays a key role in the development and formation of the nervous system and the physiological functions of the body. Stressors or pharmacological effects at this developmental stage can entail further significant structural and functional changes. It was shown that the pain and stress experi enced by newborns subsequently cause disturbances in the nervous system and significant changes in behavior (Anand, 2000). Selective serotonin reuptake inhibitors (SSRIs) are a group of drugs (fluoxetine, citalopram, fluvoxamine, paroxetine, sertraline, and others) that are widely used for the treatment of depressive and anxiety disorders. The target of SSRIs is the serotonin transporter (SERT), which is responsible for the serotonin reuptake from the synaptic cleft. Blocking of SERT leads to an increased serotoninergic neurotransmis sion. All SSRIs act by a similar mechanism, despite the differences in their chemical structure (DiavCit rin and Ornoy, 2012). Drugs of the SSRI group are recommended for the treatment of depressive disorders in pregnant and lac tating women. In addition, the use of antidepressants in child psychiatry is steadily increasing. The result is an increasing number of children are being treated with SSRIs during either fetal development or while breastfeeding or during the treatment of childhood depressions (Maciag et al., 2006a, 2006b). It is shown that drugs of this group easily penetrate across the pla cental barrier and are detected in the amniotic fluid (Casper et al., 2011). For different antidepressants in
this group, the SSRI content in the umbilical cord blood accounts for 70–86% of its content in the mother’s blood; i.e., the fetus is exposed to physiolog ically active doses of drugs (Rampono et al., 2009). Despite the increasing number of women who are taking SSRIs during pregnancy, there is an insufficient data of the impact of prenatal exposure to these drugs. Several studies showed that SSRI administration affected the outcome of pregnancy—the number of miscarriages and premature births increased and the birth weight reduced (Oliver et al., 2011). As much as 30% of infants who prenatally received SSRIs had signs of neonatal abstinence syndrome (Marsella et al., 2010). In addition, SSRI exposure during preg nancy (especially in the last trimester) reduces the neonatal Apgar scores and causes psychomotor retar dation, sleep disorders, pulmonary hypertension, and cardiovascular disorders (Casper et al., 2003, 2011; Oliver et al., 2011; DiavCitrin and Ornoy, 2012). The most frequent observations of children who prenatally received SSRI were performed mainly in early life (up to 6 years). Information about the delayed effects of prenatal exposure to SSRIs is limited due to the long duration and complexity of such studies in clinical practice (Oliver et al., 2011; Harris et al., 2012). The longterm effects of drugs of the SSRI group on the developing brain have actively been investigated in experiments with animals, primarily rodents. The critical period of development of the central nervous system (CNS) of humans, when it is most sensitive to SSRIs, is the third trimester of pregnancy. Although it is difficult to compare correctly the development of
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human and rodent brains, data on the CNS matura tion allows the last trimester of pregnancy in humans to be compared with the first week of life in rats (Kar pova et al., 2009; Thompson et al., 2009). Therefore, neonatal SSRI exposure in the first weeks of life in rats can be regarded as a model for studying the effects of prenatal exposure to drugs of this group during the third trimester of pregnancy in women (Lee, L.J. and Lee, L.J.H., 2012). Experiments have showed that chronic administra tion of SSRIs to rats and mice in the neonatal period causes longterm changes in the animal behavior. Early chronic SSRI administration subsequently increased the duration of immobilization in the “forced swimming” test (Hansen et al., 1997), reduced exploratory behavior, and increased the anxiety of mice in adulthood (Karpova et al., 2009; Lee, L.J. and Lee, L.J.H., 2012). Similar behavioral changes are characteristic of a strain of mice with genetically determined deficiency of SERT expression. In addi tion, neonatal SSRI administration was shown to affect the feeding behavior of animals and behavior under conditions of high stress load (Ansorge et al., 2004), as well as causing anhedonia (Popa et al., 2008). In adult animals that received SSRI injections in the early period of development, a decreased expression of tryptophan hydroxylase (the enzyme responsible for the synthesis of