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Neonatal tactile stimulation decreases depression-like and anxiety-like behaviors and potentiates sertraline action in young rats
Int. J. Devl Neuroscience 47 (2015) 192–197
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International Journal of Developmental Neuroscience journal homepage: www.elsevier.com/locate/ijdevneu
Neonatal tactile stimulation decreases depression-like and anxiety-like behaviors and potentiates sertraline action in young rats Daniele Freitas b , Caren T.D. Antoniazzi a , Hecson J. Segat b , Vinícia Garzella Metz c , Luciana Taschetto Vey b , Raquel C.S Barcelos a , Thiago Duarte a , Marta M.M.F. Duarte a,d , Marilise Escobar Burger a,b,c,∗ a
Programa de Pós-Graduac¸ão em Farmacologia, Universidade Federal de Santa Maria, UFSM, RS, Brazil Programa de Pós-Graduac¸ão em Bioquímica Toxicológica, UFSM, RS, Brazil c Departamento de Fisiologia e Farmacologia, UFSM, RS, Brazil d Universidade Luterana do Brasil (ULBRA), Santa Maria, Brazil b
a r t i c l e
i n f o
Article history: Received 14 August 2015 Received in revised form 29 September 2015 Accepted 29 September 2015 Keywords: Neonatal handling Tactile stimulation Forced swimming test Sertraline Anxiety-like behavior Stress
a b s t r a c t It is well known that events which occur in early life exert a significant influence on brain development, what can be reflected throughout adulthood. This study was carried out in order to assess the influence of neonatal tactile stimulation (TS) on behavioral and morphological responses related to depressionlike and anxiety-like behaviors, assessed following the administration of sertraline (SERT), a selective serotonin re-uptake inhibitor (SSRI). Male pups were submitted to daily TS, from postnatal day 8 (PND8) to postnatal day 14 (PND14), for 10 min every day. On PND50, adult animals were submitted to forced swimming training (15 min). On PND51, half of each experimental group (UH and TS) received a single sub-therapeutic dose of sertraline (SER, 0.3 mg/kg body weight, i.p.) or its vehicle (C, control group). Thirty minutes after injection, depression-like behaviors were quantified in forced swimming test (FST, for 5 min). On the following day, anxiety-like behaviors were assessed in elevated plus maze (EPM), followed by biochemical assessments. TS per se increased swimming time, decreasing immobility time in FST. Besides, TS per se was able to increase frequency of head dipping and time spent in the open arms of EPM, resulting in decreased anxiety index. In addition, groups exposed to TS showed decreased plasma levels of corticosterone per se. Interestingly, while TS exposure significantly potentiated the antidepressant activity of a subtherapeutic dose of SERT, this drug was able to exacerbate TS-induced anxiolytic activity, as observed in FST and EPM, respectively. Decreased plasma levels of both corticosterone and cortisol in animals exposed to TS and treated with SERT are able to confirm the interesting interaction between this neonatal handling and the antidepressant drug. From our results, we conclude that neonatal TS is able to exert beneficial influence on the ability to cope with stressful situations in adulthood, preventing depression and favorably modulating the action of antidepressant drugs. © 2015 Published by Elsevier Ltd. on behalf of ISDN.
1. Introduction Depression is a serious and expensive psychiatric disease (Mula, 2013) characterized by feelings of sadness, guilt, worthlessness and hopelessness (Jia et al., 2010; Petersen et al., 2001), affecting about 10–30% of women and 7–15% of men worldwide (Kessler et al., 2005). According to the World Health Organization (WHO),
∗ Corresponding author at: Universidade Federal de Santa Maria, Centro de Ciências da Saúde, Programa de Pós-Graduac¸ão em Farmacologia, 97105-900, Santa Maria, RS, Brazil. E-mail address: [email protected] (M.E. Burger). http://dx.doi.org/10.1016/j.ijdevneu.2015.09.010 0736-5748/© 2015 Published by Elsevier Ltd. on behalf of ISDN.
depression is the fourth leading cause of morbidity in the world with perspectives to become the second by 2020 (Kessler et al., 2011). Although the etiology of depression is considered multifactorial and is not well elucidated yet, its connection with deficiency of brain monoamines activity has been the mainstay for its treatment (Elhwuegi, 2004). Unfortunately, about 50% of patients who start treatment for depression do not respond to antidepressant medication (Thase, 2004), leading us to think about the existence of interferences so far unknown. Tactile stimulation (TS) is a form of neonatal handling similar to the maternal behavior of licking and affection (Schanberg and Field, 1987), which exerts solid influence on normal brain development (Chiba et al., 2012). Some studies show that TS is able
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to affect the hypothalamic–pituitary–adrenal (HPA) axis function (Katsouliet al., 2014), improving the ability of the adult organism to adapt to stressful stimuli (Stamatakis et al., 2009) and helping in the recovery from neonatal brain injuries (Richardset al., 2012; Rodrigues et al., 2004). In addition, studies by our group have shown beneficial influence of TS to prevent preference for psychostimulant drugs (Antoniazzi et al., 2014 a,b), being also able to change responsiveness to benzodiazepine drugs (Boufleur et al., 2012). Sertraline (SERT) is a usual antidepressant drug that belongs to the selective serotonin reuptake inhibitors (SSRIs) (Khan et al., 2005; Young et al., 2009), whose primary mechanism of action is inhibition of the serotonin (5-HT) transporter located in the presynaptic membrane of mesolimbic brain areas (Belmaker and Galila Agam, 2008). Indeed, 5-HT is a neurotransmitter responsible for mood regulation (Peirson and Heuchert, 2000) and for numerous functions in the central and peripheral nervous system (McGeer et al., 1988). In this sense, 5-HT is important in pathogenesis and treatment of different neuropsychiatric disorders including depression (Lucki et al., 1988). In line with this, forced swimming test (FST) is an animal model used to assess depression-like behavior (Cryan and Lucki, 2000), which was firstly described by Porsolt et al. (1978). FST involves two sequential sessions of exposure to deep water, from where animals cannot escape. The first session is in fact a stressor, assumed to induce a state of behavioral despair (Porsolt et al., 1978; Borsini and Meli, 1988; Castagne et al., 2009) interpreted as depression-like symptoms. Considering the promising benefits of TS in early life, the current study was developed to assess the influence of this neonatal handling on the antidepressant activity of SERT in rats. Behaviors involving depression and anxiety-like symptoms were assessed, as well as the activity of HPA axis.
2. Material and methods 2.1. Animals and experimental procedure Seven pregnant female Wistar rats from the breeding facility at Universidade Federal de Santa Maria (UFSM), RS, Brazil, were housed in Plexiglas cages with free access to food and water. Animals were kept in a room with controlled temperature (22–23 ◦ C) on a 12-h light/dark cycle with lights on at 7:00 a.m. The experimental protocol was approved by the Animal Ethics Committee (Universidade Federal de Santa Maria), affiliated to the National Council for the Control of Animal Experimentations (CONCEA), following international norms of care and animal maintenance. All efforts were made to minimize animal suffering and to reduce the number of animals used in experiments. The pups date of birth (postnatal day 0-PND0) was monitored, and gender distinguished by larger genital papilla and longer anogenital distance in male vs. female pups (Liu et al., 2008). On the same day, litters were culled to seven pups (four males and three females to ensure adequate nutritional status (Boufleur et al., 2012; Vazquez et al., 2006)), being only male rats designated to the current experimental protocol. In order to avoid using littermates in the same experimental group, on postnatal day one (PND1) male pups were taken from several litters and randomly assigned to experimental groups (n = 14, each group), separated into unhandled (UH) and tactile stimulation (TS). TS groups received stimulus from PND8 to 14, according to the protocol described below. On PND22, litters were weaned and left undisturbed up to 50 days of age, when they were submitted to training of FST, being immersed in water for 15 min. This procedure works as a stressor which is thought to induce a state of behavioral despair (Porsolt et al., 1978; Castagne et al., 2009) related to a “depressive state” (Borsini and Meli, 1988). On PND51, half (n = 7)
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of each experimental group (UH and TS) received a single dose of sertraline (SERT, 0.3 mg/kg body weight i.p.), which corresponds to a sub-therapeutic dose (following pilot studies at our laboratory), or its vehicle (C-control group). SERT (Pharmanostra, India,) was dissolved in NaCl 0.9% plus 0.05 mL Tween 80 (Sigma–Aldrich, Brazil);vehicle (C) consisted of NaCl 0.9% plus 0.05 mL Tween 80. SERT was the antidepressant drug chosen because it has been frequently reported in studies involving animal models (Ulloa et al., 2010; Mikail et al., 2012; Kaygisiz et al., 2014). Thirty minutes following the injections, animals were submitted to forced swimming test (FST). One hour after FST, anxiety-like symptoms were observed in elevated plus maze (EPM). Following the EPM paradigm, all animals were weighed, anesthetized with sodium pentobarbital (80 mg/kg, i.p.), and euthanized by exsanguination. Blood was collected by cardiac puncture (Fig. 1).
2.2. Neonatal handling Tactile stimulation (TS) consisted of gently stimulating pups’ dorsal surface individually with the index finger from rostral to caudal direction for 10 min (each day) out of the nest by the same experimenter (Antoniazzi et al., 2014a,b; Boufleur et al., 2012; 2013; Rodrigues et al., 2004). TS was applied daily between 12:00 and 14:00 from PND8 until PND14, according to previous studies by our group (data submitted), since most beneficial influence of TS has been observed in pups. After the procedures, pups were returned to their litters, staying the remaining time with their mothers until weaning.
2.3. Behavioral assessments 2.3.1. Forced swimming test (FST) Behavioral responses related to depression-like symptoms are experimentally assessed in FST, whose method has been described in several studies (Porsoltet al., 1978; Wieland and Lucki1990; Castagne et al., 2009). On the first day (PND50) rats were forced to swim for a 15-min period (pretest session), and carefully dried before returning to their home cages. Twenty-four hours following the test (PND51), all rats were injected with SER or vehicle, as described above, and again submitted to FST for 5 min. Immobility, climbing and swimming times were quantified by trained raters blinded to handling and treatment. In this sense, immobility is no additional activity other than the required to keep the head above water, whereas climbing is defined as upward struggling movements of the forepaws at the side of the cylinder. Movements around the swimming cylinder (Porsolt et al., 1978) are indicative of swimming time.