serotonin) in the dorsal raphe nucleus and SERT in the medial prefrontal cortex and the pri mary somatosensory cortex was detected (Maciag et al., 2006b). Therefore, neonatal SSRI administration to animals leads to further changes in the activity of the serotonergic system in the brain, which may be one of the mechanisms of the development of delayed behavioral effects of these drugs. Fluvoxamine (FA) is a modern antidepressant of the SSRI group, which in its pharmacological proper ties is similar to fluoxetine but exhibits a high effi ciency and selectivity (Hrdina, 1991). Fluvoxamine enhances the serotonergic transmission and reduces the serotonin turnover. The effects of neonatal FA administration have not been studied earlier. Heptapeptide semax (MEHFPGP) is an analogue of the adrenocorticotropic hormone fragment ACTH(410), which has a prolonged neurotropic action (Ashmarin et al., 1995; Dolotov et al., 2006). Currently, semax is used in medicine as a nootropic and neuroprotective drug (Ashmarin et al, 1997). Experiments have shown that chronic semax adminis tration to neonatal rat pups later reduces their anxiety and improves the animal learning ability (Sebentsova et al., 2005). Semax increases the concentration of serotonin and its metabolite in the brain, which testi fies to an increase in the functional activity of the sero tonergic system in the brain (Eremin et al., 2005). The aim of this study was to investigate the delayed effects of chronic neonatal FA administration to white rat pups and assess the possibility of correcting these effects with semax. BIOLOGY BULLETIN
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MATERIALS AND METHODS This study was carried out in outbred white rat pups of both sexes. In total, we used ten litters of animals (34–38 rats in each group). Each litter was divided into three groups. Rats of the control group received intraperitoneal injections of water (2 mL/kg body weight) on postnatal days 1–14 and intranasal injec tions of water (0.1 mL/kg body weight) on postnatal days 15–28. Rats of the FA group received intraperito neal injections of FA (fluvoxamine maleate, Sigma, United States) at a dose of 10 mg/kg body weight on postnatal days 1–14 and intranasal injections of water on postnatal days 15–28. Rats of the FA–semax group received intraperitoneal injections of FA at a dose of 10 mg/kg on postnatal days 1–14 and intranasal injec tions of semax at a dose of 0.05 mg/kg body weight on postnatal days 15–28 (Fig. 1). To assess the physical development of rat pups, the eyeopening age, and the body weight of animals were recorded once a day during the first month of life and once a week during the second month. To assess the level of anxiety, the rats were tested in an “elevated plus maze” on postnatal day 31. To assess their learning ability, the acquisition of the foodreinforcing task in a complex maze was studied in them on postnatal days 42–46. “Elevated plus maze” test. The experimental cham ber of the maze consisted of four arms diverging from the center (sleeve length, 50 cm; width, 15 cm; and wall height, 30 cm). Two opposite arms were darkened and closed with walls at their ends, and the other two arms remained illuminated and open. The maze was elevated to a height of 55 cm above the floor. A rat was placed in the center of the maze, and the total time spent on the open arms and the number of exits from the closed arms of the maze were recorded for 3 min. “Acquisition of the foodreinforcing task in a complex maze”. The complex maze is a square chamber divided with five transparent partitions into six corridors. Each partition had a rectangular hole. Before the experi ment, the animals underwent a 24h food deprivation. On the first day, the rats were placed in the maze for adaptation for 30 min. In the next four days, the ani mals were placed in the maze five consecutive times daily; the duration of each time did not exceed 3 min. The latent period of exit from the starting compart ment, the number of correct trials (the number of cases when the animals found food reinforcement within 3 min), and the number of grooming episodes were recorded daily. The animals received food once a day after the experiment. The results were processed statistically using the Statistica software package. The intragroup mean val ues and the deviations were determined. The differ ences between the groups were assessed using the ANOVA and MANOVA methods and the Mann– Whitney Utest. Data in the figures are presented as
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the mean value ± standard error of the mean. Differ ences were considered significant at p < 0.05.