2.3.2. Elevated plus maze (EPM) One hour after FST, anxiety-like symptoms were assessed in the EPM paradigm, which is based on the innate fear rodents have for open and elevated spaces (Montgomery, 1955). The apparatus was made of wood and consisted of a plus-shaped platform elevated 50 cm from the floor. Two opposite arms (50 cm × 10 cm) were enclosed by 40 cm high walls whereas the other two arms had no walls. The four arms had a central platform (10 cm × 10 cm) at their intersection, which gave access to any of the four arms. At the beginning of each test, each animal was individually placed in the central platform facing an open arm. Time spent and number of entries in open and closed arms were monitored during a fiveminute test. A shorter time spent (expressed as a percentage of total length of the test) and fewer entries in the open arms of the maze indicated anxiety (Hlavacova et al., 2010). Total activity in the maze (total number of arm entries) and number of open and closed arms
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Fig. 1. Experimental design: Neonatal handlings were conducted from PND8 until PND14 (n = 14). On PND51 animals received one dose of sertraline (SERT; i.p., n = 7) and were submitted to behavioral assessments. Abbreviations: PND, post natal day; FST, forced swimming test; EPM, elevated plus-maze.
entries was used as anxiety index (Cohen et al., 2007; Mazor et al., 2007). Anxiety index was calculated as follows:
Anxiety index = 1 − n
Time spent in the open arms Total time on the maze
Number of open arm entries +
Total entries on the maze
2
Anxiety index values range from 0 to 1 where an increase in the index expresses increased anxiety-like behavior (Cohen et al., 2007; Mazor et al., 2007). The apparatus was cleaned with a 20% alcohol solution using wet sponge and paper towel before the introduction of each animal. Observers were blinded to treatment during all behavioral observations. 2.4. Biochemical assessments 2.4.1. Corticosterone and cortisol assessments Blood aliquots were centrifuged at 4 ◦ C at 1500×g for 15 min (n = 5). Plasma aliquots were frozen at −80 ◦ C until their analysis. Corticosterone and cortisol plasma levels were analyzed through enzyme immunoassay (ELISA) using a commercial kit according to the manufacturer’s instructions (Immuno Biological Laboratories). 2.5. Statistical analysis Levene’s test was used in order to verify data distribution. Two-way ANOVA followed by Duncan’s post-hoc test analyzed parametric data, using Statistica 8.0 software. Values of P < 0.05 were considered statistically significant for all comparisons made. 3. Results 3.1. Forced swimming test (FST) Two-way ANOVA of FST revealed a significant main effect of handling on climbing, swimming and immobility time[F(1,24) = 10.04, 11.26 and 37.87, p < 0.05 respectively], as well as a significant main effect of drug on swimming and immobility time [F(1,24)=10.54 and 6.27, p<0.05, respectively]. TS per se increased swimming time (Fig. 1B), decreasing immobility time (Fig. 1C) in FST of both control and SERT treated groups in comparison to UH group. Interestingly, SERT administration was able to increase swimming time in UH group when compared to its respective control (Fig. 1B). In fact, among SERT treated groups, animals exposed to TS showed higher climbing time and lower immobility time in FST (Fig. 2A–C). 3.2. Elevated plus maze (EPM) Two-way ANOVA of EPM revealed a main effect of handling[F(1,24) = 92.22; 5.55; 33.66 and 24.08, p < 0.05] and a main effect of drug [F(1,24) = 57.48; 6.78; 16.77 and 18.34, p < 0.05]on time spent, number of entries and frequency of head dipping in
the open arms as well as on anxiety index, respectively. Two-way ANOVA also revealed a significant handling × drug interaction on time spent and frequency of head dipping in the open arms as well as on anxiety index [F(1,24) = 21.99; 4.19 and 0.96, p < 0.05], respectively. TS exposure per se was able to increase time spent (Fig. 2A) and frequency of head dipping (Fig. 2C) in the open arms of EPM, decreasing the anxiety index (Fig. 2D) in relation to UH group. On the other hand, SERT treatment was able to increase time spent (Fig. 2A), number of entries (Fig. 2B) and frequency of head dipping (Fig. 2C) in the open arms, decreasing the anxiety index (Fig. 2D) in TS in comparison to its correspondent control group. Among SERT-treated groups, TS exposure exacerbated all these behavioral parameters observed in EPM, when compared to UH group (Fig 3A–D). No influence of UH or TS groups on number of total entries in both open and closed arms of EPM was observed (Data no shown)
3.3. Corticosterone and cortisol Two-way ANOVA revealed a main effect of handling [F(1,24) = 66.06 and 12.59; p < 0.05], drug [F(1,24) = 3.83; 157.89 and 73.91; p < 0.05] on plasma levels of corticosterone and cortisol, respectively, and a significant handling × drug interaction [F(1,24) = 11.38; p < 0.05] on plasma levels of corticosterone, respectively. While TS exposure per se was able to decrease plasma levels of corticosterone, cortisol was similar to UH group. On the other hand, SERT administration decreased plasma levels of both corticosterone and cortisol in relation to their respective controls, regardless of handling. In fact, the effects of SERT on rats exposed to TS were exacerbated, since this experimental group showed lower corticosterone and cortisol than UH group, also treated with SERT (Table 1).
Table 1 Influence of a subtherapeutic dose of sertraline (SERT) on corticosterone (ng/mL) and cortisol (ng/mL) plasma levels in rats previously submitted to tactile stimulation (TS) in comparison to unhandled (UH) animals. Group
Corticosterone (ng/mL)
Cortisol (ng/mL)
UH-C TS-C UH-SERT TS-SERT
83,40 ± 2,29 73,25 ± 1,18* 64,20 ± 2,72+ 41,20 ± 1,07*,+
190,00 ± 3,27 182,50 ± 3,42 162,80 ± 4,66+ 142,60 ± 3,77*,+
Abbreviations: UH, unhandled; TS, tactile stimulation; SERT, Sertraline; C, Control. Data are expressed as mean ± SEM. (n = 5). * Significant difference from UH group in the same treatment. + Significant difference from Control in the same handling (p < 0.05).