The measurement of the body weight of the new born rats showed no initial differences between the animals of the three experimental groups (F2, 131 = 0.43, p > 0.60). The changes in the body weight of rats were recorded from postnatal days 1 to 63. It was shown that, during the first two weeks of life (against the background of FA administration), the body weight of rats in the “FA” and “FA–semax” groups increased slowly compared to the control (F13, 1235 = 4.81, p < 0.001 and F13, 1092 = 6.72, p < 0.001, respec tively). No significant differences between the groups “FA” and “FA–semax” have been found (F13, 1079 = 0.63, p > 0.80) (Fig. 2a). On postnatal days 15–28, sig nificant differences in the body weight between the three groups of animals were not observed (F26, 1391 = 0.10, p > 0.95) (Fig. 2b). The measurement of the body weight of rats during the second month of life (Fig. 2c) showed that, on postnatal days 28–63, the weight of animals of the “FA” group was significantly lower than in the control (F5, 185 = 3.05, p < 0.02). In the “FA– semax” group, the body weight of rats did not differ significantly from the control (F5, 190 = 0.55, p > 0.75); however, on postnatal days 35–56, their weight was greater compared to the animals of the “FA” group (F3, 105 = 3.07, p < 0.04).
RESULTS AND DISCUSSION The assessment of the effect of neonatal FA admin istration on the eyeopening age showed a significant effect of the factor “group” on this index (F2, 88 = 4.50, p < 0.02). Further analysis showed that, in both the “FA” and the “FA–semax” groups, the eyeopening age slightly, though significantly, decreased relative to control values (Fig. 1). On postnatal day 16, eyes were opened in 83.3% of animals in the groups of rats administered with FA, whereas in the control group this index was 65.1% (p < 0.03). Significant differences between the “FA” and “FA–semax” groups with respect to this index were not found.
The anxiety level of animals was assessed in the “elevated plus maze” on postnatal day 31. In the rats of the “FA” group, a significant decrease in the time spent in the open arms of the maze and in the number of exits from the closed arms of the maze relative to control values was detected (Fig. 3). Such changes indicate an increase in the anxiety level in these rats. In the “FA–semax” group, the tested parameters did not differ significantly from the control but were signifi cantly higher than in the “FA” group. Thus, the administration of FA in postnatal days 1–14 led to increased anxiety levels in the rats aged 31 days, and subsequent administration of semax on postnatal days 15–28 abolished the effect of FA.
1 2 3
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15 Fig. 1. Effect of fluvoxamine on the eyeopening age. Des ignations for Figs. 1–4: (1), control; (2), “FA” group; (3), “FA–semax” group. * Significant differences from the control (p < 0.05).
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Fig. 3. Behavioral parameters of rats in the “elevated plus maze” test: (a) time spent in the open arms of the maze and (b) the number of exits from the closed arms. Here and in Fig. 4, signs * and # indicate significant (p < 0.05) differences from the control and “FA” group, respectively.