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Fig. 2. Antidepressant activity of a subtherapeutic dose of sertraline (SERT) in rats previously submitted to tactile stimulation (TS) in comparison to unhandled (UH) animals in forced swimming test (FST). Depression-like behavior were assessed using climbing time (A), swimming time (B) and immobility time (C). Data are expressed as mean ± S.E.M. *Significant difference from UH group in the same treatment (p < 0.05). **Significant difference from vehicle in the same handling (p < 0.05).
4. Discussion The current study was developed to assess the influence of neonatal TS per se on behavioral, biochemical and morphological parameters related to anxiety and depression-like behavior, as well as the possible influence of this neonatal handling on the effects of SERT, which is a frequently used therapeutic tool to treat neuropsychiatric conditions such as dysthymia and major depression. TS may prevent emotional disturbances in response to early adversities (Imanaka et al., 2008), being also able to change the hypothalamic–pituitary–adrenal (HPA) axis function, modifying
the ability of the organism to respond, cope and adapt to stressful stimuli (Ladd et al., 2000; Levine, 1957). Our research group has recently shown that animals exposed to neonatal TS were less anxious and more resistant to stressful events (Boufleur et al., 2013), indicating that TS may facilitate the activity of benzodiazepine drugs, particularly diazepam (Boufleur et al., 2012). Interestingly, the current study shows that neonatal TS was able to decrease depression-like behavior, as observed by the increased swimming time and the decreased immobility time in FST. This animal model has been widely used to assess potential activity of antidepressant drugs (Cryan et al., 2005; Shaw et al., 2007; Mikail
Fig. 3. Influence of a subtherapeutic dose of sertraline (SERT) on anxiety-like behavior in rats previously submitted to tactile stimulation (TS) in comparison to unhandled (UH) animals in elevated plus maze (EPM). Anxiety-like behavior were assessed by time spent (sec) in the open arms (A), number of entries in the open arms (B), frequency of head dipping (C) and anxiety index (D). Data are expressed as mean ± S.E.M. *Significant difference from UH group in the same treatment (p<0.05). **Significant difference from vehicle in the same handling (p < 0.05).
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et al., 2012). In this sense, decreased swimming time together with increased immobility time in FST are indicators of depressive behavior, which may be reversed by antidepressant drugs (Cryan et al., 2005; Porsolt et al., 1977). Taken together, our results suggest that TS in early life is able to exert beneficial influence per se on depressive parameters, thus facilitating the activity of SERT in adulthood. Additionally, our findings also demonstrated that animals exposed to neonatal TS showed reduced anxiety-like behavior per se, as observed by increased time spent and frequency of head dipping in the open arms of EPM, thus resulting in a lower anxiety index in the EPM task. In opposition, when decreased, these behaviors are indicators of fear and anxiety, resulting in an increased anxiety index, as previously described (Hlavacona et al., 2010). Indeed, the beneficial influences of TS on anxiety parameters, as observed in the current study, are not innovative findings since previous studies by our group evidenced similar behaviors in rats exposed to neonatal TS (Antoniazzi et al., 2014a,b; Boufleur et al., 2013), consistently reinforcing the anxiolytic properties of this neonatal handling. Neonatal TS has an antidepressant-like activity per se, also facilitating the activity of SERT, and this drug apparently contributes with lower TS-induced anxiety and emotionality, since (i) time spent, number of entries and frequency of head dipping in the open arms of EPM were significantly higher in TS group treated with SERT, indicating a favorable interactive modulation between drug and neonatal handling; (ii) decreased hormone secretion from HPA axis following SERT administration in TS-exposed group was also indicative of lower emotionality and stress; (iii) TS per se decreased plasma corticosterone levels, confirming beneficial influence of this neonatal handling on neuroendocrine function. While manipulations during early life may affect adrenocortical function in adult rats (Daskalakis et al., 2009), increased adrenal weight and elevated levels of circulating glucocorticoids may be considered markers of stress (Heiderstadt et al., 2000), as previously described. Of particular importance, these changes may intensify central and peripheral pathological symptoms such as anxiety and depression-like behaviors (Korte, 2001; Mitchell and O’Keane, 1998; McEwen, 2000; Erickson et al., 2003). Furthermore, an experimental study has shown that rats exposed to maternal separation presented increased basal corticosterone and decreased corticotrophin (ACTH) levels following acute stress (Marais et al., 2008). Our study has included both corticosterone and cortisol, which are regulated in the same way and simultaneously released, indicating more reliability and consistence (Saito et al., 1992). In fact, studies involving FST have shown a relationship between increased secretion of glucocorticoids and depressive behavior in rodents, reversed by antidepressant drugs (Chiba et al., 2012; VegaRivera et al., 2014). In conclusion, our findings show for the first time that neonatal TS is able to facilitate the activity of antidepressant drugs, particularly SERT, which shows its therapeutic action in a subtherapeutic dose. In addition, hormonal markers of stress such as corticosterone and cortisol, and anxiety-like behaviors decreased with neonatal TS and exacerbated with TS together with a subtherapeutic dose of SERT. Therefore, neonatal TS may be an innovative and promising tool to prevent depressive-like behaviors, ameliorating pharmacological activity of antidepressant drugs.
Acknowledgments The authors are grateful to CNPq, CAPES, FAPERGS and PRPGPUFSM (PROAP) for fellowships and financial support. Authors report no conflicts of interest.
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International Journal of Developmental Neuroscience journal homepage: www.elsevier.com/locate/ijdevneu
Neonatal tactile stimulation decreases depression-like and anxiety-like behaviors and potentiates sertraline action in young rats Daniele Freitas b , Caren T.D. Antoniazzi a , Hecson J. Segat b , Vinícia Garzella Metz c , Luciana Taschetto Vey b , Raquel C.S Barcelos a , Thiago Duarte a , Marta M.M.F. Duarte a,d , Marilise Escobar Burger a,b,c,∗ a
Programa de Pós-Graduac¸ão em Farmacologia, Universidade Federal de Santa Maria, UFSM, RS, Brazil Programa de Pós-Graduac¸ão em Bioquímica Toxicológica, UFSM, RS, Brazil c Departamento de Fisiologia e Farmacologia, UFSM, RS, Brazil d Universidade Luterana do Brasil (ULBRA), Santa Maria, Brazil b
a r t i c l e
i n f o
Article history: Received 14 August 2015 Received in revised form 29 September 2015 Accepted 29 September 2015 Keywords: Neonatal handling Tactile stimulation Forced swimming test Sertraline Anxiety-like behavior Stress
a b s t r a c t It is well known that events which occur in early life exert a significant influence on brain development, what can be reflected throughout adulthood. This study was carried out in order to assess the influence of neonatal tactile stimulation (TS) on behavioral and morphological responses related to depressionlike and anxiety-like behaviors, assessed following the administration of sertraline (SERT), a selective serotonin re-uptake inhibitor (SSRI). Male pups were submitted to daily TS, from postnatal day 8 (PND8) to postnatal day 14 (PND14), for 10 min every day. On PND50, adult animals were submitted to forced swimming training (15 min). On PND51, half of each experimental group (UH and TS) received a single sub-therapeutic dose of sertraline (SER, 0.3 mg/kg body weight, i.p.) or its vehicle (C, control group). Thirty minutes after injection, depression-like behaviors were quantified in forced swimming test (FST, for 5 min). On the following day, anxiety-like behaviors were assessed in elevated plus maze (EPM), followed by biochemical assessments. TS per se increased swimming time, decreasing immobility time in FST. Besides, TS per se was able to increase frequency of head dipping and time spent in the open arms of EPM, resulting in decreased anxiety index. In addition, groups exposed to TS showed decreased plasma levels of corticosterone per se. Interestingly, while TS exposure significantly potentiated the antidepressant activity of a subtherapeutic dose of SERT, this drug was able to exacerbate TS-induced anxiolytic activity, as observed in FST and EPM, respectively. Decreased plasma levels of both corticosterone and cortisol in animals exposed to TS and treated with SERT are able to confirm the interesting interaction between this neonatal handling and the antidepressant drug. From our results, we conclude that neonatal TS is able to exert beneficial influence on the ability to cope with stressful situations in adulthood, preventing depression and favorably modulating the action of antidepressant drugs. © 2015 Published by Elsevier Ltd. on behalf of ISDN.
1. Introduction Depression is a serious and expensive psychiatric disease (Mula, 2013) characterized by feelings of sadness, guilt, worthlessness and hopelessness (Jia et al., 2010; Petersen et al., 2001), affecting about 10–30% of women and 7–15% of men worldwide (Kessler et al., 2005). According to the World Health Organization (WHO),
∗ Corresponding author at: Universidade Federal de Santa Maria, Centro de Ciências da Saúde, Programa de Pós-Graduac¸ão em Farmacologia, 97105-900, Santa Maria, RS, Brazil. E-mail address: [email protected] (M.E. Burger). http://dx.doi.org/10.1016/j.ijdevneu.2015.09.010 0736-5748/© 2015 Published by Elsevier Ltd. on behalf of ISDN.
depression is the fourth leading cause of morbidity in the world with perspectives to become the second by 2020 (Kessler et al., 2011). Although the etiology of depression is considered multifactorial and is not well elucidated yet, its connection with deficiency of brain monoamines activity has been the mainstay for its treatment (Elhwuegi, 2004). Unfortunately, about 50% of patients who start treatment for depression do not respond to antidepressant medication (Thase, 2004), leading us to think about the existence of interferences so far unknown. Tactile stimulation (TS) is a form of neonatal handling similar to the maternal behavior of licking and affection (Schanberg and Field, 1987), which exerts solid influence on normal brain development (Chiba et al., 2012). Some studies show that TS is able
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to affect the hypothalamic–pituitary–adrenal (HPA) axis function (Katsouliet al., 2014), improving the ability of the adult organism to adapt to stressful stimuli (Stamatakis et al., 2009) and helping in the recovery from neonatal brain injuries (Richardset al., 2012; Rodrigues et al., 2004). In addition, studies by our group have shown beneficial influence of TS to prevent preference for psychostimulant drugs (Antoniazzi et al., 2014 a,b), being also able to change responsiveness to benzodiazepine drugs (Boufleur et al., 2012). Sertraline (SERT) is a usual antidepressant drug that belongs to the selective serotonin reuptake inhibitors (SSRIs) (Khan et al., 2005; Young et al., 2009), whose primary mechanism of action is inhibition of the serotonin (5-HT) transporter located in the presynaptic membrane of mesolimbic brain areas (Belmaker and Galila Agam, 2008). Indeed, 5-HT is a neurotransmitter responsible for mood regulation (Peirson and Heuchert, 2000) and for numerous functions in the central and peripheral nervous system (McGeer et al., 1988). In this sense, 5-HT is important in pathogenesis and treatment of different neuropsychiatric disorders including depression (Lucki et al., 1988). In line with this, forced swimming test (FST) is an animal model used to assess depression-like behavior (Cryan and Lucki, 2000), which was firstly described by Porsolt et al. (1978). FST involves two sequential sessions of exposure to deep water, from where animals cannot escape. The first session is in fact a stressor, assumed to induce a state of behavioral despair (Porsolt et al., 1978; Borsini and Meli, 1988; Castagne et al., 2009) interpreted as depression-like symptoms. Considering the promising benefits of TS in early life, the current study was developed to assess the influence of this neonatal handling on the antidepressant activity of SERT in rats. Behaviors involving depression and anxiety-like symptoms were assessed, as well as the activity of HPA axis.