The foodreinforcing task in the complex maze was acquired on postnatal days 42–47. In the “FA” group, the recorded parameters differed significantly from the control (F3, 168 > 4.25, p < 0.01). Subsequent analysis showed that the number of correct trials in the animals of this group on the first and second days of training was smaller and the latency of exit from the starting compartment and the number of grooming events were higher than in the control (Fig. 4). These changes indicate deterioration of learning and an increased anxiety level of the animals. In the animals of the “FA–semax” group, the learning parameters did not differ significantly from those of the control animals (F3, 177 < 1.2, p > 0.30). The number of grooming events in the “FA–semax” group was significantly smaller than in the “FA” group. In addition, in the first day of training the number of correct trials tended to increase and the latency tended to decrease in the ani mals of the “FA–semax” group compared to the “FA” group (p < 0.10). Thus, FA administration impaired the learning ability and increased the level of anxiety of animals in the complex maze, and the administration of semax attenuated but not completely eliminated the effects of FA. Summarizing all above, we can conclude that the administration of FA on postnatal days 1–14 led to slowed somatic growth of rats. This effect had a bipha sic pattern. During the first two weeks of life, against the background of FA administration, the rat pups that received the drug lagged behind the control animals in body weight. After termination of drug administration, no significant difference in the body weight between the groups was observed. Therefore, this slowdown was due to the acute effects of FA. However, after postnatal day 35, this lag in the body weight relative to the con trol increased and on postnatal day 56 exceeded 25 g BIOLOGY BULLETIN
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(>10% body weight). Note that, at an age of 31 days, the pups were weaned from their mothers and switched to independent feeding. According to pub lished data, neonatal SSRI administration affects the feeding behavior of animals (Ansorge et al., 2004) and causes anhedonia (Popa et al., 2008). It can be assumed that the growth deceleration observed in the rat pups administered with FA was also associated with a change in the feeding behavior in rats. Chronic intranasal administration of semax in postnatal days 15–28 attenu ated the effect of FA on the animal body weight. This effect of semax may be associated with its adaptogenic effect, which leads to stimulation of the feeding behavior of animals. The rat pups treated with FA opened eyes at an ear lier age than the control animals. It is known that the SSRI administration increases the time during which serotonin remains in the synaptic cleft, thus increasing its effect on the receptors (Maciag et al., 2006a). Dur ing the period of active development of the nervous system, monoamines function as trophic factors. Serotonin is a signaling factor in the processes of cell proliferation and differentiation in the CNS (Verney et al., 2002). In the critical period of brain develop ment (in rats from prenatal day 18 to postnatal day 22), the content of serotonin and tryptophan hydroxylase in the raphe nucleus increases 35 times (Rind et al., 2000). It can be assumed that the increase in the sero toninergic system activity in this period leads to accel erated development. Probably, this explains the earlier eye opening in the group of rats treated with FA. The eyeopening age in the rat pups almost coincided with the beginning of semax administration, which deter mined the absence of the influence of this peptide on the effect of FA.
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Based on data obtained, we can conclude that chronic intraperitoneal administration of the selective serotonin reuptake inhibitor FA to white rat pups on postnatal days 1–14 decelerates the somatic animal growth, increases the level of anxiety, and reduces the ability of animals to learn. Chronic intranasal admin istration of semax on postnatal days 15–28 largely compensates the negative effects of FA administration.
5 Number of correct trails
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This study was supported by the program of the Presidium of the Russian Academy of Sciences “Molecular and Cell Biology,” the program “Leading Scientific Schools” (project no. NSh2628.2012.4), and the Russian Foundation for Basic Research (project no. 140401913).
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ACKNOWLEDGMENTS 1 2 3
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Fig. 4. Behavioral parameters of rats in the test “acquisi tion of the foodreinforcing task in a complex maze.”
In the rats that received neonatal FA injections, subsequent increase in the anxiety level and an impaired ability to learn with positive reinforcement were observed. The administration of semax almost completely normalized the emotional state of the ani mals, disturbed by FA injections, and had a positive effect on the learning ability of the rats. According to the published data, the animals that were treated with SSRIs during early development later showed a decrease in the serotonin system activ ity—reduced tryptophan hydroxylase expression in the dorsal raphe nucleus and SERT in the medial pre frontal cortex and the primary somatosensory cortex (Maciag et al., 2006b). Apparently, the neonatal FA administration has a similar effect. Previously it was shown that semax increases the content of serotonin and its metabolite in the brain; i.e., it increases the functional activity of the serotonergic system in the brain (Eremin et al., 2005). It can be assumed that the compensatory effect of semax on the negative conse quences of chronic neonatal FA administration is determined by its activating effect on the brain sero tonergic system.
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Translated by M. Batrukova