2. Material and methods 2.1. Animals and experimental procedure Seven pregnant female Wistar rats from the breeding facility at Universidade Federal de Santa Maria (UFSM), RS, Brazil, were housed in Plexiglas cages with free access to food and water. Animals were kept in a room with controlled temperature (22–23 ◦ C) on a 12-h light/dark cycle with lights on at 7:00 a.m. The experimental protocol was approved by the Animal Ethics Committee (Universidade Federal de Santa Maria), affiliated to the National Council for the Control of Animal Experimentations (CONCEA), following international norms of care and animal maintenance. All efforts were made to minimize animal suffering and to reduce the number of animals used in experiments. The pups date of birth (postnatal day 0-PND0) was monitored, and gender distinguished by larger genital papilla and longer anogenital distance in male vs. female pups (Liu et al., 2008). On the same day, litters were culled to seven pups (four males and three females to ensure adequate nutritional status (Boufleur et al., 2012; Vazquez et al., 2006)), being only male rats designated to the current experimental protocol. In order to avoid using littermates in the same experimental group, on postnatal day one (PND1) male pups were taken from several litters and randomly assigned to experimental groups (n = 14, each group), separated into unhandled (UH) and tactile stimulation (TS). TS groups received stimulus from PND8 to 14, according to the protocol described below. On PND22, litters were weaned and left undisturbed up to 50 days of age, when they were submitted to training of FST, being immersed in water for 15 min. This procedure works as a stressor which is thought to induce a state of behavioral despair (Porsolt et al., 1978; Castagne et al., 2009) related to a “depressive state” (Borsini and Meli, 1988). On PND51, half (n = 7)
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of each experimental group (UH and TS) received a single dose of sertraline (SERT, 0.3 mg/kg body weight i.p.), which corresponds to a sub-therapeutic dose (following pilot studies at our laboratory), or its vehicle (C-control group). SERT (Pharmanostra, India,) was dissolved in NaCl 0.9% plus 0.05 mL Tween 80 (Sigma–Aldrich, Brazil);vehicle (C) consisted of NaCl 0.9% plus 0.05 mL Tween 80. SERT was the antidepressant drug chosen because it has been frequently reported in studies involving animal models (Ulloa et al., 2010; Mikail et al., 2012; Kaygisiz et al., 2014). Thirty minutes following the injections, animals were submitted to forced swimming test (FST). One hour after FST, anxiety-like symptoms were observed in elevated plus maze (EPM). Following the EPM paradigm, all animals were weighed, anesthetized with sodium pentobarbital (80 mg/kg, i.p.), and euthanized by exsanguination. Blood was collected by cardiac puncture (Fig. 1).
2.2. Neonatal handling Tactile stimulation (TS) consisted of gently stimulating pups’ dorsal surface individually with the index finger from rostral to caudal direction for 10 min (each day) out of the nest by the same experimenter (Antoniazzi et al., 2014a,b; Boufleur et al., 2012; 2013; Rodrigues et al., 2004). TS was applied daily between 12:00 and 14:00 from PND8 until PND14, according to previous studies by our group (data submitted), since most beneficial influence of TS has been observed in pups. After the procedures, pups were returned to their litters, staying the remaining time with their mothers until weaning.
2.3. Behavioral assessments 2.3.1. Forced swimming test (FST) Behavioral responses related to depression-like symptoms are experimentally assessed in FST, whose method has been described in several studies (Porsoltet al., 1978; Wieland and Lucki1990; Castagne et al., 2009). On the first day (PND50) rats were forced to swim for a 15-min period (pretest session), and carefully dried before returning to their home cages. Twenty-four hours following the test (PND51), all rats were injected with SER or vehicle, as described above, and again submitted to FST for 5 min. Immobility, climbing and swimming times were quantified by trained raters blinded to handling and treatment. In this sense, immobility is no additional activity other than the required to keep the head above water, whereas climbing is defined as upward struggling movements of the forepaws at the side of the cylinder. Movements around the swimming cylinder (Porsolt et al., 1978) are indicative of swimming time.
2.3.2. Elevated plus maze (EPM) One hour after FST, anxiety-like symptoms were assessed in the EPM paradigm, which is based on the innate fear rodents have for open and elevated spaces (Montgomery, 1955). The apparatus was made of wood and consisted of a plus-shaped platform elevated 50 cm from the floor. Two opposite arms (50 cm × 10 cm) were enclosed by 40 cm high walls whereas the other two arms had no walls. The four arms had a central platform (10 cm × 10 cm) at their intersection, which gave access to any of the four arms. At the beginning of each test, each animal was individually placed in the central platform facing an open arm. Time spent and number of entries in open and closed arms were monitored during a fiveminute test. A shorter time spent (expressed as a percentage of total length of the test) and fewer entries in the open arms of the maze indicated anxiety (Hlavacova et al., 2010). Total activity in the maze (total number of arm entries) and number of open and closed arms
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Fig. 1. Experimental design: Neonatal handlings were conducted from PND8 until PND14 (n = 14). On PND51 animals received one dose of sertraline (SERT; i.p., n = 7) and were submitted to behavioral assessments. Abbreviations: PND, post natal day; FST, forced swimming test; EPM, elevated plus-maze.
entries was used as anxiety index (Cohen et al., 2007; Mazor et al., 2007). Anxiety index was calculated as follows:
Anxiety index = 1 − n
Time spent in the open arms Total time on the maze
Number of open arm entries +
Total entries on the maze
2
Anxiety index values range from 0 to 1 where an increase in the index expresses increased anxiety-like behavior (Cohen et al., 2007; Mazor et al., 2007). The apparatus was cleaned with a 20% alcohol solution using wet sponge and paper towel before the introduction of each animal. Observers were blinded to treatment during all behavioral observations. 2.4. Biochemical assessments 2.4.1. Corticosterone and cortisol assessments Blood aliquots were centrifuged at 4 ◦ C at 1500×g for 15 min (n = 5). Plasma aliquots were frozen at −80 ◦ C until their analysis. Corticosterone and cortisol plasma levels were analyzed through enzyme immunoassay (ELISA) using a commercial kit according to the manufacturer’s instructions (Immuno Biological Laboratories). 2.5. Statistical analysis Levene’s test was used in order to verify data distribution. Two-way ANOVA followed by Duncan’s post-hoc test analyzed parametric data, using Statistica 8.0 software. Values of P < 0.05 were considered statistically significant for all comparisons made. 3. Results 3.1. Forced swimming test (FST) Two-way ANOVA of FST revealed a significant main effect of handling on climbing, swimming and immobility time[F(1,24) = 10.04, 11.26 and 37.87, p < 0.05 respectively], as well as a significant main effect of drug on swimming and immobility time [F(1,24)=10.54 and 6.27, p<0.05, respectively]. TS per se increased swimming time (Fig. 1B), decreasing immobility time (Fig. 1C) in FST of both control and SERT treated groups in comparison to UH group. Interestingly, SERT administration was able to increase swimming time in UH group when compared to its respective control (Fig. 1B). In fact, among SERT treated groups, animals exposed to TS showed higher climbing time and lower immobility time in FST (Fig. 2A–C). 3.2. Elevated plus maze (EPM) Two-way ANOVA of EPM revealed a main effect of handling[F(1,24) = 92.22; 5.55; 33.66 and 24.08, p < 0.05] and a main effect of drug [F(1,24) = 57.48; 6.78; 16.77 and 18.34, p < 0.05]on time spent, number of entries and frequency of head dipping in
the open arms as well as on anxiety index, respectively. Two-way ANOVA also revealed a significant handling × drug interaction on time spent and frequency of head dipping in the open arms as well as on anxiety index [F(1,24) = 21.99; 4.19 and 0.96, p < 0.05], respectively. TS exposure per se was able to increase time spent (Fig. 2A) and frequency of head dipping (Fig. 2C) in the open arms of EPM, decreasing the anxiety index (Fig. 2D) in relation to UH group. On the other hand, SERT treatment was able to increase time spent (Fig. 2A), number of entries (Fig. 2B) and frequency of head dipping (Fig. 2C) in the open arms, decreasing the anxiety index (Fig. 2D) in TS in comparison to its correspondent control group. Among SERT-treated groups, TS exposure exacerbated all these behavioral parameters observed in EPM, when compared to UH group (Fig 3A–D). No influence of UH or TS groups on number of total entries in both open and closed arms of EPM was observed (Data no shown)
3.3. Corticosterone and cortisol Two-way ANOVA revealed a main effect of handling [F(1,24) = 66.06 and 12.59; p < 0.05], drug [F(1,24) = 3.83; 157.89 and 73.91; p < 0.05] on plasma levels of corticosterone and cortisol, respectively, and a significant handling × drug interaction [F(1,24) = 11.38; p < 0.05] on plasma levels of corticosterone, respectively. While TS exposure per se was able to decrease plasma levels of corticosterone, cortisol was similar to UH group. On the other hand, SERT administration decreased plasma levels of both corticosterone and cortisol in relation to their respective controls, regardless of handling. In fact, the effects of SERT on rats exposed to TS were exacerbated, since this experimental group showed lower corticosterone and cortisol than UH group, also treated with SERT (Table 1).
Table 1 Influence of a subtherapeutic dose of sertraline (SERT) on corticosterone (ng/mL) and cortisol (ng/mL) plasma levels in rats previously submitted to tactile stimulation (TS) in comparison to unhandled (UH) animals. Group
Corticosterone (ng/mL)
Cortisol (ng/mL)
UH-C TS-C UH-SERT TS-SERT
83,40 ± 2,29 73,25 ± 1,18* 64,20 ± 2,72+ 41,20 ± 1,07*,+
190,00 ± 3,27 182,50 ± 3,42 162,80 ± 4,66+ 142,60 ± 3,77*,+
Abbreviations: UH, unhandled; TS, tactile stimulation; SERT, Sertraline; C, Control. Data are expressed as mean ± SEM. (n = 5). * Significant difference from UH group in the same treatment. + Significant difference from Control in the same handling (p < 0.05).
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Fig. 2. Antidepressant activity of a subtherapeutic dose of sertraline (SERT) in rats previously submitted to tactile stimulation (TS) in comparison to unhandled (UH) animals in forced swimming test (FST). Depression-like behavior were assessed using climbing time (A), swimming time (B) and immobility time (C). Data are expressed as mean ± S.E.M. *Significant difference from UH group in the same treatment (p < 0.05). **Significant difference from vehicle in the same handling (p < 0.05).
4. Discussion The current study was developed to assess the influence of neonatal TS per se on behavioral, biochemical and morphological parameters related to anxiety and depression-like behavior, as well as the possible influence of this neonatal handling on the effects of SERT, which is a frequently used therapeutic tool to treat neuropsychiatric conditions such as dysthymia and major depression. TS may prevent emotional disturbances in response to early adversities (Imanaka et al., 2008), being also able to change the hypothalamic–pituitary–adrenal (HPA) axis function, modifying
the ability of the organism to respond, cope and adapt to stressful stimuli (Ladd et al., 2000; Levine, 1957). Our research group has recently shown that animals exposed to neonatal TS were less anxious and more resistant to stressful events (Boufleur et al., 2013), indicating that TS may facilitate the activity of benzodiazepine drugs, particularly diazepam (Boufleur et al., 2012). Interestingly, the current study shows that neonatal TS was able to decrease depression-like behavior, as observed by the increased swimming time and the decreased immobility time in FST. This animal model has been widely used to assess potential activity of antidepressant drugs (Cryan et al., 2005; Shaw et al., 2007; Mikail
Fig. 3. Influence of a subtherapeutic dose of sertraline (SERT) on anxiety-like behavior in rats previously submitted to tactile stimulation (TS) in comparison to unhandled (UH) animals in elevated plus maze (EPM). Anxiety-like behavior were assessed by time spent (sec) in the open arms (A), number of entries in the open arms (B), frequency of head dipping (C) and anxiety index (D). Data are expressed as mean ± S.E.M. *Significant difference from UH group in the same treatment (p<0.05). **Significant difference from vehicle in the same handling (p < 0.05).
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et al., 2012). In this sense, decreased swimming time together with increased immobility time in FST are indicators of depressive behavior, which may be reversed by antidepressant drugs (Cryan et al., 2005; Porsolt et al., 1977). Taken together, our results suggest that TS in early life is able to exert beneficial influence per se on depressive parameters, thus facilitating the activity of SERT in adulthood. Additionally, our findings also demonstrated that animals exposed to neonatal TS showed reduced anxiety-like behavior per se, as observed by increased time spent and frequency of head dipping in the open arms of EPM, thus resulting in a lower anxiety index in the EPM task. In opposition, when decreased, these behaviors are indicators of fear and anxiety, resulting in an increased anxiety index, as previously described (Hlavacona et al., 2010). Indeed, the beneficial influences of TS on anxiety parameters, as observed in the current study, are not innovative findings since previous studies by our group evidenced similar behaviors in rats exposed to neonatal TS (Antoniazzi et al., 2014a,b; Boufleur et al., 2013), consistently reinforcing the anxiolytic properties of this neonatal handling. Neonatal TS has an antidepressant-like activity per se, also facilitating the activity of SERT, and this drug apparently contributes with lower TS-induced anxiety and emotionality, since (i) time spent, number of entries and frequency of head dipping in the open arms of EPM were significantly higher in TS group treated with SERT, indicating a favorable interactive modulation between drug and neonatal handling; (ii) decreased hormone secretion from HPA axis following SERT administration in TS-exposed group was also indicative of lower emotionality and stress; (iii) TS per se decreased plasma corticosterone levels, confirming beneficial influence of this neonatal handling on neuroendocrine function. While manipulations during early life may affect adrenocortical function in adult rats (Daskalakis et al., 2009), increased adrenal weight and elevated levels of circulating glucocorticoids may be considered markers of stress (Heiderstadt et al., 2000), as previously described. Of particular importance, these changes may intensify central and peripheral pathological symptoms such as anxiety and depression-like behaviors (Korte, 2001; Mitchell and O’Keane, 1998; McEwen, 2000; Erickson et al., 2003). Furthermore, an experimental study has shown that rats exposed to maternal separation presented increased basal corticosterone and decreased corticotrophin (ACTH) levels following acute stress (Marais et al., 2008). Our study has included both corticosterone and cortisol, which are regulated in the same way and simultaneously released, indicating more reliability and consistence (Saito et al., 1992). In fact, studies involving FST have shown a relationship between increased secretion of glucocorticoids and depressive behavior in rodents, reversed by antidepressant drugs (Chiba et al., 2012; VegaRivera et al., 2014). In conclusion, our findings show for the first time that neonatal TS is able to facilitate the activity of antidepressant drugs, particularly SERT, which shows its therapeutic action in a subtherapeutic dose. In addition, hormonal markers of stress such as corticosterone and cortisol, and anxiety-like behaviors decreased with neonatal TS and exacerbated with TS together with a subtherapeutic dose of SERT. Therefore, neonatal TS may be an innovative and promising tool to prevent depressive-like behaviors, ameliorating pharmacological activity of antidepressant drugs.
Acknowledgments The authors are grateful to CNPq, CAPES, FAPERGS and PRPGPUFSM (PROAP) for fellowships and financial support. Authors report no conflicts of interest.
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